activation of the plasma kallikrein-kinin system...
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ACTIVATION OF THE PLASMA KALLIKREIN-KININ SYSTEM ON
RESPIRATORY EPITHELIUM AND PLEURAL MESOTHELIUM
Julius Francesco Varano della Vergiliana
This thesis is presented for the degree of Doctor of Philosophy in the Discipline of
Microbiology and Immunology, the School of Biomedical, Biomolecular and Chemical
Sciences from the University of Western Australia
2010
i
DECLARATION
The work presented in this thesis was performed solely by the author except where
otherwise stated and has not been submitted previously for any other degrees.
Julius Francesco Varano della Vergiliana (Student)
Geoffrey A. Stewart (Coordinating supervisor)
ii
ACKNOWLEDGMENTS
Firstly, I would like to acknowledge my supervisors, Prof. Geoffrey Stewart, Dr.
Nithiananthan Asokananthan and Dr. Anthony Bakker. Your guidance and support
throughout the duration of my PhD is greatly appreciated.
I would like to thank Prof Y C Gary Lee, Dr. Sally Lansley and Ms. Ai Ling Tan for their
guidance and support with my pleural mesothelial cell project. Thanks also to Jenette
Creaney for providing the pleural effusion and serum samples, and Dr Bahareh Badrian and
Ms. Hui Min Cheah for providing the primary human mesothelioma cells.
Thanks to the staff of the Centre of Microscopy, Characterisation and Analysis, in
particular Kathy Heel and Tracey Lee-Pullen for their practical guidance and advice with
my flow cytometry and fluorescence microscopy work.
Great thanks to Peng Kai Soh, Siew Ping Lai, Tina Chan, Grace Chen, Saima Majeed and
Royce Ng. Your friendship has made my time in this lab truly enjoyable and memorable.
Lastly, I would to thank my family and friends, especially Sofia, Andrew and Trina, for all
their support.
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SUMMARY
The plasma kallikrein-kinin system (KKS) is a cell-associated proteolytic mechanism of
activation resulting in the release of the inflammatory peptide, bradykinin (BK). This
process involves assembly of high molecular weight kininogen (HK) and plasma
prekallikrein (PPK), and activation of the HK-PPK complex by prolylcarboxypeptidase
(PRCP) or heat shock protein 90 (HSP90). Activation of the HK-PPK complex results in
conversion of PPK to plasma kallikrein (PK) which, in turn, liberates BK from HK. This
system has been comprehensively described on endothelial cells and more recently other
cells have been shown to possess such a system. However, the significance of the plasma
KKS on respiratory epithelium and pleural mesothelium is unclear. As such, this thesis
describes the assembly and activation of the plasma KKS on these cell types.
The A549 and BEAS-2B respiratory epithelial cell lines were shown to express known
endothelial HK receptor-associated proteins namely, urokinase plasminogen activator
receptor (uPAR), cytokeratin 1 (CK1) and gC1qR, but not Mac-1. Additionally, A549 cells
bound FITC-labeled HK, which was inhibited by EDTA or 50-fold molar excess unlabeled
HK. However, binding of FITC-HK was only weakly inhibited following pre-treatment
with individual antibodies against uPAR, gC1qR or CK1, although about 45% inhibition
was achieved when the antibodies were used in combination. In addition, sodium chlorate
treatment had no effect on FITC-HK binding, indicating sulphated proteoglycans were not
involved. PK activity was generated following sequential treatment of A549 and normal
human bronchial epithelial (NHBE) cells with HK and PPK, resulting in the release of BK
from HK. Similar results were also observed using A549 cell-free matrix and lysate.
Activation of PPK on A549 and NHBE cells was inhibited by cysteine, BK, protamine
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sulphate and the HSP90 inhibitor, novobiocin. However, the lack of activity of other
protease inhibitors including antipain, leupeptin and AEBSF and the known PRCP
substrate inhibitor, angiotensin II (ANG II), indicated this protease may not be involved in
activating PPK on respiratory epithelium. Ex vivo, activated Factor XII (FXIIa) has been
shown to directly activate PPK, but a neutralising antibody against FXIIa had negligible
effect on PPK activation. Plasma KKS activation was also demonstrated on epithelial cells
derived from the prostate and gut, as well as murine myoblasts, myotubes, human
fibroblasts, monocytes and mast cells.
In a parallel study, the significance of the plasma KKS on mesothelial cells was examined.
BK was detected in pleural effusions from patients with a variety of clinical conditions, and
concentrations were higher in a large proportion of effusions compared to matched serum
samples, indicating the presence of localised kinin production in the pleural space. BK
concentrations in pleural effusions did not differ between disease groups, but were
significantly elevated in patients with exudative effusions. Plasma KKS activation was
demonstrated using benign and transformed mesothelial cells lines, and primary cells
obtained from mouse omentum and from patients with malignant mesothelioma. Incubation
of the MeT-5A mesothelial cell line with pleural effusions generated PK activity,
suggesting the presence of plasma KKS components. PPK activation was moderately
sensitive to inhibition by antipain, leupeptin, 2-ME and AEBSF, but strongly inhibited by
cysteine, BK, protamine sulphate and novobiocin. MeT-5A, NCI-H28 and NCI-H2052
human mesothelial cell lines expressed cell surface HSP90, but not PRCP or FXII.
Furthermore, BK, but not des-Arg9-BK, induced calcium mobilisation in mesothelial cells,
but neither had any effect on cytokine or chemokine release.
v
As kallikreins are known to directly activate B2R and protease activated receptors (PARs),
the role of kallikreins as signaling molecules on mesothelial cells was also assessed.
Porcine tissue kallikrein, an ortholog of human tissue kallikrein, but not PK or trypsin-
activated PPK, induced calcium mobilisation in MeT-5A cells. These cells were shown to
express all four PARs, and calcium mobilisation was induced by thrombin, trypsin and
agonist peptides (APs) of PAR1 and PAR2, but not PAR3 or PAR4. Additionally, BK-
induced calcium mobilisation was inhibited by the B2R antagonists, Hoe 140, indicating the
presence of functional B2R on MeT-5A cells. Porcine kallikrein-induced calcium
mobilisation was not inhibited by pre-treatment with Hoe 140, and cross-desensitisation
was not observed using BK, indicating a lack of involvement of B2R. However, cross-
desensitisation between porcine kallikrein and trypsin and PAR2 AP was demonstrated,
indicating the protease signals through PAR2 on MeT-5A cells.
In summary, the studies reported in this thesis indicate that the respiratory epithelium and
pleural mesothelium can function as sites of local BK formation. Therefore, plasma KKS
activation on these tissues may contribute to inflammatory disease given the biological
significance of kinins and their relevance within the lung.
vi
PUBLICATIONS RESULTING FROM THESIS
1. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. (2010).
Activation of the plasma kallikrein-kinin system on human lung epithelial cells.
Biol Chem 391; [Epub adhead of print].
2. Varano della Vergiliana, J.F., Lansley, S., Tan, A.L., Creaney, J., Lee, Y.C.G. and
Stewart, G.A. (2010). Activation of the plasma kallikrein-kinin system on pleural
mesothelial cells.
To be submitted to European Respiratory Journal.
3. Varano della Vergiliana, J.F. and Stewart, G.A. (2010). Kallikrein activates
protease-activated receptor 2 (PAR2) on pleural mesothelial cells.
In preparation.
POSTER AND ORAL PRESENTATIONS
1. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Binding of
high molecular weight kininogen to respiratory epithelial cells. Combined
Biological Science Meeting, Perth, Western Australia, 2007. Poster presentation.
2. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Binding of
high molecular weight kininogen to respiratory epithelial cells. University of
Western Australia, School of Biomedical, Biomolecular and Chemical Sciences
vii
Research Symposium, Perth, Western Australia, 2007. Poster presentation. Best
poster prize for first year PhD student.
3. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Activation of
the plasma kallikrein-kinin system on respiratory epithelial cells. Lung Institute of
Western Australia meeting, Perth, Western Australia, 2008. Oral presentation.
4. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Binding of
high molecular weight kininogen to respiratory epithelial cells. European
Respiratory Society annual congress, Berlin, Germany, 2008. E-communication
poster presentation.
5. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Activation of
the plasma kallikrein kinin system on respiratory epithelial cells. Joint University of
Western Australia and HeJie University of Science and Technology meeting, China,
2008. Oral presentation performed by G. A. Stewart.
6. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. The plasma
kallikrein-kinin system on respiratory epithelium. Lung Institute of Western
Australia lung club meeting, Perth, Western Australia, 2009. Oral presentation.
7. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Activation of
the plasma kallikrein-kinin system on respiratory epithelial cells. European
Respiratory Society annual congress, Barcelona, Spain, 2010. Poster presentation.
viii
8. Varano della Vergiliana, J.F., Asokananthan, N. and Stewart, G.A. Activation of
the plasma kallikrein-kinin system on respiratory epithelial and pleural mesothelia
cells. Lung Institute of Western Australia lung club meeting, Perth, Western
Australia, 2010. Oral presentation to be performed on August 27th
.
ix
ABBREVIATIONS
°C Degrees Celsius
2-ME 2-mercaptoethanol
AA Arachidonic acid
AEBSF 2-aminoethyl benzenesulphonyl fluoride
ACE Angiotensin converting enzyme
ALI Acute lung injury
AM Acetoxymethyl
AMP Anti-microbial peptides
ANG 1-7 Angiotensin fragment 1-7
ANG I Angiotensin I
ANG II Angiotensin II
AP Agonist peptide
APS Ammonium persulphate
ARDS Acute respiratory distress syndrome
B1R Bradykinin receptor subtype 1
B2R Bradykinin receptor subtype 2
BAL Bronchoalveolar lavage
BEGM Bronchial epithelial cell growth medium
BK Bradykinin
BK 1-5 Bradykinin fragment 1-5
BK 1-7 Bradykinin fragment 1-7
BSA Bovine serum albumin
C1-INH C1 inhibitor
x
CaCl2 Calcium chloride
CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane regulator
CK1 Cytokeratin 1
COPD Chronic obstructive pulmonary disease
CP Control peptide
CPN Carboxypeptidase N
CTI Corn trypsin inhibitor
DAB 3, 3’ Diaminobenzidine
DAG Diacylglycerol
DAPI 4’,6-Diamidino-2-phenylindole
DC Dendritic cells
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethyl sulphoxide
DTT Dithiothreitol
EDTA Ethylenediamine tetra-acetic acid
EGFR Epidermal growth factor receptor
EIA Enzyme immunoassay
ELISA Enzyme-linked immunosorbant assay
ERK Extracellular signal-regulated kinase
FCS Fetal calf serum
FITC Fluorescein isothiocyanate
FXI Factor XI
FXIa Activated Factor XI
FXII Factor XII
xi
FXIIa Activated Factor XII
FXIIf Factor XII fragment
g Gram
GPCR G-protein-coupled receptor
HBBS Hank’s balanced salt solution
HCl Hydrochloric acid
HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid
hK Human kallikrein
HK High molecular weight kininogen
HKa Two-chain high molecular weight kininogen
Hoe 140 Icatibant acetate
HRP Horse radish peroxidase
HSP90 Heat shock protein 90
HUVEC Human umbilical vein endothelial cells
Ig Immunoglobulin
IgG Immunoglobulin gamma
IL-6 Interleukin-6
IL-8 Interleukin-8
KCl Potassium chloride
kDa Kilodalton
KH2PO4 Potassium dihydrogen orthophosphate
KKS Kallikrein-kinin system
KLK Kallikrein-related peptidase
L Litre
LBTI Lima bean trypsin inhibitor
xii
LHC-9 Laboratory of Human Carcinogenesis medium-9
LK Low molecular weight kininogen
LPS Lipopolysaccharide
M Molar
MCP-1 Monocyte chemotactic protein-1
mDC monocyte-derived dendritic cells
MgCl2 Magnesium chloride
MgSO4 Magnesium sulphate
min Minute
ml Millilitre
mM Millimolar
MW Molecular weight
NA Not available
Na2CO3 Sodium carbonate
Na2HPO4 Di-sodium hydrogen orthophosphate
NaCl Sodium chloride
NaHCO3 Sodium hydrogen carbonate
NaOH Sodium hydroxide
NEP Neutral endopeptidase
NFκB Nuclear factor κ B
ng Nanogram
NHBE Normal human bronchial epithelial
NH4OH Ammonium hydroxide
nM Nanomolar
nm Nanometer
xiii
NO Nitric oxide
O/N Overnight
PAGE Polyacrylamide gel electrophoresis
PAMP Pathogen-associated molecular pattern
PAR Protease-activated receptor
PBS Phosphate buffered saline
pg Picogram
PGE2 Prostaglandin E2
PGI2 Prostaglandin I2
PK Plasma kallikrein
PKC Protein kinase C
PLC Phospholipase C
PMA Phorbol myristate acetate
PMSF Phenylmethylsulphonyl fluoride
pNA p-nitroaniline
pNNP p-nitrophenylphosphate
PPK Plasma prekallikrein
PRCP Prolylcarboxypeptidase
RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute
RT Room temperature
SDS Sodium dodecyl sulphate
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
sec Second
SEM Standard error of the mean
xiv
TEMED N, N, N’, N’ tetramethylethylenediamine
TGF Transforming growth factor
TK Tissue kallikrein
TLR Toll-like receptor
TMB Tetramethylbenzidine
TNF- Tumour necrosis factor-alpha
tPA Tissue plasminogen activator
TPCK N-tosyl-L-phenylalanine chloromethyl ketone
uPA Urokinase plasminogen activator
uPAR Urokinase plasminogen activator receptor
UV Ultraviolet
v/v volume per volume
w/v weight per volume
x Times
ZnCl2 Zinc chloride
2M Alpha-2 macroglobulin
μg Microgram
μl Microlitre
μM Micromolar
μm Micrometer
xv
TABLE OF CONTENTS
CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.1.1 The respiratory epithelium ............................................................................. 2
1.1.2 The pleural mesothelium ................................................................................ 3
1.1.3 The kallikrein-kinin systems .......................................................................... 4
1.2.1 High and low molecular weight kininogen .................................................... 5
1.2.2 PPK ................................................................................................................ 6
1.4.1 Activation of PPK by FXIIa .......................................................................... 9
DECLARATION ................................................................................................................ i
ACKNOWLEDGMENTS ................................................................................................ ii
SUMMARY ...................................................................................................................... iii
PUBLICATIONS RESULTING FROM THESIS ........................................................ vi
POSTER AND ORAL PRESENTATIONS ................................................................... vi
ABBREVIATIONS .......................................................................................................... ix
TABLE OF CONTENTS ................................................................................................ xv
LIST OF FIGURES ....................................................................................................... xxi
LIST OF TABLES ....................................................................................................... xxiv
1.1 Introduction ................................................................................................................. 1
1.2 Kininogens and PPK ................................................................................................... 5
1.3 HK binds receptors on cell surfaces .......................................................................... 7
1.4 Activation of PPK on cellular surfaces ..................................................................... 9
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1.4.2 Activation of PPK requires cell-bound HK ................................................. 10
1.4.3 Activation of PPK by prolylcarboxypeptidase ............................................. 10
1.4.4 Activation of PPK by heat shock protein 90 ................................................ 11
1.8.1 The plasma KKS and anti-thrombotic activities .......................................... 15
1.8.2 The plasma KKS and pro-fibrinolytic activities .......................................... 16
1.8.3 The plasma KKS and inflammatory mediator synthesis .............................. 16
1.8.4 The plasma KKS and vasodilation ............................................................... 18
1.8.5 The plasma KKS and vascular permeability ................................................ 19
1.8.6 The plasma KKS and cellular migration ...................................................... 20
1.8.7 The plasma KKS and cellular maturation and differentiation ..................... 21
1.8.8 The plasma KKS and anti-microbial effects ................................................ 21
1.8.9 The direct effects of kallikreins.................................................................... 23
CHAPTER 2 MATERIALS
2.2.1 General buffers ............................................................................................. 29
2.2.2 Acid phosphatase assay ................................................................................ 31
1.5 Proteolysis of HK....................................................................................................... 12
1.6 Kinin metabolism ...................................................................................................... 13
1.7 BK receptors .............................................................................................................. 13
1.8 Biological effects of the plasma KKS ....................................................................... 14
1.9 Plasma KKS assembly and activation on other cell types ..................................... 23
1.10 The plasma KKS and the respiratory epithelium and pleural mesothelium ..... 24
1.11 Aims of the thesis ..................................................................................................... 26
2.1 General chemicals and specific reagents ................................................................. 29
2.2 Buffers and solutions ................................................................................................ 29
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2.2.3 Cell culture reagents ..................................................................................... 32
2.2.4 Chromogenic assays for kallikrein proteases ............................................... 33
2.2.5 Enzyme-linked immunosorbant assay (ELISA) .......................................... 34
2.2.6 Calcium mobilisation ................................................................................... 34
2.2.7 Immunocytochemistry studies ..................................................................... 35
2.2.8 Matrix preparation ........................................................................................ 37
2.2.9 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)38
CHAPTER 3 METHODS
3.1.1 Deparrifinisation and rehydration of tissue sections .................................... 42
3.1.2 Antigen retrieval........................................................................................... 42
3.1.3 Immunohistochemistry and immunocytochemistry ..................................... 42
3.1.4 Co-localisation studies using sequential indirect immunofluorescence ...... 44
3.3.1 Spectrophotometric analysis of calcium mobilisation ................................. 45
3.3.2 Flow cytometric analysis of calcium mobilisation ...................................... 45
2.3 Antibodies .................................................................................................................. 39
2.4 Mammalian cells........................................................................................................ 40
2.5 Human lung tissue sections ...................................................................................... 41
2.6 PAR agonist (AP) and control (CP) peptides ......................................................... 41
3.1 Immunocytochemistry and immunocytochemistry ............................................... 42
3.2. Flow cytometric analysis of receptor expression ................................................... 44
3.3 Analysis of calcium mobilisation ............................................................................. 45
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3.8.1. Activation on cell surfaces .......................................................................... 49
3.8.2. PPK activation by cell lysates ..................................................................... 50
3.8.3 PPK activation by cell-free matrices ............................................................ 51
3.9.1 Competitive BK EIA .................................................................................... 51
3.9.2 IL-6 and IL-8 enzyme-linked immunosorbant assay (ELISA) .................... 52
3.9.3 TNF- and monocyte chemotactic protein (MCP)-1 ELISA ...................... 53
3.10.1 Cell culture conditions ............................................................................... 54
3.10.2 Pre-coating of tissue culture plates with rat tail collagen and gelatin ........ 54
3.10.3 Propagation of cell lines ............................................................................. 54
3.10.4 Myotube differentiation of C2C12 cells ...................................................... 55
3.10.5 Isolation of human and murine primary mesothelial cells ......................... 55
3.10.6 Determination of cell count and viability................................................... 56
3.10.7 Storage of cells by freezing ........................................................................ 57
3.11.1 Serum starvation and stimulation ............................................................... 57
3.11.2 Determination of cell viability ................................................................... 57
3.4 Determination of protein concentration.................................................................. 46
3.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ...... 46
3.6 Fluorescein isothiocyanate (FITC) conjugation o f HK......................................... 47
3.7 FITC-HK binding to cells ......................................................................................... 48
3.8 Activation of PPK ...................................................................................................... 49
3.9 Enzyme immunoassays (EIA) .................................................................................. 51
3.10 Cell culture ............................................................................................................... 54
3.11 Cell stimulation ....................................................................................................... 57
3.12 Pleural fluid samples ............................................................................................... 58
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3.12.1 Subjects ...................................................................................................... 58
3.12.2 Pleural fluid collection ............................................................................... 59
CHAPTER 4 ACTIVATION OF THE PLASMA KALLIKREIN-KININ
SYSTEM ON RESPIRATORY EPITHELIAL CELLS
4.2.1 Expression and co-localisation of the known HK receptor proteins on
respiratory epithelial cells ..................................................................................... 63
4.2.2 Binding of FITC-labeled HK to respiratory epithelial cells......................... 63
4.2.3 Inhibition of FITC-labeled HK binding to respiratory epithelial cells ........ 64
4.2.4 PPK activation and liberation of BK ............................................................ 64
4.2.5 Inhibition of PPK activation ........................................................................ 65
4.2.5.1 Inhibition of PPK activation on respiratory epithelial cells ..................... 65
4.2.5.2 Inhibition of PPK activation on A549 cell-free matrix and lysate ............ 67
4.2.5.3 Inhibition of trypsin-activated PPK activity ............................................. 67
4.2.6 Plasma KKS activation on epithelia derived from tissues other than human
lung and non-epithelial cells ................................................................................. 67
CHAPTER 5 ACTIVATION OF THE PLASMA KALLIKREIN-KININ
SYSTEM ON PLEURAL MESOTHELIAL CELLS
5.2.1 BK in pleural effusions ................................................................................ 79
5.2.2 PPK activation on mesothelial cells ............................................................. 80
5.2.3 Inhibition of PPK activation on mesothelial cells ........................................ 81
3.13 Statistical analysis ................................................................................................... 59
4.1 Introduction ............................................................................................................... 60
4.2 Results ........................................................................................................................ 63
4.3 Discussion ................................................................................................................... 69
4.4 Summary .................................................................................................................... 77
5.1 Introduction ............................................................................................................... 77
5.2 Results ........................................................................................................................ 79
xx
5.2.4 Mesothelial cells express HSP90, but not PRCP or FXII ............................ 81
5.2.5 The effect of BK and des-Arg9-BK on calcium mobilisation, and cytokine and
chemokine release from mesothelial cells............................................................. 82
CHAPTER 6 THE ROLE OF THE B2R AND PROTEASE-ACTIVATED
RECEPTORS IN KALLIKREIN SIGNALING IN
MESOTHELIAL CELLS
6.2.1 The effect of tissue and plasma KKS associated enzymes on calcium
mobilisation in MeT-5A cells ............................................................................... 92
6.2.2 Expression and functionality of PARs and B2R on MeT-5A cells .............. 92
6.2.3 Specificity of PAR1 and PAR2 on mesothelial cells .................................... 93
6.2.4 The effect of porcine kallikrein on B2R and PARs ...................................... 93
CHAPTER 7 GENERAL DISCUSSION AND FUTURE PERSPECTIVES
APPENDIX I COHORT 1 AND 2 PATIENT CHARACTERISTICS
APPENDIX II PUBLICATIONS
5.3 Discussion ................................................................................................................... 83
5.4 Summary .................................................................................................................... 90
6.1 Introduction ............................................................................................................... 90
6.2 Results ........................................................................................................................ 92
6.3 Discussion ................................................................................................................... 94
6.4 Summary .................................................................................................................. 100
7.1 General discussion and future perspectives .......................................................... 101
REFERENCES .............................................................................................................. 108
CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
1
1.1 Introduction
The respiratory epithelium extends as a monolayer of cells throughout the airways and
directly interfaces with the external environment to provide a protective barrier to the
underlying parenchyma. In addition, the respiratory epithelium coordinates various
pulmonary functions including mucocilliary clearance, surfactant and mucous secretion,
and gas exchange (Thompson et al., 1995). As with the respiratory epithelium, the
mesothelium consists of a monolayer of specialised epithelial cells of mesodermal origin,
which line the pleural, peritoneal and pericardial cavities. Within the pleural space, the
mesothelium secretes glycosaminoglycans and surfactant to provide a non-adhesive
membrane to allow the free movement of apposing organs, while supporting the passage of
fluid, cells and particulates across the pleura (Mutsaers and Wilkosz, 2007).
Both the respiratory epithelium and pleural mesothelium also play a role in regulating
inflammatory events within the lung. By modulating the activity of luminal, parenchymal
and vascular cells, the epithelium and mesothelium help preserve the structural and
functional integrity of this tissue. Therefore, these cells represent key effectors in various
pathophysiological processes and contribute to inflammation associated with a variety of
clinical conditions. Despite this realisation, the mechanisms by which the epithelium and
mesothelium contribute to inflammatory lung disease require further examination. In this
regard, the kallikrein-kinin systems (KKS) have emerged as important inflammatory
pathways responsible for the generation of kinins. As such, the participation of KKS on the
epithelium and mesothelium is of interest, given the significance of kinins in pulmonary
disease (Akbary et al., 1996, Saleh et al., 1997, Saleh et al., 1998), and the detection of
KKS components in airway (Proud et al., 1983, Baumgarten et al., 1986, Christiansen et
2
al., 1992) and pleural (Uchida et al., 1983) fluids. With regard to the lung, the tissue KKS
is the most studied kinin-forming system, whereas little attention has been directed to
identifying a role for the plasma KKS. This, then, forms the central aim of the thesis.
1.1.1 The respiratory epithelium
As an indirect consequence of inhalation, the human lung is exposed to a wide array of
environmental or chemical insults, including fumes, gases and biological and particulate
matter and, thus, the respiratory epithelium has developed numerous strategic functions to
maintain pulmonary homeostasis (Bals and Hiemstra, 2004). The presence of tight junction
proteins enables the epithelium to form a continuous lining within the lung, which results in
an impermeable barrier to infectious and other material. Thus, the epithelium protects the
underlying cells, while allowing directed and controlled movement of ions and fluids across
the apical surface. Simultaneously, mechanical clearance of inhaled and aspirated matter is
mediated by cough (distal airways) or ciliary activity on mucus-trapped particulates
(proximal airway). However, the respiratory epithelium also plays a role in initiating
immune function and actively participates in protecting the host against infectious agents,
particularly via the innate arm (Thompson et al., 1995).
The immunological competence of the respiratory epithelium is exemplified by the plethora
of cytokines (Cromwell et al., 1992), chemokines (Stellato et al., 1997, Sauty et al., 1999)
and lipid mediators (Asokananthan et al., 2002, Richardson et al., 2005) released in
response to a host of stimuli. Additionally, the epithelium expresses a variety of receptors
that allow it to detect and respond to extracellular ligands (e.g., microbial products,
proteases, cytokines), as well as supporting the development of inflammatory foci through
3
leukocyte adhesion (Suzuki et al., 2008). As the respiratory epithelium interfaces with the
external environment, the innate immune response is of critical importance, and a rapid and
sturdy response may be produced. However, these responses must be tightly regulated to
avert the detrimental effects of uncontrolled responses to pathogens and innocuous
material. Failure to maintain the immunological integrity of the epithelium may form the
basis of various pathological conditions, including asthma (Holgate, 2007, Prefontaine and
Qutayba, 2007), chronic obstructive pulmonary disease (COPD) (Thorley and Tetley, 2007)
and adult respiratory distress syndrome (Wang et al., 2007).
1.1.2 The pleural mesothelium
The mesothelium is also a metabolically dynamic membrane which co-ordinates numerous
inflammatory events essential to maintaining homeostasis (Mutsaers, 2002). Although the
pleural mesothelium is intimately connected with the underlying lung parenchyma, unlike
the respiratory epithelium, it does not interface with the external environment. Despite this,
however, it plays a role in the innate defense of the pleural space and, as such, participates
in inflammatory responses following the introduction of environmental or chemical insults
to, or mechanical disruption of, the mesothelial layer (Jantz and Antony, 2008). As with the
respiratory epithelium, the mesothelium produces a variety of inflammatory molecules
including cytokines (Lanfrancone et al., 1992), chemokines (Li et al., 1998, Visser et al.,
1998), lipid mediators (Hott et al., 1994, Topley et al., 1994) and growth factors (Jayne et
al., 2000), and expresses an array of receptors (Hussain et al., 2008) and adhesion
molecules (Jonjic et al., 1992). As with the epithelium, dysfunction of the pleural
mesothelium contributes to various diseases, including effusion development (Jantz and
Antony, 2008), serosal adhesions and mesothelioma (Mutsaers, 2004).
4
1.1.3 The kallikrein-kinin systems
The tissue and plasma KKS are the two major pathways of kinin formation. The tissue
KKS represents the least complex kinin-forming system as it involves only two
components namely, human kallikrein (hK)1 and low molecular weight kininogen (LK).
hK1 is secreted from a variety of glandular tissues following intracellular processing of the
hK1 zymogen. Following this, hK1 cleaves LK to generate the decapeptide, Lys-BK, also
known as kallidin (Kaplan et al., 2002) (Section 1.2.1). In contrast, the plasma KKS
involves the assembly and activation of multiple plasma-derived factors involved in the
liberation of the pro-inflammatory nonapeptide, bradykinin (BK). Activation of this system
involves a series of proteolytic events mediated by high molecular weight kininogen (HK),
plasma prekallikrein (PPK) and Factor XII (FXII). It was first demonstrated on negatively
charged, non-physiological surfaces, such as glass, silica and kaolin, but activation as now
been demonstrated on cell surfaces (Colman and Schmaier, 1997).
In the ex vivo system, initiation of kinin formation follows autoactivation of FXII to form
activated FXII (FXIIa) which, in turn, activates the serine protease zymogen of plasma
kallikrein (PK), PPK. PK cleaves HK to release BK and reciprocally activates FXII to
increase the rate and extent of the reaction (Miller et al., 1979, Wiggins and Cochrane,
1979, Silverberg et al., 1980, Rojkjaer et al., 1998). In addition, PK or FXIIa cleave
components of the complement pathway (DiScipio, 1982, Ghebrehiwet et al., 1983) and
activate FXI (FXIa), which then plays a role in the intrinsic pathway of coagulation
(Bowma and Griffin, 1977, Wiggins et al., 1979, Brunnee et al., 1993) (Figure 1.1).
Despite these extensive findings, a convincing physiologic, negatively charged surface has
yet been described. In contrast to the ex vivo system, the cell-based system involves
Figure 1.1 Ex vivo model of plasma KKS activation
FXII auto-activates on negatively charged, artificial surfaces to form FXIIa (1) which then
catalyses the conversion of PPK to active PK (2). PK then cleaves HK to liberate BK (3)
and reciprocally activates FXII (4), which may also activate FXI (5).
FXII
FXIIa
1
FXIa
FXI
PPK
PK
HK BK
2
4
3
5
FXII
FXIIa
PPK
PK
HK BK
FXI
FXIa
5
assembly of HK and PPK as a complex on the cell surface. Following this, activated PK
liberates BK, with activation of FXII only occurring as a secondary event (Schmaier, 2000).
1.2 Kininogens and PPK
1.2.1 High and low molecular weight kininogen
HK, previously known as Fitzgerald Factor (Waldmann et al., 1975), is a plasma
glycoprotein of approximately 120 kDa and circulates at a concentration of about 80 μg/ml
(Colman and Schmaier, 1997) as a complex with PPK (Mandle and Kaplan, 1977) or FXI
(Thompson et al., 1977). In its native form, HK is a single-chain molecule composed of six
domains, each with different functions. Proteolysis of HK by PK liberates the BK moiety to
generate a two-chain form of HK consisting of a disulphide-linked 65 kDa N-terminal
heavy chain and a 56-62 kDa C-terminal light chain (Section 1.5). The heavy chain of HK
comprises domains 1-3, which possess cysteine protease inhibitory activity (Higashiyama
et al., 1986). The BK sequence (Arg1-Pro
2-Pro
3-Gly
4-Phe
5-Ser
6-Pro
7-Phe
8-Arg
9) is derived
from domain 4 (Weisel et al., 1994), whereas domains 5 and 6, forming part of the light
chain, mediate the interaction of HK with cellular surfaces (Hasan et al., 1995a) and PPK
or FXI (Tait and Fujikawa, 1987), respectively (Figure 1.2). In addition to PK, HK is also a
substrate for FXIIa (Wiggins, 1983) and FXIa (Scott et al., 1985).
LK is the other plasma kininogen, and both HK and LK are transcribed from the same gene,
which is located on chromosome 3 (Fong et al., 1991). LK is a 66 kDa glycoprotein
(Colman and Schmaier, 1997) and the amino acid sequence from the N-terminus to 12
amino acid residues beyond the C-terminus of BK is identical to the corresponding region
Figure 1.2 Structure of HK
HK is a single-chain glycoprotein consisting of an N-terminal heavy chain, the BK moiety
and a C-terminal light chain. Domains (D)1-3 are contained within the heavy chain and
possess inhibitor activity against cysteine proteases. The BK sequence resides within D4,
while D5 and D6 comprise the light chain and mediates HK binding to cell surfaces and to
PPK or FXI. Adapted form Weisel et al. (1994).
D4: BK
sequence
D5
D6
D1
D2
D3
-COOH
NH2-
PPK or
FXI binding
Cell surface
binding
Cysteine protease
inhibitor activity
6
of HK. LK is also cleaved to produce heavy and light chains, but the light chain moieties of
HK and LK differ due to alternative splicing of the kininogen gene (Kitamura et al., 1985).
In contrast to HK, LK is preferably cleaved by hK1, rather than PK. hK1, also known as
pancreatic/renal tissue kallikrein (TK), belongs to a family of 15 homologous kallikrein-
related peptidases (KLK) which exhibit tryptic or chymotryptic activity (Bhoola et al.,
1992, Yousef and Diamandis, 2001). Compared to other KLKs (Deperthes et al., 1997,
Charlesworth et al., 1999), hK1 demonstrates kininogenase activity and selectively cleaves
LK to generate the decapeptide Lys-BK, also known as kallidin (Lys1-Arg
2-Pro
3-Pro
4-Gly
5-
Phe6-Ser
7-Pro
8-Phe
9-Arg
10). However, both hK1 and PK are capable of cleaving either HK
or LK (Colman and Schmaier, 1997), and PK can effectively liberate BK from LK in the
presence of neutrophil elastase (Sato and Nagasawa, 1988).
It should be emphasised that for the purpose of this thesis, the term “TK” is used to
encompass all 15 members of the KLK family. The term “hK1” strictly refers to the TK
with kininogenase activity as its primary physiological function. Thus, the use of the term
“TK” does not necessarily imply the protease exhibits kininogenase activity against HK or
LK.
1.2.2 PPK
PPK, also known as Fletcher Factor (Hathaway et al., 1965), is the single-chain zymogen
of PK, and is transcribed from a gene located on chromosome 4 (Beaubien et al., 1991).
PPK exists in two glycosylated forms of 85 and 88 kDa (Mandle and Kaplan, 1977) and
circulates at a concentration of approximately 50 μg/ml (Colman and Schmaier, 1997). Ex
vivo, the conversion of PPK to PK is catalysed by FXIIa on negatively charged, non-
7
physiological surfaces (Miller et al., 1979, Silverberg et al., 1980), a process augmented by
the presence of HK (Griffin, 1978). However, on cellular surfaces, activation of PPK
occurs independently of FXIIa when PPK is bound to HK (Rojkjaer et al., 1998) (Section
1.4). Activation of PPK by FXIIa involves cleavage between the Arg371
-Ile372
bond (Hooley
et al., 2007) and generates a disulphide-linked two-chain form, which consists of an N-
terminal 56 kDa heavy chain and a C-terminal 33 or 36 kDa light chain (Mandle and
Kaplan, 1977).
The majority of PPK circulates as a noncovalent complex with HK (Mandle et al., 1976)
and this interaction is mediated by the N-terminal heavy chain of PPK (Page and Colman,
1991, Herwald et al., 1993, Page et al., 1994). This portion of the molecule comprises four
repeats of 90 or 91 amino acid residues (Chung et al., 1986), termed apple domains and
apple domain 1 (Phe56
-Gly86
) and 4 (Lys266
-Gly295
) mediate kininogen binding (Page et al.,
1994). The light chain of PPK contains the active catalytic site, comprising His415
, Asp464
and Ser559
(Chung et al., 1986), and interacts with the protease inhibitors, C1 inhibitor (C1-
INH) and alpha 2-macroglobulin (2M) (van de Graaf et al., 1983), both of which account
for the majority of kallikrein inhibition in vivo (Figure 1.3). Additional substrates for PK
include FXII (Cool et al., 1985) and pro-urokinase (Ichinose et al., 1986, Hauert et al.,
1989).
1.3 HK binds receptors on cell surfaces
In addition to binding extracellular matrix (Motta et al., 2001, Moreira et al., 2002)
including laminin (Schousboe and Nystron, 2009), HK also binds to cell surfaces and was
first demonstrated on platelets (Gustafson et al., 1986), and then on endothelial cells
Figure 1.3 Structure of PPK
PPK is a single chain zymogen, which exists in two glycosylated forms consisting of a 56
kDa N-terminal heavy chain and a 33 or 36 kDa C-terminal light chain. The N-terminal
portion of the molecule mediates binding to HK and is composed of four apple domains
(A), each comprising 90-91 amino acid residues. The C-terminal light chain contains the
active catalytic site of PK and interacts with C1-INH and 2M. Red: FXIIa activation site
on PPK; Yellow: Amino acid residues comprising the catalytic domain of PPK.
A1
A2
A3
A4
NH
2-
-COOH
Arg371
–Ile372
His415
Asp464
Ser559
8
(Schmaier et al., 1988). Binding to cells occurs with high affinity and is a zinc-dependent,
reversible and saturatable process (Reddigari et al., 1993a, Hasan et al., 1995b) involving
regions of the heavy and light chains of HK (Reddigari et al., 1993a). Subsequently, three
proteins on the cell surface were found to bind HK on endothelial cells namely, cytokeratin
(CK)1 (Hasan et al., 1998), urokinase plasminogen activator (uPA) receptor (uPAR)
(Colman et al., 1997) and the receptor that binds the globular region of the complement
component C1q, gC1qR (Joseph et al., 1996). These proteins, which are now regarded as
forming the HK receptor, recognise three non-contiguous regions of HK, spanning domains
3 (Herwald et al., 1995), 4 (Hasan et al., 1994) and 5 (Hasan et al., 1995a).
The exact relationship between the three receptors in relation to HK binding to endothelial
cells is ambiguous (Shariat-Madar et al., 2002). Though direct evidence is presently not
available, data from co-localisation studies indicate CK1, uPAR and gC1qR form a tri-
molecular receptor complex, which then may act in concert to bind HK. These data are
supported by observations showing that antibodies against CK1, uPAR (Mahdi et al., 2001)
and gC1qR block HK binding to endothelial cells, and the total additive inhibition exceeds
100% (Joseph et al., 1999a). However, uPAR and gC1qR only partially co-localise on
endothelial cells, suggesting the existence of CK1-uPAR and gC1qR-CK1 bimolecular
complexes capable of binding HK (Mahdi et al., 2001).
Unlike endothelium, antibody blocking studies have also demonstrated that HK binds to
Mac-1 (CD11b/CD18) on neutrophils (Wachtfogel et al., 1994) and macrophages, as well
as uPAR and gC1qR (Barbasz et al., 2008), indicating multi-receptor complexes
comprising Mac-1, uPAR and gC1qR may be involved in this process. However, direct or
indirect evidence from co-localisation studies is presently unavailable. Other molecules
9
thought to be involved in binding HK include heparan sulphate proteoglycans on
endothelial cells (Renne et al., 2000) and thrombospondin-1 (DeLa Cadena et al., 1998),
glycoprotein 1b (Joseph et al., 1999b) and the glycoprotein 1b-IX-V complex (Bradford et
al., 1997).
1.4 Activation of PPK on cellular surfaces
1.4.1 Activation of PPK by FXIIa
In vitro, FXII auto-activates to form FXIIa (Miller et al., 1979, Silverberg et al., 1980), but
has yet to be convincingly described in vivo. In this regard, at least one report indicates
auto-activation of FXII on endothelial cells (Reddigari et al., 1993b), but these data have
not been confirmed by others (Rojkjaer et al., 1998). For example, treatment of endothelial
cells (Motta et al., 1998) or cell matrices (Motta et al., 2001, Moreira et al., 2002) with HK
and PPK generates PK activity independent of FXII. Similarly, PK activity on endothelial
cells has been demonstrated following incubation with FXII-deficient plasma, but not PPK-
deficient plasma (Rojkjaer et al., 1998). Additionally, the activity of a partially purified
endothelial cell PPK activator was not inhibited by neutralising antibodies against FXII,
and demonstrated negligible activity against FXIIa substrates (Shariat-Madar et al., 2002).
Collectively, these data raise doubts about the significance of FXII auto-activation as a
physiological phenomenon or as the initiating mechanism of plasma KKS activation on
biological surfaces. Rather, FXII activation occurs following PK formation and contributes
to the rate and extent of plasma KKS activation by activating PPK directly (Rojkjaer et al.,
1998) (Figure 1.4).
Figure 1.4 Cell-based model of plasma KKS activation
HK and PPK assemble on multi-receptor complexes on cellular surfaces (1), after which
PRCP or HSP90 associate with the HK-PPK complex and catalyse the conversion of PPK
to PK (2). PK or PPK cleave HK to liberate the BK moiety (3) and activates FXII (4).
FXIIa may then activate PPK directly to increase the rate and extent of the reaction (5).
HK PPK
HK PPK
HK PK
PRCP/
HSP90
BK PK
FXII FXIIa
PK
1
2
3
4
5
10
1.4.2 Activation of PPK requires cell-bound HK
In the absence of HK on the cell surface, endothelial cells do not bind PPK, a conclusion
supported by the observation that a monoclonal antibody directed against the PPK binding
site on HK inhibits PPK binding and PK formation (Motta et al., 1998). Similar results are
also observed using peptides which compete with HK to bind PPK (Lin et al., 1997) or
following incubation of endothelial cells with HK-deficient plasma (Joseph et al., 2001a).
Once complexed with cell-bound HK, activated PPK liberates BK from HK (Nishikawa et
al., 1992) (Section 1.5).
1.4.3 Activation of PPK by prolylcarboxypeptidase
A number of studies have suggested that the endothelial PPK activator is
prolylcarboxypeptidase (PRCP; angiotensinase C) (Shariat-Madar et al., 2002), a serine
protease known to potentiate the hypotensive effect of BK by formation of angiotensin 1-7
(ANG (1-7)) from ANG I and ANG II (Oliveira et al., 1999). This conclusion was drawn
from observations showing that PPK activation can be inhibited by PRCP-neutralising
antibodies or by substrate inhibitors including BK and ANG II (Shariat-Madar et al., 2002).
Additionally, overexpression of PRCP in transfected cells induced PK activity, which was
abolished when cells were treated with small interfering RNA to PRCP (Shariat-Madar et
al., 2005). In contrast to FXIIa, PRCP does not directly activate PPK, but involves a
stoichiometric interaction with the HK-PPK complex to activate PPK (Shariat-Madar et al.,
2004). Although originally described as being highly concentrated in lysosomes, confocal
microscopy has shown the constitutive expression of a membrane-bound form of PRCP on
endothelial cells (Shariat-Madar et al., 2002), which co-localised with the proteins
11
comprising the HK receptor (Shariat-Madar et al., 2004). Thus, PRCP is capable of
participating in proteolysis of extracellular substrates, including PPK.
The precise mechanism by which PRCP activates PPK is unclear. Since FXIIa cleaves the
Arg371
-Ile372
bond of PPK (Hooley et al., 2007), it has been argued that this same site is
utilised by PRCP in vitro (Shariat-Madar et al., 2004). However, cleavage of this bond by
PRCP is unexpected since it should preferentially cleave the C-terminal penultimate proline
residue of PPK (Skidgel and Erdos, 1998). An alternative mechanism of activation has
been proposed by Hooley and others (2007), whereby cleavage of the C-terminus of PPK
by PRCP produces a labile form of PPK, capable of auto-activation. However, conclusive
experimental data are still lacking.
1.4.4 Activation of PPK by heat shock protein 90
In addition to PRCP, it has been proposed that both and β isoforms of heat shock protein
90 (HSP90) can act as PPK activators on endothelial cells (Joseph et al., 2002). In this
regard, endothelial cell cytosol preparations subject to affinity chromatography using corn
trypsin inhibitor (CTI) as a ligand identified HSP90 as the fraction responsible for PPK
activation (Joseph et al., 2002). Although HSP90 is not known to possess proteolytic
activity (Kaplan, 2008), its affinity for CTI may suggest otherwise and, as such, may
directly participate in PPK proteolysis. HSPs are known to maintain homeostasis of a
variety of client proteins by formation of multi-protein chaperone complexes (Mahalingam
et al., 2009). While the majority of known HSP90 client proteins reside within the cytosol
and nucleoplasm (Picard, 2002), the chaperone is expressed on the surface of cell
membranes (Picard, 2004) and is capable of interacting with extracellular substrates
12
(Eustace et al., 2004). Thus, enzymatic activity within HK may be stabilised, or an active
site within PPK exposed following a conformational change in the proteins upon
interaction with HSP90. Similar to PRCP, a stoichiometric interaction between HSP90 and
the HK-PPK complex is necessary for PPK activation (Joseph et al., 2002).
1.5 Proteolysis of HK
HK is initially cleaved at the C-terminal portion of the BK moiety (Arg371
-Ser372
) by PK to
generate a disulphide-linked 64 kDa heavy chain and 56 kDa light chain heterodimer
containing the BK sequence attached to the C-terminus of the heavy chain. This
heterodimer then undergoes further proteolysis by PK at the N-terminal portion of BK
(Lys362
-Arg363
) to liberate the 0.9 kDa BK moiety and an intermediate kinin-free kininogen
of similar molecular size to that of the heterodimer. Lastly, a third cleavage by PK liberates
a 7 kDa peptide which produces a stable, kinin-free, two-chain HK (HKa) comprising a 64
kDa heavy chain and 45 kDa light chain (Mori and Nagasawa, 1981) (Figure 1.5).
Although occurring at a slower rate, PPK also activates HK to liberate BK with a similar
pattern of proteolysis as PK, but without conversion of the zymogen. Proteolysis of HK by
PPK requires assembly of the HK-PPK complex and involves a separate active site to that
seen in PK, as demonstrated by the inhibition of PPK by CTI, which does not inhibit PK
(Joseph et al., 2009). Additionally, FXIIa liberates BK from HK, and the pattern of
proteolysis is indistinguishable from that observed with PK (Wiggins, 1983). In contrast,
FXIa cleaves HK to generate a 76 kDa heavy chain and 45 kDa light chain heterodimer.
Subsequent cleavage by FXIa produces a heavy chain similar in mass to that generated by
Figure 1.5 Proteolysis of HK by PK
PPK cleaves HK at the C-terminus of the BK moiety, generating a “nicked” HK comprising
a 64 kDa heavy chain and 56 kDa light chain (1). A subsequent cleavage at the C-terminus
of the heavy chain liberates BK and produces an intermediate kinin-free kininogen (2). A
third cleavage liberates a 7 kDa peptide and produces a stable, kinin-free HKa, composed
of a 64 kDa heavy chain and 45 kDa light chain (3).
BK H 64 kDa L 56 kDa
S S
BK H 64 kDa L 56 kDa
S S
BK H 64 kDa L 56 kDa
S S
BK H 64 kDa L 45 kDa
S S
1
2
3
13
PK or FXIIa (i.e., 64 kDa). However, further proteolysis of the light chain occurs, which
produces inactive fragments (Scott et al., 1985).
1.6 Kinin metabolism
BK and Lys-BK, the initial kinins liberated, are rapidly catabolised by carboxypeptidase N
(CPN), which removes the C-terminal arginine residue to yield des-Arg9-BK and des-
Arg10
-Lys-BK, respectively (Erdos and Sloane, 1962, Sheikh and Kaplan, 1986a). Also, the
N-terminal lysine residue of Lys-BK and des-Arg10
-Lys-BK can be cleaved by an
aminopeptidase to generate BK and des-Arg9-BK, respectively (Hopsu-Havu et al., 1966).
BK is also metabolised by angiotensin converting enzyme (ACE), which removes the C-
terminal Phe8-Arg
9 residues to generate the heptapeptide, BK fragment 1-7 (BK 1-7) (Arg
1-
Pro2-Pro
3-Gly
4-Phe
5-Ser
6-Pro
7). Subsequent cleavage by ACE removes the C-terminal Ser
6-
Pro7 residues to yield the pentapeptide, BK fragment 1-5 (BK 1-5) (Arg
1-Pro
2-Pro
3-Gly
4-
Phe5). Additionally, ACE metabolises des-Arg
9-BK to remove the Ser
6-Pro
7-Phe
8 residues
(Sheikh and Kaplan, 1986b). BK is also metabolised by neutral endopeptidase (NEP) and
aminopeptidase P, which remove the Phe8-Arg
9 (Roques et al., 1993) and Arg
1-Pro
2
residues (Simmons and Orawski, 1992), respectively (Figure 1.6).
1.7 BK receptors
The diverse biological effects of kinins are mediated through two seven-transmembrane G-
protein-coupled receptors (GPCR) namely, B1R (Menke et al., 1994) and B2R (McEachern
et al., 1991). B1R possesses a high affinity for des-Arg9-BK and des-Arg
10-Lys-BK (Menke
et al., 1994), whereas BK and Lys-BK are the preferred B2R ligands (McEachern et al.,
Figure 1.6 Pathways of kinin formation and metabolism.
BK and Lys-BK are generated following liberation from HK and HK by the actions of PK
and hK1, respectively. The N-terminal lysine residue is removed from Lys-BK by an
aminopeptidase to yield BK. CPN cleaves the C-terminal arginine residues of BK and Lys-
BK to produce des-Arg9-BK and des-Arg10-Lys-BK, respectively. NEP and ACE remove
the C-terminal Phe-Arg from BK to generate BK 1-7. ACE acts on des-Arg9-BK or BK 1-7
to yield BK 1-5. Green: high affinity B1R agonists; Red: high affinity B2R agonists; Blue:
inactive kinin fragments.
Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe–Arg
Lys-Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe–Arg
LK
hK1 HK
PK
Lys-BK
BK
Lys-Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe
des-arg10-lys-BK
CPN
Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe
des-Arg9-BK
Arg–Pro–Pro–Gly–Phe–Ser–Pro
Bradykinin 1-7
Arg–Pro–Pro–Gly–Phe
Bradykinin 1-5
ACE
CPN
ACE
ACE/NEP
14
1991). Stimulation of BK receptors induces intracellular calcium mobilisation and protein
kinase C (PKC) activation following diacylglycerol (DAG) formation and phosphoinositol
hydrolysis by phospholipase C (PLC) (Francel and Dawson, 1988, Portilla et al., 1988,
Smith et al., 1995, Zhang et al., 2001).
The B2R is constitutively expressed under non-pathological conditions (Leeb-Lundberg et
al., 2005) and undergoes rapid internalisation and homologous desensitisation following
BK stimulation (Smith et al., 1995). In contrast, extracellular B1R expression on cells is
low due to constitutive endocytosis of the receptor in the absence of ligand (Enquist et al.,
2007). However, the expression of B1R on the cell surface is significantly increased in
response to inflammatory stimuli including B1R and B2R ligands (Schanstra et al., 1998,
Phagoo et al., 1999), and IL-1β (Tsukagoshi et al., 1999, Phagoo et al., 2000, Phagoo et al.,
2001), and is insensitive to desensitisation (Mathis et al., 1996, Austin et al., 1997). Thus,
the B2R represents the dominant receptor subtype in normal physiological settings and is
associated with transient responses to BK and Lys-BK stimulation. In contrast, persistent
B1R-mediated responses may dominate in inflammatory conditions due to the limited
desensitisation potential of the receptor and a shift in the BK receptor repertoire towards
this subtype.
1.8 Biological effects of the plasma KKS
One of the most foremost consequences of plasma KKS activation along biological
membranes is the generation of kinins capable of activating cells and initiating kinin-
dependent processes. In this regard, BK is a potent inducer of prostaglandin (PG)I2
synthesis (Yamasaki et al., 2000), tissue plasminogen activator (tPA) release (Brown et al.,
15
1999) and nitric oxide (NO) formation (Palmer et al., 1987). Additionally, kinins stimulate
cytokine and chemokine release (Tiffany and Burch, 1989, Koyama et al., 2000, Ailberti et
al., 2003) and cellular differentiation (Vancheri et al., 2005, Monteiro et al., 2006, Matus et
al., 2008). In addition, kinins are chemotactic per se (Ifuku et al., 2007) and possess anti-
microbial properties (Kowalska et al., 2002).
1.8.1 The plasma KKS and anti-thrombotic activities
Thrombin is the coagulation factor responsible for catalysing the conversion of fibrinogen
to fibrin (Walker and Royston, 2002), but it also affects cells through the activation of
protease-activated receptor (PAR)1 (Vu et al., 1991), PAR3 (Ishihara et al., 1997) and
PAR4 (Xu et al., 1998). The inhibition of thrombin by activated and non-activated
components of the plasma KKS has been demonstrated using thrombin-induced platelet
activation as an experimental model. Following thrombin stimulation, calpain is
externalised on platelets and activates extracellular receptors necessary for fibrinogen
binding and platelet aggregation (Schmaier et al., 1990). HK and LK inhibit thrombin-
induced platelet aggregation by inhibiting the activity of calpain (Puri et al., 1987, Puri et
al., 1989) and this interaction is mediated by the kininogen heavy chain (Bradford et al.,
1990). Additionally, HK and LK directly prevent thrombin binding to high affinity sites on
cells (Meloni and Schmaier, 1991, Bradford et al., 1997) through domain 3 of the
kininogen heavy chain (Jiang et al., 1992). Furthermore, BK and BK 1-5 inhibit the ability
of thrombin to proteolytically activate PAR1 (Hasan et al., 1996) and PAR4 (Nieman et al.,
2005).
16
1.8.2 The plasma KKS and pro-fibrinolytic activities
Fibrinolysis involves the conversion of the serine protease zymogen plasminogen into
plasmin which, in turn, participates in fibrin degradation. The activation of plasminogen is
mediated systemically by tPA, and locally by uPA (Mondino and Blasi, 2004). The
degradation of fibrin prevents its extracellular deposition and participation in an array of
biological activities, including thrombosis, cellular and matrix interactions and
inflammation (Mosesson, 2005). Plasma KKS activation induces fibrinolysis by several
mechanisms. For example, FXIIa (Goldsmith et al., 1978) and PK (Colman, 1969) can
directly activate plasminogen. Second, BK is a potent inducer of tPA release from
endothelial cells (Brown et al., 1999), thus, augmenting systemic plasma fibrinolysis.
Lastly, PK can catalyse the conversion of pro-uPA to uPA (Ichinose et al., 1986), allowing
its participation in cellular fibrinolysis. The generation of plasmin by PK or FXIIa may also
contribute to the fibrin-independent effects of plasmin, including cell migration (Syrovets et
al., 1997, Tarui et al., 2002), arachidonate and lipid mediator release (Chang et al., 1993,
Weide et al., 1994) and cytokine and chemokine expression (Syrovets et al., 2001, Burysek
et al., 2002, Li et al., 2007).
1.8.3 The plasma KKS and inflammatory mediator synthesis
BK induces the synthesis and release of various inflammatory mediators including
interleukin (IL)-1 (Paegelow et al., 1995, Pan et al., 1996), IL-6, IL-8, transforming growth
factor (TGF)-β (Koyama et al., 2000, Rodgers et al., 2002), tumour necrosis factor (TNF)-
(Ferreira et al., 1993) and prostanoids (Nakao et al., 2000, Yamasaki et al., 2000). The
effects of BK on cytokine release may follow nuclear factor κB (NFκB) activation (Pan et
17
al., 1996, Xie et al., 2000), a transcription factor responsible for the expression of
numerous cytokines (Mercurio and Manning 1999). Furthermore, BK-induced cytokine
secretion may be dependent on autocrine responses to TNF- (Ferreira et al., 1993) and
PGE2 (Rodgers et al., 2002). Interestingly, both B1R and B2R agonists act synergistically
with cytokines to augment the release of inflammatory mediators. In this regard, BK
potentiates cytokine-induced IL-6 (Modeer et al., 1998) and IL-8 (Brunius et al., 2005)
release and PGE2 biosynthesis (Sundqvist and Lerner, 1996, Ransjo et al., 1997, Brechter
and Lerner, 2007).
As with BK, Lys-BK and des-Arg9-BK also induce cytokine (Modeer et al., 1998) and
prostaglandin release (Ljunggren and Lerner, 1990), and act with cytokines to potentiate
other mediator release (Lerner and Modeer, 1991, Modeer et al., 1998). However, evidence
describing an active role for des-Arg10
-Lys-BK in mediator release is limited. For example,
des-Arg10
-Lys-BK demonstrates negligible effect on PGE2 release from lung epithelial cells
(Saunders et al., 1999) which is not influenced by IL-1β pre-treatment (Newton et al.,
2002). In contrast, however, stimulation of alveolar macrophages with des-Arg10
-Lys-BK
induces TNF- release, but only from IL-1β pre-treated cells (Tsukagoshi et al., 1999).
Thus, des-Arg10
-Lys-BK may only induce a cytokine response following IL-1β-mediated
up-regulation of the B1R. However, des-Arg9-BK can stimulate cytokine and prostanoid
release from cells without prior cytokine pre-treatment, albeit with less potency than B2R
agonists (Ljunggren and Lerner, 1990, Pang and Knox, 1997, Modeer et al., 1998). Thus,
BK and Lys-BK are the dominant kinins with regard to cytokine release, in contrast to des-
Ag9-BK and des-Ag
10-lys-BK. With regard to prostaglandin release, the order of potency of
18
BK ≥ Lys-BK > des-Ag9-BK > des-Ag
10-Lys-BK implicates the B2R as the primary
mediator.
HKa has also been shown to stimulate the release of cytokines from monocytes including
IL-1β, IL-6 and TNF-, and chemokines, including IL-8 and MCP-1. Interestingly, HKa
stimulates cytokine release from monocytes through uPAR, gC1qR and Mac-1 (Khan et al.,
2006), proteins thought to be involved in the binding of HK (Wachtfogel et al., 1994,
Barbasz et al., 2008). Furthermore, proteases of the kinin-cascade have been shown to have
a direct effect on the expression and release of inflammatory mediators. FXIIa has been
shown to stimulate IL-1β and IL-6 secretion by monocytes by an unknown mechanism.
However, in the presence of lipopolysaccharide (LPS), FXII and FXII fragment (FXIIf)
also induce IL-1β (Toossi et al., 1992). Likewise, arachidonic acid (AA) release is induced
by hK1 and PK in cells stably transfected to express B2R, which is inhibited by B2R
antagonism by icatibant acetate (Hoe 140), inactive in cells lacking the B2R (Hecquet et al.,
2000) and independent of kininogen (Biyashev et al., 2006).
1.8.4 The plasma KKS and vasodilation
BK is a potent inducer of vasodilation (Fox et al., 1961) due to its ability to stimulate PGI2
synthesis (Yamasaki et al., 2000) and NO formation (Palmer et al., 1987) in endothelial
cells. Systemic activation of the plasma KKS following administration of dextran sulphate
increases circulating kinin levels and is accompanied by arterial hypotension (Siebeck et
al., 1994, Schmid et al., 1998). Likewise, transgenic mice over-expressing hK1 are
hypotensive and blood pressure is restored by kallikrein inhibition or B2R antagonism by
Hoe 140 (Wang et al., 1994, Song et al., 1996). Similarly, B2R over-expression causes
19
sustained hypotension in transgenic mice, which is reversed by Hoe 140 (Wang et al.,
1997). Furthermore, gene delivery of hK1 (Xiong et al., 1995) or kallikrein binding protein
(Ma et al., 1995) re-establishes a normotensive state in hypertensive rats and hK1 over-
expressing transgenic mice, respectively. Collectively, these studies highlight the
importance of the kinin-forming cascades in regulating blood pressure and are supported by
the clinical efficacy of ACE inhibitors for the treatment of hypertension (Brown et al.,
1998).
1.8.5 The plasma KKS and vascular permeability
BK-induced vascular permeability is mediated by enhanced transcellular fluid passage
across the endothelium (Riethmuller et al., 2006, Jungmann et al., 2007), combined with
PGI2- and NO-induced vasodilation (Feletou et al., 1996). The significance of the plasma
KKS in the induction of vascular permeability is illustrated in patients with hereditary and
acquired angio-oedema, which are both characterised by intermittent oedema due a
deficiency in functional C1-INH (Davis, 2003). C1-INH homozygous and heterozygous
mice display increased vascular permeability, which is enhanced by treatment with ACE
inhibitors and reversed by inhibition of PK and B2R (Han et al., 2002). Also, patients with
hereditary, acquired and captopril-induced angio-oedema have elevated plasma BK levels
during episodes of oedema, which is lowered by administration of C1-INH (Nussberger et
al., 1998). Thus, excessive kinin production as a result of uninhibited plasma KKS
activation contributes to the increased vascular permeability characteristic of these diseases.
20
1.8.6 The plasma KKS and cellular migration
In addition to inducing chemokine release, kinins are chemotactic per se. For example,
migration of immature monocyte-derived dendritic cells (mDC) can be induced by BK, but
not des-Arg10
-Lys-BK (Bertram et al., 2007). In contrast, microglial cells (Ifuku et al.,
2007) and neutrophils (Paegelow et al., 2002) migrate in response to des-arg10
-Lys-BK, in
addition to BK and des-Arg9-BK. Chemotaxis of immature mDCs and neutrophils in
response to BK is exclusively mediated by the B2R, as chemotaxis is inhibited by Hoe 140,
but not the B1R antagonist, Lys-des-Arg9-Leu
8-BK (Bertram et al., 2007). However, the
opposite is true for microglial cells and BK-induced migration in these cells is mimicked by
B1R agonists (Ifuku et al., 2007). Studies also demonstrate a lack of migration by
neutrophils in response to des-arg10
-BK and des-arg9-BK in the absence of IL-1β up-
regulation of the B1R (Ehrenfeld et al., 2006). However, migration has also been shown to
occur in unprimed neutrophils (Paegelow et al., 2002). The absence of IL-1β priming in
immature mDCs does not explain the lack of effect of B1R agonists on migration, as mDCs
express the B1R (Bertram et al., 2007).
Conversely, HKa has been show to inhibit the migration of endothelial (Katkade et al.,
2005) and prostate epithelial cells (Liu et al., 2009). This process is mediated through
kininogen domain 5 and binding of HKa to uPAR disrupts formation of a ternary complex
formed with the epidermal growth factor receptor (EGFR), and integrins. In turn, signal
transduction pathways necessary for migration are interrupted, including phosphorylation
of EGFR, extracellular signal-regulated kinase (ERK) and Akt (Katkade et al., 2005, Liu et
al., 2009). In addition, HKa binding to uPAR interferes with uPA-mediated uPAR
internalisation and regeneration of unoccupied uPAR, thereby disrupting uPAR-mediated
21
cellular migration (Liu et al., 2008). In parallel to its effects on migration, the interaction
between HKa and uPAR inhibits cell adhesion and induces apoptosis by disruption of uPA-
uPAR and uPAR-integrin signal complex activation (Cao et al., 2004, Liu et al., 2008).
1.8.7 The plasma KKS and cellular maturation and differentiation
Activated components of the plasma KKS play a role in the differentiation of various cell
types. For example, kinins induce the differentiation of keratinocytes (Matus et al., 2008),
fibroblasts (Vancheri et al., 2005), DCs (Monteiro et al., 2006), neuronal progenitor cells
(Martins et al., 2008) and adipose-derived mesenchymal stem cells (Kim et al., 2008). In
addition, PK is known to mediate adipogenesis via activation of the plasminogen cascade
(Selvarajan et al., 2001, Lilla et al., 2009). Furthermore, administration of hK1 induces
neurogenesis in a model of ischemia in infarction-susceptible rats (Ling et al., 2008).
1.8.8 The plasma KKS and anti-microbial effects
Digestion of kininogen by host or microbial proteases generates peptides with anti-
microbial activity. For example, BK (Kowalska et al., 2002) and fragments derived from
the structural domains of kininogens (Nordahl et al., 2005, Frick et al., 2006) possess anti-
microbial activity against a variety of bacterial species. For example, neutrophil elastase
degrades protease-sensitive regions of HK and LK (Vogel et al., 1988) to yield anti-
microbial peptides (AMPs) derived from kininogen domain 3 (Frick et al., 2006) and
domain 5 (Nordahl et al., 2005). Likewise, the combined actions of mast cell tryptase and
neutrophil elastase generate a vascular permeability enhancing peptide (E-kinin) from
kininogen (Imamura et al., 1996, Imamura et al., 2002), which has bactericidal activity
22
against Pseudomonas aeruginosa and Staphylococcus aureus (Nordahl et al., 2005). In
addition, plasma KKS activation occurs on bacterial surfaces (Ben Nasr et al., 1996,
Mattsson et al., 2001) and AMPs related to domain 3, but not domain 5, are liberated by
this process (Frick et al., 2006). Furthermore, proteolysis of HK by bacterial proteases
release AMPs derived from domain 5 (Nordahl et al., 2005).
In addition, inhibition of plasma KKS activation has been shown to augment dissemination
of Streptococcus pyogenes infection in a mouse model (Frick et al., 2006). However,
bacterial infection may result in widespread and unrestrained plasma KKS activation,
resulting in bacteremia, sepsis and septic shock associated with the consumption of plasma
KKS components (Aasen et al., 1980, Aasen et al., 1983, Martinez-Brotons et al., 1987)
and the release of BK (Mattsson et al., 2001), which contributes to the hypovolemic
hypotension and coagulopathy associated with severe infection (Oehmcke and Herwald,
2009).
In a mouse model of Trypanosoma cruzi infection, the B2R was shown to cooperatively
interact with toll-like receptor (TLR)2, a receptor involved in the recognition of conserved
pathogen-associated molecular patterns (PAMPs). In this model, macrophage TLR2
recognised PAMPs derived from T. cruzi and inflammatory mediators were released, which
induced the extravasation of plasma kininogen. The T. cruzi protease, cruzipain, then
digested kininogen and liberated BK. Following this, B2R signaling stimulated maturation
of DCs to release IL-12 and induced type 1 polarisation of naïve T-cells (Monteiro et al.,
2006). Thus, elimination of T. cruzi involves activation of a TLR2/B2R axis, in which BK
intersects innate and adaptive immune responses.
23
1.8.9 The direct effects of kallikreins
Similar to BK, kallikrein induces the redistribution and internalisation of B2R, a process
blocked by Hoe 140 and the kallikrein inhibitor aprotinin (Hecquet et al., 2002). However,
the lack of heterogenous desensitisation by BK and kallikrein suggest kallikreins may
activate the B2R differently than BK (Hecquet et al., 2000, Biyashev et al., 2006). It has
been proposed that kallikreins activate B2R via a process similar to that observed for the
proteolytic activation of PARs (Hecquet et al., 2000), which kallikreins also activate
(Oikonomopoulou et al., 2006, Ramsay et al., 2008, Stefansson et al., 2008, Gratio et al.,
2010). However, the notion that B2R functions as a PAR has been disputed by Houle et al.
(2003) who proposed local kinin release by low dose hK1 was responsible for B2R
activation. However, in the same study, high dose hK1 was shown to cleave B2R and
stimulate AA release. Likewise, contractile responses were also observed in rabbit jugular
vein by submicromolar levels of hK1 without cross-desensitisation by BK. More recently,
kallikrein-mediated activation of B2R was observed in serum-starved cells and was
unaffected by the absence of zinc necessary for kininogen absorption on cellular surfaces
(Biyashev et al., 2006). Thus, direct receptor proteolysis represents an alternative
mechanism by which kallikreins mediate their effects on B2R.
1.9 Plasma KKS assembly and activation on other cell types
Although plasma KKS activation on biological surfaces has been most studied on
endothelial cells, other cells are known to possess such a system. For example, HK binding,
the initiating step in kinin formation, was originally shown to occur on platelets (Gustafson
et al., 1986) and, after endothelium, on monocytes (Barbasz et al., 2008) and astrocytes
24
(Fernando et al., 2003). Similarly, neutrophils have been shown to bind HK, PPK and FXII
(Gustafson et al., 1989). On astrocytes, binding is mediated by the endothelial HK
receptors, uPAR, gC1qR and CK1 (Fernando et al., 2003), while on monocytes and
neutrophils Mac-1 is involved (Wachtfogel et al., 1994, Barbasz et al., 2008). Although
experimental data are lacking, the localisation of HK to these cells is likely to serve as a
binding site for PPK and result in PK formation and liberation of BK. Likewise, plasma
KKS assembly and activation occurs on vascular smooth muscle (Fernando et al., 2005)
and macrophage-like cells (Barbasz and Kozik, 2009). However, the receptors responsible
for binding HK and the contribution of PRCP or HSP90 to PPK activation were not
explored. Nevertheless, such data suggest that plasma KKS activation is not limited to
endothelium and the cell types discussed above, but supported by a diverse range of cell
types.
1.10 The plasma KKS and the respiratory epithelium and pleural mesothelium
Previous studies have identified activated components of the plasma KKS within
bronchoalveolar lavage (BAL) and nasal and serosal fluids, but the underlying mechanism
of activation within these tissues is unclear. For example, an influx of HK and PPK was
observed in nasal secretions from allergic individuals challenged with allergen and was
accompanied by PPK activation and kinin formation (Proud et al., 1983, Baumgarten et al.,
1985, Baumgarten et al., 1986). Additionally, allergen challenge can induce kinin
formation in the airways of allergic subjects (Baumgarten et al., 1986, Baumgarten et al.,
1992, Christiansen et al., 1992, Wihl et al., 1995) and similar results may be observed
following methacholine challenge of non-allergic individuals (Baumgarten et al., 1992).
Likewise, increased PK and kinin levels are found in BAL fluid from patients with acute
25
pneumonia, chronic bronchitis (Zhang et al., 1997), sarcoidosis and pulmonary fibrosis
(Baumgarten et al., 1992). Furthermore, HK and PPK activation occurs in pleural and
peritoneal exudates in models of pleurisy (Uchida et al., 1983, Majima et al., 1992) and
pancreatitis (Ruud et al., 1982, Ruud et al., 1984, Ruud et al., 1985, Waldner et al., 1993),
respectively.
A parallel increase in albumin or 2M is observed with HK and PPK influx in the airway
(Baumgarten et al., 1985, Baumgarten et al., 1986, Zhang et al., 1997), and kinin
generation (Baumgarten et al., 1986, Baumgarten et al., 1992), indicating that exudation of
bulk plasma proteins is responsible for the appearance of plasma KKS components within
these tissues. As such, luminal entry of plasma across the respiratory epithelium is an
established inflammatory process (Persson et al., 1998) and is a common feature in both
asthma and COPD (Persson and Uller, 2009). Likewise, pleural disease is associated with
plasma exudation across the pleura (Jantz and Antony, 2008) which, therefore, may
contribute to the passage of high molecular weight proteins across the mesothelium (Asseo
and Tracopoulos, 1981, Alexandrakis et al., 2000). Such leakage may, thus, contribute to a
HK and PPK pool within the airway lumen and pleural space, allowing plasma KKS
activation to proceed along the epithelium and mesothelium.
Currently, the tissue KKS is the most studied kinin system within the airway (Baumgarten
et al., 1986, Christiansen et al., 1987, Christiansen et al., 1989, Christiansen et al., 1992,
Proud and Vio, 1993, Schenkels et al., 1995, O'Riordan et al., 2003, Sexton et al., 2009).
Indeed, hK1 has been described as the foremost kininogenase in various inflammatory lung
disorders, including asthma (Christiansen et al., 1987) and chronic bronchitis (Zhang et al.,
1997). However, PK represents the dominant kallikrein in acute lung disease (Zhang et al.,
26
1997) and pleuritis (Uchida et al., 1983, Fujie et al., 1993, Costa et al., 2002), indicating a
significant role for the plasma KKS within these tissues. Furthermore, localisation of PK to
the respiratory epithelium (Hermann et al., 1999, Fink et al., 2007, Chee et al., 2008) and
pleural mesothelium (Chee et al., 2007) suggest they may participate in local kinin
formation through plasma KKS activation.
1.11 Aims of the thesis
Given these findings, it was proposed that the respiratory epithelium and pleural
mesothelium support plasma KKS activation and, therefore, may be sites of local BK
formation. Establishing a role for plasma KKS activation in these tissues may be of
particular significance given the biological effects of kinins and their relevance in
pulmonary and pleural disease. The pathological significance of kinins in these tissues is
evident by the attenuation of bronchial symptoms (Akbary et al., 1996) and pleural
inflammation (Saleh et al., 1997, Saleh et al., 1998) by BK receptor antagonists. BK has
several actions within the airway and pleural space, including bronchoconstriction
(Ichinose and Barnes, 1990, Polosa and Holgate, 1990, Polosa et al., 1994), vasodilation
(Yamawaki et al., 1994), microvascular leakage (Katori et al., 1978, Ichinose and Barnes,
1990, Hayashi et al., 2002), airway hypersecretion (Davis et al., 1982, Wells et al., 1993,
Nagaki et al., 1995, Nagaki et al., 1996) and the production of pro-inflammatory mediators
by epithelial (Koyama et al., 1998) and mesothelial (van de Veld et al., 1986) cells.
It is on this background that the work described in thesis was initiated. Particular attention
was given to determining a potential role for the plasma KKS on respiratory epithelial cells,
with an emphasis on determining the involvement of the known endothelial HK receptor
27
associated proteins and characterisation of the activator responsible for PPK activation. In
addition, parallel studies were performed to explore a role for the plasma KKS on
mesothelial cells. Finally, the role of kallikreins as signaling molecules on pleural
mesothelial cells was investigated.
The specific aims of this thesis were:
1. To determine whether respiratory epithelium and pleural mesothelium support
assembly and activation of the plasma KKS, including binding of HK, assembly of
PPK, formation of PK and liberation of BK.
2. To determine whether plasma KKS activation is a common feature of epithelia per
se and of cells of non-epithelial origin.
3. To characterise the activator responsible for HK-PPK complex activation on
respiratory epithelium and pleural mesothelium and compare to those previously
described on endothelial cells.
4. To determine the biological effects of kinins on respiratory epithelium and
mesothelium, in particular the release of inflammatory mediators.
5. To determine the direct effect of kallikreins on calcium mobilisation and the role of
the B2R and PARs in kallikrein signaling.
CHAPTER 2
MATERIALS
29
2.1 General chemicals and specific reagents
General chemicals were obtained from BDH (Kilsyth, Victoria, Australia), Calbiochem (La
Jolla, CA, USA) or Ajax Finechem (Auckland, New Zealand). The specific reagents used
and their suppliers are shown in Table 2.1. The location of the suppliers is listed in Table
2.2.
2.2 Buffers and solutions
Unless otherwise stated, all buffers and solutions were prepared in distilled water and
stored at RT.
2.2.1 General buffers
Hank’s balance salt solution (HBBS), calcium- and magnesium-free, pH 7.4
KCl 5.4 mM
KH2PO4 0.4 mM
NaCl 137 mM
Na2HPO4 0.3 mM
NaHCO3 4.2 mM
Glucose 5.6 mM
The solution was prepared in MilliQ water and the pH adjusted using HCl. The solution
was sterilised by filtration through a 0.2 μm filter.
Table 2.1b List of specific reagents used and their suppliers
Reagent Supplier
Hematoxylin Sigma-Aldrich
HOE-140 Sigma-Aldrich
Horse Serum Invitrogen
Human high molecular weight kininogen Innovative Research
Human plasma kallikrein Innovative Research
Human plasma prekallikrein Innovative Research
Hydrogen peroxide BDH
Ionomycin Sigma-Aldrich
Interleukin-6 BD Pharmingen
Interleukin-8 BD Pharmingen
Leupeptin Sigma-Aldrich
N,N,N’,N’ Tetramethylethylenediamine Bio-Rad Laboratories
Non-enzymatic dissociation medium Sigma Aldrich
Novobiocin Sigma-Aldrich
Paraformaldehyde BDH
Phosphate buffered saline tablets Oxoid
PD-10 desalting columns GE Healthcare
Phorbol myristate acetate Sigma-Aldrich
Phosphoramidon Sigma-Aldrich
Plummer’s inhibitor Calbiochem
Pluronic F-127 Invitrogen
Porcine pancreatic kallikrein Sigma-Aldrich
Protamine sulphate (grade X) Sigma-Aldrich
Table 2.1a List of specific reagents used and their suppliers
Reagent Supplier
2-Aminoethyl benzenesulphonyl fluoride Roche Diagnostics
2-Mercaptoethanol Sigma-Aldrich
4-Nitrophenyl phosphate Sigma-Aldrich
Angiotensin 1-7 Sigma-Aldrich
Angiotensin II Sigma-Aldrich
Antipain Sigma-Aldrich
Apstatin AnaSpec
Ammonium persulphate Bio-Rad Laboratories
Benzamidine Sigma-Aldrich
Bovine serum albumin Serologicals
Bradykinin Sigma-Aldrich
Bradykinin fragment 1-5 Sigma-Aldrich
Bradykinin fragment 1-7 Sigma-Aldrich
Bradford reagent Sigma-Aldrich
Broad range molecular weight standards Bio-Rad Laboratories
Bromophenol blue Sigma-Aldrich
Captopril Sigma-Aldrich
Des-Arg9-bradykinin Sigma-Aldrich
Diaminobenzidine tablets Sigma-Aldrich
Fluorescein isothiocyanate Sigma-Aldrich
Fluo-4/Acetoxymethyl ester Invitrogen
Fura-2/Acetoxymethyl ester Biotium
Gelatin Difco Laboratories
Table 2.1c List of specific reagents used and their suppliers
Reagent Supplier
Protease inhibitor cocktail Sigma-Aldrich
Rat tail collagen Sigma-Aldrich
Saponin Sigma-Aldrich
S-2266 DiaPharma
S-2302 DiaPharma
Thrombin Sigma-Aldrich
N-Tosyl-L-phenylalanine chloromethyl ketone -treated trypsin Sigma-Aldrich
Tris/glycine/SDS buffer (10x) Bio-Rad Laboratories
Triton® X-100 Sigma-Aldrich
Trypan blue Sigma-Aldrich
Tween® 20 Sigma-Aldrich
Table 2.2 Location of suppliers
Supplier Location
AnaSpec San Jose, CA, USA
BDH Victoria, Australia
BD Pharmingen San Diego, CA, USA
Biotium Hayward, CA,
USA
Bio-Rad Laboratories Hercules, CA, USA
Calbiochem La Jolla, CA, USA
DiaPharma West Chester, OH,
USA
Difco Laboratories Detroit, MI,
USA
GE Healthcare Uppsala, Sweden
Innovative Research Novi, MI, USA
Invitrogen Melbourne,
Australia
Oxoid Hampshire, England
Roche Diagnostics Mannheim,
Germany
Sigma-Aldrich St Louis, MO, USA
30
HBBS with calcium and magnesium, pH 7.4
As described above with the addition of:
CaCl2 1.3 mM
MgCl2 0.5 mM
MgSO4 0.6 mM
HEPES buffer containing EDTA, pH 7.4
NCl 137 mM
KCl 4 mM
Glucose 11 mM
N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid (HEPES) 10 mM
CaCl2 1 mM
Bovine serum albumin (BSA) (Serologicals) 0.5 mg/ml
Ethylenediamine tetra-acetic acid (EDTA) 10 mM
The solution was prepared in MilliQ water and the pH adjusted using HCl. The solution
was sterilised by filtration using a 0.2 μm filter. The solution was stored at 4°C.
HEPES buffer containing zinc, pH 7.4
As described above without EDTA, but with the addition of:
ZnCl2 50 μM
HEPES buffer containing sodium azide, pH 7.4
As described above with the addition of:
Sodium azide 0.1% (w/v)
31
Phosphate buffered saline (PBS), pH 7.4
One PBS tablet (Oxoid) was dissolved in 100 ml distilled water to obtain a phosphate
buffer containing 0.2 g/L KCl, 8 g/L NaCl, 1.15 g/L Na2HPO4 and 0.2 g/L KH2PO4.
Sodium carbonate buffer, pH 9.3
Na2CO3 100 mM
The pH was adjusted using HCl.
Sodium chlorate stock solution, pH 7.4
NaClO3 100 mM
The solution was prepared in PBS and the pH adjusted using HCl. The solution was
sterilised by filtration using a 0.2 μm filter and stored at 4°C.
2.2.2 Acid phosphatase assay
Acid phosphatase substrate solution
4-nitrophenylphosphate (pNNP) (Sigma-Aldrich) 1 mg/ml
Sodium acetate buffer, pH 5 0.1 M
Triton® X-100 (Sigma-Aldrich) 0.1% (v/v)
Sodium hydroxide working stock solution
NaOH 1 M
32
2.2.3 Cell culture reagents
Unless otherwise stated, all cell culture reagents were obtained from Invitrogen
(Melbourne, Australia).
Antibiotic/antimycotic-treated media
Penicillin 100 U/ml
Streptomycin 100 μg
Amphotericin B 0.25 μg
Antibiotic/antimycotic was prepared in basal medium.
Cell freezing medium
Dimethyl sulphoxide (DMSO) 10% (v/v)
Fetal calf serum (FCS) 90% (v/v)
The reagent was stored at -20°C until required.
FCS
FCS was incubated at 56°C for 30 min to inactivate serum complement components,
aliquoted and stored at -20°C.
Gelatin solution
Gelatin (Difco Laboratories) 0.1% (w/v)
Gelatin solution was prepared in PBS and filter sterilised through a 0.2 μm filter. The
solution was stored at 4°C.
33
Penicillin/streptomycin
Penicillin 100 U/ml
Streptomycin 100 μg
Penicillin/streptomycin was prepared in basal medium.
Trypsin-EDTA dissociation medium
Trypsin 0.0625% (v/v)
EDTA 0.25 mM
Trypsin/EDTA was prepared in PBS and stored at 4°C
2.2.4 Chromogenic assays for kallikrein proteases
Chromogenix S-2266 assay buffer, pH 8.2
Tris base 40 mM
The pH was adjusted using HCl.
Chromogenix S-2266 substrate stock solution for hK1
S-2266 (DiaPharma) 1 mM
The solution was prepared in MilliQ water and stored at 4ºC in the dark. The substrate was
stable for approximately 6 months.
Chromogenix S-2303 substrate stock solution for PK
S-2302 (DiaPharma) 8 mM
The solution was prepared in MilliQ water and stored at 4ºC in the dark. The substrate was
stable for approximately 6 months.
34
2.2.5 Enzyme-linked immunosorbant assay (ELISA)
Alkaline carbonate buffer, pH 9.6
NaHCO3 0.1 M
Na2CO3 0.1 M
The pH was adjusted using HCl and the buffer stored at 4°C.
ELISA blocking diluent
BSA 1% (w/v)
Tween® 20 (Sigma-Aldrich) 0.05% (v/v)
The solution was prepared in PBS and stored at 4ºC.
ELISA wash buffer
Tween® 20 0.05% (v/v)
The solution was prepared in PBS.
2.2.6 Calcium mobilisation
Pluronic F-127 stock solution
Pluronic F-127 (Invitrogen) 20% (w/v)
The solution was prepared in 100% (v/v) DMSO.
35
Fluo-4/acetoxymethyl (AM) working stock solution
Fluo-4/AM (Invitrogen) 500 μM
The solution was prepared in equal volumes of 100% (v/v) DMSO and 20% (w/v) Pluronic
F-127, and stored at 4°C in the dark.
Fura-2/AM working stock solution
Fura-2/AM (Biotium) 500 μM
The solution was prepared in equal volumes of 10% (v/v) DMSO and 20% (w/v) Pluronic
F-127, and stored at -20°C in the dark.
2.2.7 Immunocytochemistry studies
Immunocytochemistry blocking diluent
Horse serum (Invitrogen) 4% (v/v)
Gelatin 1% (w/v)
Tween® 20 0.05% (v/v)
The solution was prepared in PBS and stored at 4°C.
Immunocytochemistry blocking diluent with saponin
As described above with the addition of:
Saponin (Sigma-Aldrich) 0.5% (w/v)
36
3, 3’ diaminobenzidine (DAB) working stock solution
One brown tablet and one white tablet (Sigma-Aldrich) were dissolved in MilliQ water to
give a working solution containing 0.7 mg/ml DAB, 1.6 mg/ml urea and 0.06 M Tris. The
solution was used immediately.
Hydrogen peroxide working stock solution
H2O2 0.3% (v/v)
The solution was prepared in MilliQ water and used immediately.
Metal enhancer for DAB solution
NiCl2 0.3% (w/v)
The solution was prepared in DAB working stock solution.
Paraformaldehyde fixative
Paraformaldehyde 4% (w/v)
The solution was prepared in PBS and heated to completely dissolve the paraformaldehyde.
The pH was adjusted to 7.4 and aliquots stored at -20°C until required.
Scott’s tap water
MgSO4 2% (w/v)
NaHCO3 0.35% (w/v)
The solution was prepared in tap water and used within 2 weeks.
37
Sodium citrate buffer, pH 6
Na3C6H5O7 10 mM
Tween® 20 0.05% (v/v)
The pH was adjusted using HCl.
2.2.8 Matrix preparation
Ammonium hydroxide solution
NH4OH 0.025 M
The solution was prepared in MilliQ water.
Tris buffer, pH 7.4
Tris base 0.02 M
NaCl 0.15 M
Tween® 20 0.5% (v/v)
The solution was prepared in MilliQ water and the pH adjusted using HCl.
Permeablisation buffer
Triton® X-100 (Sigma-Aldrich) 0.5% (v/v)
The solution was prepared in PBS.
38
2.2.9 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
44% acrylamide/bis-acrylamide (37:1) solution
Acrylamide 44% (w/v)
Bis-acrylamide 1.2% (w/v)
The solution was stored at 4°C in the dark.
Ammonium persulphate (APS)
APS (Bio-Rad Laboratories) 10% (w/v)
The solution was prepared in MilliQ water and stored at 4ºC for no longer than 1-2 days.
SDS-PAGE de-staining solution
Glacial acetic acid 6% (v/v)
SDS-PAGE resolving gel buffer, pH 8.8
Tris base 1.5 M
Sodium dodecyl sulphate (SDS) 2% (w/v)
The solution was prepared in MilliQ water and the pH adjusted using HCl. The solution
was stored at 4ºC.
39
SDS-PAGE electrophoresis buffer, pH 8.3
Tris base 25 mM
Glycine 192 mM
SDS 0.1% (w/v)
The solution was prepared in MilliQ water and the pH adjusted using HCl. The solution
was stored at 4ºC.
5 x SDS-PAGE sample buffer
Tris base 250 mM
SDS 10% (w/v)
Glycerol 30% (v/v)
Bromophenol blue (Sigma-Aldrich) 0.02% (v/v)
2-mercaptoethanol (2-ME) (Sigma-Aldrich) 5% (v/v)
SDS-PAGE stacking gel buffer, pH 6.8
Tris base 0.5 M
SDS 0.4% (w/v)
The solution was prepared in MilliQ water and the pH adjusted using HCl. The solution
was stored at 4ºC.
2.3 Antibodies
Antibodies were obtained from Abcam (Sydney, Australia), Santa Cruz Biotechnology
(Santa Cruz, CA, USA), R&D Systems (Minneapolis, MN, USA), Affinity Bioreagents
(Rockford, IL, USA) or BD Pharmingen (San Diego, CA, USA). Biotin-conjugated
40
secondary antibodies were obtained from BD Pharmingen or Santa Cruz Biotechnology.
Alexa Fluor®-conjugated secondary antibodies were obtained from Invitrogen (Melbourne,
Australia). The monoclonal and polyclonal antibodies used are listed in Tables 2.3 and 2.4,
respectively.
2.4 Mammalian cells
Mammalian cell lines were originally obtained from the American Type Culture Collection
(ATCC) (Manassas, VA, USA) and are listed in Table 2.5. C2C12 and HT-29 cells were
kindly provided by Dr Anthony Bakker and Professor Hartman, respectively (School of
Biomedical, Biomolecular and Chemical Sciences, University of Western Australia). MeT-
5A, MSTO-211H, NCI-H2052 and NCI-H28 cells were kindly provided by Professor Y C
Gary Lee (Lung Institute of Western Australia, University of Western Australia). PC3 cells
were provided by Professor Leedman (Western Australian Institute of Medical Research,
University of Western Australia).
Normal human bronchial epithelial (NHBE) cells were obtained from Lonza (Basel,
Switzerland). Primary murine peritoneal mesothelial (MPM) cells and human
mesothelioma cells were isolated from tissue specimens as described in Sections 3.10.5.
Primary cells are listed in Table 2.6.
Table 2.3 List of monoclonal antibodies used and their specificities
Antibody Immunogen Specificity
Anti-CK1 Truncated human CK1 NA
Anti-FXII Full length native human Amino acid residues 336-364 of
FXII light chain human FXII light chain
Anti-gC1qR Bacterial-expressed gC1qR Amino acid residues 76-93 within
the N-terminal region of human
gC1qR that binds C1q
Anti-IL-6 Recombinant IL-6 NA
Anti-IL-8 Recombinant IL-8 NA
Anti-Mac Rheumatoid synovial cells Human CD11b cell surface
and human monocytes glycoprotein
Anti-PAR1 Synthetic peptide of PAR1 Amino acid residues 42-55 of
human PAR1
Anti-PAR2 Synthetic peptide of PAR2 Amino acid residues 37-50 of
human PAR2
Anti-PAR3 Synthetic peptide of PAR3 Amino acid residues 31-47 of
human PAR3
Anti-uPAR Recombinant human uPAR The region of human uPAR that
binds uPA
NA: not available.
Table 2.4 List of polyclonal antibodies used and their specificities
Antibody Immunogen Specificity
Anti-HSP90 Synthetic peptide of Amino acid residues 2-12 of
truncated HSP90 human HSP90
Anti-HSP90β Synthetic peptide of Amino acid residues 2-13 of
Truncated HSP90β truncated human HSP90β
Anti-PAR4 C-terminus of PAR4 NA
Anti-PRCP Peptide within the internal NA
region of PRCP
NA: not available.
Table 2.5 Mammalian cell lines used and their origin
Cell line Origin ATTC code
A549 Human alveolar epithelial cells initiated from an CCL-185
explant culture of lung carcinomatous tissue
BEAS-2B Normal human bronchial epithelial cells CRL-9609
immortalised with an adenovirus 12-SV40 virus
hybrid
C2C12 Mouse mitogenic myoblasts subcloned from a CRL-1772
mouse myoblast cell line
CFT1 Human tracheal epithelial cells from a CF patient NA
carrying the homozygous Δ508 mutation in the gene
encoding the CFTR and immortalised with HPV
18/E6/E7
HMC-1 Human immature mast cell line derived from NA
peripheral blood mononuclear cells of a patient
with mast cell leukemia
HT-29 Human large intestinal epithelial cells initiated HTB-38
from a colorectal adenocarcinoma
MeT-5A Normal human mesothelial cells obtained from a CRL-9444
benign pleural fluid and immortalised with a
pRSV-T plasmid and cloned
MSTO-211H Human mesothelioma cells derived from a pleural CRL-2081
Effusion
NCI-H2052 Human mesothelioma cells derived from a pleural CRL-5915
effusion
NCI-H28 Human mesothelioma cells derived from a pleural CRL-5820
Effusion
PC3 Human prostate epithelium initiated from a bone CRL-1435
metastasis of a grade IV prostatic adenocarcinoma
U-937 Human monocytes derived from a pleural effusion CRL-1593
of a patient with histiocytic lymphoma
NA: Not available.
Table 2.6 Primary cells used and their origin
Cell Origin
Murine peritoneal mesothelial cells Normal mesothelial cells derived
from mouse omentum
Normal human bronchial epithelial cells Normal human bronchial epithelium
Human pleural mesothelioma cells Mesothelioma cells isolated from a
malignant pleural effusion of a 68
year old male diagnosed with pleural
mesothelioma
41
2.5 Human lung tissue sections
Human lung tissue sections were obtained from Zyagen Laboratories (San Diego, CA,
USA). Sections were rapidly harvested and fixed in 10% (v/v) neutral buffered formalin.
Lung tissue was then embedded in paraffin and provided as 5-7 μm sections on slides.
2.6 PAR agonist (AP) and control (CP) peptides
Synthetic PAR APs and CPs were obtained from Proteonomics International (Perth,
Western Australia) and synthesised with amidated C-termini as described previously
(Asokananthan et al., 2002). The sequences of the PAR APs and CPs, respectively, were as
follows: frog PAR1, TFLLRN-NH2 and FTLLRN-NH2; human PAR2, SLIGKV-NH2 and
LSIGKV-NH2; human PAR3, TFRGAP-NH2 and FTRGAP-NH2; human PAR4, GYPGQV-
NH2 and GYPGVQ-NH2.
CHAPTER 3
METHODS
42
3.1 Immunohistochemistry and immunocytochemistry
3.1.1 Deparrifinisation and rehydration of tissue sections
Tissue sections were deparraffinised in two changes of 100% (v/v) xylene for 5 min each
and then washed with 50% (v/v) xylene/50% (v/v) ethanol for 5 min. The sections were
then rehydrated sequentially in 100% (v/v), 95% (v/v), 70% (v/v) and 50% (v/v) ethanol for
5 min each. Following this, the sections were rinsed in two changes of PBS for 5 min each.
3.1.2 Antigen retrieval
Deparraffinised and rehydrated sections were incubated in sodium citrate buffer, pH 6, in a
water bath pre-heated to 95°C for 30 min. Sections were then allowed to cool to RT in
buffer and rinsed in two changes of PBS for 5 min each. Following this, sections were
processed as normal for immunocytochemistry (Section 3.1.3).
3.1.3 Immunohistochemistry and immunocytochemistry
Immunocytochemistry was performed on cells seeded into 8-well chamber slides (Nalge
Nunc International; Naperville, IL, USA). At approximately 70% confluence, adherent cells
were rinsed in PBS and fixed in 4% (w/v) paraformaldehyde for 20 min at RT. Endogenous
peroxidase activity was quenched by incubation in 3% (v/v) H2O2 for 5 min. Non-specific
binding was blocked by the addition of 3 drops of blocking agent (DAKO Biotin Blocking
System; Glostrup, Denmark), according to the manufacturer’s instructions, followed by
incubation with blocking diluent at 4ºC O/N. The cells were then incubated with the
43
primary antibody or the appropriate isotype control O/N at 4ºC. Cells were rinsed three
times in PBS for 5 min each and incubated with the biotinylated secondary antibody for 1
hr at RT. Following this, cells were incubated with a 1:1000 dilution of horse radish
peroxidase (HRP)-labeled streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg,
MD) for 30 min at RT in the dark and visualised by addition of DAB. Cells were
counterstained in Gills No. 1 hematoxylin for 1 min, and Scott’s tap water for 45 sec. The
cells were then dehydrated in one change of 70% (v/v) ethanol and three changes of 100%
(v/v) ethanol for 1 min each, followed by fixation in three changes of xylene for 1 min
each. Slides were mounted in DePeX medium (BDH, Australia) and image acquisition
performed using a digital camera mounted on a light microscope (TE2000-U; Nikon Corp.,
Chiyoda-ku, Tokyo, Japan).
For immunofluorescence studies, cells were prepared as described above and incubated
with the appropriate Alexa Fluor®-conjugated antibody for 1 hr at RT in the dark. The cells
were washed in PBS and counterstained with 300 nM 4’,6-diamidino-2-phenylindole
(DAPI) for 5 min at RT to reveal nuclei. The cells were mounted in Immun-MountTM
(Thermo Shandon; Pittsburgh, PA, USA) and stored at 4ºC in the dark until required. The
cells were visualised using a digital camera mounted on a fluorescence microscope
(TE2000-U; Nikon Corp., Chiyoda-ku, Tokyo, Japan). Untreated specimens were also
assessed for autofluorescence. Post-image acquisition processing was performed using
Adobe Photoshop CS2 (Chatswood, Sydney, Australia).
44
3.1.4 Co-localisation studies using sequential indirect immunofluorescence
For co-localisation studies, an indirect, sequential immunofluorescence method was
employed as all of the primary antibodies being used were raised in the same species.
Following blocking, the cells were incubated with the first primary antibody or appropriate
isotype control O/N at 4ºC. The cells were rinsed three times in PBS for 5 min each and
incubated with the appropriate Alexa fluor® 594-conjugated antibody for 1 hr at RT in the
dark. The serum block was repeated, followed by incubation with the second primary
antibody or isotype control O/N at 4ºC. Cells were then incubated with the appropriate
Alexa Fluor® 488-conjugated antibody for 1 hr at RT. Samples were then processed as
described in Section 3.1.3.
3.2. Flow cytometric analysis of receptor expression
Cells (1 x 105) were detached from culture flasks using a non-enzymatic dissociation
medium (Sigma-Aldrich; St Louis, MO, USA) and a suspension containing 2-5 x 105 cells
was fixed in an equivalent volume of 4% (w/v) paraformaldeyhyde for 20 min at RT. Cells
were rinsed in PBS by centrifugation at 1,800 rpm for 5 min at RT and incubated with the
primary antibody either O/N at 4ºC or 2 hr at 37ºC. Following this, cells were incubated
with the appropriate Alexa Fluor® 488-conjugated antibody for 1 hour at RT. The rinse
step was repeated and the cells resuspended in 200 µl PBS and stored at 4ºC in the dark
until required. For all experiments, untreated cells, and cells treated in the absence of the
primary antibody, were included as negative controls. Data were acquired and analysed
using a BD FACSCanto II with FACSDiva version 5 software (BD Bioscience; San Jose,
CA, USA). Laser excitation was set at 488nm and green fluorescence detected through a
45
530/30 bandpass filter. Unless stated otherwise, the fluorescence of 10,000 events was
recorded for each sample and data analysed using FlowJo software (Ashland, OR, USA).
3.3 Analysis of calcium mobilisation
3.3.1 Spectrophotometric analysis of calcium mobilisation
Spectrophotometric analysis of calcium mobilisation was performed as previously
described (Asokananthan et al., 2002). Cells were grown to confluence on glass coverslips
and loaded with 6 μM Fura-2/AM for 30 min at 37°C in calcium and magnesium-free
HBBS. The cells were washed three times and incubated in a final volume of 1 ml HBSS
(containing calcium and magnesium ions) and incubated for a further 30 min to allow
complete de-esterification of the dye. Measurements of calcium mobilisation were
performed using a spectrophotometer (Cairn, Faversham, UK) attached to an
epifluorescence microscope (Nikon; Tokyo, Japan). The ratio of fluorescence emission at
510 nm was measured following excitation at 340 and 380 nm. Baseline fluorescence of the
cells was monitored for 100 sec and they were then stimulated with agonists, and
fluorescence intensity recorded.
3.3.2 Flow cytometric analysis of calcium mobilisation
Cells (1 x 106) were resuspended in calcium and magnesium-free HBBS and loaded with 1
μM Fluo-4/AM for 30 min at 37C. The cells were washed three times and resuspended in
a final volume of 1.2 ml HBSS (containing calcium and magnesium ions) and incubated for
a further 30 min to allow complete de-esterification of the dye. Data were acquired and
46
analysed using a BD FACSCalibur with FACSDiva version 5 software (BD Bioscience;
San Jose, CA, USA). Laser excitation of Fluo-4/AM was set at 488nm and green
fluorescence detected using a 530/30 bandpass filter. Baseline fluorescence of the cells was
monitored for 2 min and they were then stimulated with agonists, and fluorescence intensity
recorded. For all experiments, cells were stimulated with 1 μM ionomycin following
agonist stimulation to confirm cellular responsiveness. Baseline fluorescence was defined
as the threshold and data presented as the percentage of responding cells over this value
using FlowJo software.
3.4 Determination of protein concentration
Samples (10 μl) were mixed with 200 µl Bradford reagent (Bio-Rad Laboratories;
Hercules, CA, USA) in a 96-well microtitre plate and incubated for 5 min at RT. The
absorbances were determined at 595 nm using a SpectraMax 190 spectrophotometer with
SoftMax Pro 4.8 software (Molecular Devices; CA, USA). Total protein concentration was
interpolated from a standard curve constructed using BSA over the concentration range of
1–0.0625 mg/ml.
3.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE analysis of proteins was performed using a 10% polyacrylamide gel under
reducing conditions. The resolving gel buffer was allowed to polymerise for 45 min at RT.
Following this, the stacking gel buffer was loaded into the casting plates with the combs
and incubated for 45 min at RT to polymerise. The combs were removed and the gel placed
in SDS-PAGE tank buffer in the electrophoresis tank. Samples were diluted 1:5 with SDS-
47
PAGE sample buffer containing 5% (v/v) 2-ME and boiled for 5 min at 95ºC prior to
loading into the gel. Samples (25 μl) were loaded into the wells and subject to
electrophoresis at 130 V for approximately 2 hr or until the dye front had run through to the
bottom of the gel. Following electrophoresis, the gel was removed from the casting plates,
rinsed of SDS in distilled water and stained using a commercial Coomasie blue stain (Bio-
Rad Laboratories) according the manufacturer’s instructions. Gels were then destained in
6% (v/v) acetic acid until the desired resolution of the protein bands was obtained. Gels
were then photographed and post-acquisition image processing performed using Adobed
Photoshop CS2.
3.6 Fluorescein isothiocyanate (FITC) conjugation of HK
PD-10 desalting columns (GE Healthcare; Uppsala, Sweden) were equilibrated with 25 ml
100 mM sodium carbonate buffer (pH 9.3) and the 1 mg protein loaded onto the column in
a final volume of 2.5 ml. The flow through was discarded, followed by elution of the
protein with 250 µl aliquots of 100 mM sodium carbonate (pH 9.4). The fractions (200 μl)
were collected and the absorbances read at 280 nm. Fractions containing 90% of the total
absorbance were pooled and the protein concentration determined using a commercial
Bradford reagent (Section 3.3). Following this, FITC stock solution (5 mg/ml) was freshly
prepared and 100 µg added per 1 mg of desalted protein. After incubation for 90 min at RT
with gentle agitation, the conjugated protein was desalted through a PD-10 column
equilibrated with PBS. Again, fractions containing 90% of the total absorbance at 280 nm
were pooled and the conjugate aliquoted and stored at -80ºC. Protein concentration and the
FITC-protein ratio of the labeled protein were determined using the following equation:
48
Protein concentration (mg/ml) = (A280 – (A495 x 0.35)) / 1.4, where 0.35 is a correction
factor for absorbance by FITC at 280 nm
Labeled proteins were subject to reducing 10% SDS-PAGE to examine the integrity of the
protein (Section 3.5) (Figure 3.1).
3.7 FITC-HK binding to cells
Binding of FITC-HK to respiratory epithelial cells was performed as previously described
for endothelial cells (van Iwaarden et al., 1988, Reddigari et al., 1993a, Zhao et al., 2001).
All incubations were performed at 37ºC in 10 mM HEPES buffer (pH 7.4) containing 0.1%
(w/v) sodium azide to minimise ligand internalisation. Cells were incubated with FITC-
labeled HK diluted in 10 mM HEPES buffer (containing either 50 µM ZnCl2 or 10 mM
EDTA) for various intervals of up to 3 hr. The cells were washed with PBS and fixed in 2%
(v/v) paraformaldehyde and analysis of binding was determined by flow cytometry (Section
3.2). For all experiments, untreated cells in buffer alone were included as a negative
control. Additionally, monoclonal antibodies were used in studies to determine the
specificity of FITC-HK to receptor proteins previously shown to play a role in binding
(Colman et al., 1997, Joseph et al., 1999a). Cells were pre-treated with antibodies in the
range of 1-20 µg/ml for 1 hr, followed by incubation with 8.3 nM FITC-HK for 2 hr. Cells,
pre-treated with similar concentrations of isotype control antibody, were used as a negative
control. The role of sulphated proteoglycans in FITC-HK binding on A549 cells was
examined by culturing cells in medium supplemented with 5 mM or 10 mM sodium
chlorate (Safaiyan et al., 1999) for 1, 2 or 4 days prior to incubation with 8.3 nM FITC-HK.
FITC-HK binding was determined using a BD FACSCalibur with FACSDiva version 5
Figure 3.1 SDS-PAGE analysis of FITC-labeled HK
Purified HK was labeled with FITC and subjected to 10% reducing SDS-PAGE. Lane 1:
SDS-PAGE molecular weight markers; Lane 2: unlabeled HK; Lane 3: FITC-labeled HK.
Numbers on left refer to the size of the molecular weight markers in kDa.
1 2 3
97
55
116
45
45
49
software (BD Bioscience; San Jose, CA, USA) and data expressed as a percentage of cells
positive for FITC when gated using untreated cells. Total and non-specific binding was
defined as the binding of FITC-HK occurring in the presence of 50 µM ZnCl2 or 10 mM
EDTA, respectively. Specific binding was determined by subtracting the amount of non-
specific binding from total binding. For inhibition studies using monoclonal antibodies and
sodium chlorate, the results obtained were compared against those in the presence of the
IgG1 isotype control antibody or cells cultured in medium lacking sodium chlorate, both of
which were defined as 100% binding.
3.8 Activation of PPK
Unless otherwise stated, all incubations were performed at 37°C in 5% (v/v) CO2. Cells,
lysates and matrices were incubated with the PK-selective chromogenic substrate S-2302
(0.8 mM final concentration). PK activity was determined by monitoring the absorbance at
405 nm over a 90 min interval using a SpectraMax 190 spectrophotometer with SoftMax
Pro 4.8 software (Molecular Devices; CA, USA).
3.8.1. Activation on cell surfaces
PPK activation assays using cultured cells were performed as previously described (Zhao et
al., 2001). Briefly, confluent cell monolayers in 96-well microtitre plates were incubated
with 20 nM HK for 1 hr in 10 mM HEPES buffer (containing 50 µM ZnCl2). The cells
were washed of free HK, then incubated with 20 nM PPK in the same buffer alone or buffer
in the presence of inhibitors for 1 hr. Following this, supernatants were collected and stored
at -80ºC for subsequent measurement of BK release (Section 3.8.2), and PK activity then
50
measured using the S-2302 substrate. For suspension cell lines, HMC-1 and U-937, 100 μl
containing 2.5 x 104 cells were incubated concomitantly with HK (20 nM final
concentration), PPK (20 nM final concentration) and S-2302 (0.8 mM final concentration),
and absorbances measured immediately. For all assays, untreated cells and cells treated
with HK and PPK alone were included as controls. For inhibition studies, absorbances were
blanked using untreated cells, and PK activity compared against data obtained in the
absence of inhibitors, which was defined as 0% inhibition. For antibody neutralisation
experiments, cells treated with the isotype control antibody were defined as 0% inhibition.
For experiments using pleural effusions, confluent MeT-5A cells were incubated with 100
μl pleural effusions in duplicate for 1 hr, thoroughly washed in HEPES buffer, and PK
activity monitored for 4 hr in the presence of 0.8 mM S-2302. Absorbances were blanked
using untreated cells.
3.8.2. PPK activation by cell lysates
Confluent 75 cm2 culture flasks were washed with ice-cold PBS and adherent cells
removed in 10 mM HEPES buffer, pH 7.4, (containing 50 μM ZnCl2) using a cell scraper.
The lysate was collected in 1.5 ml microfuge tubes and flasks rinsed with an additional 0.5
ml aliquot of 10 mM HEPES buffer and pooled. The pooled lysate was then disrupted
further by passage through a 21-gauge needle and the tubes centrifuged at 13,000 rpm for
10 min at 4C to pellet any insoluble cell debris. The protein concentration was determined
using the Bradford assay (Section 3.3) and the concentration adjusted in 10 mM HEPES
buffer. The lysate was mixed with HK (20 nM final concentration), PPK (20 nM final
concentration) and S-2302 (0.8 mM final concentration) in microtitre plates and PK activity
monitored. For all assays, untreated lysate and lysate treated with HK or PPK alone were
51
included. For inhibition studies, absorbances were blanked using untreated lysate, and PK
activity compared against data obtained in the absence of inhibitors, which was defined as
0% inhibition.
3.8.3 PPK activation by cell-free matrices
The preparation of matrices for PPK activation assays were performed as previously
described for endothelial cells (Motta et al., 2001, Moreira et al., 2002) using cells seeded
in 96-well microtitre plates. Upon confluency, cells were washed with PBS three times and
then treated with 0.5% (v/v) Triton X-100 in PBS for 15 min. The wells were washed with
PBS and cells incubated with 0.025 M NH4OH for 10 min. Following this, cells were
washed five times with 0.02 M Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and
0.05% (v/v) Tween 20, followed by washing five times with 10 mM HEPES buffer. The
absence of cells was confirmed by light microscopy and the wells were sequentially treated
with 20 nM HK and PPK for 1 hr each, and PK activity determined following the addition
of the S-2302 substrate. For all assays, untreated matrix and matrix treated with HK or PPK
alone were included.
3.9 Enzyme immunoassays (EIA)
3.9.1 Competitive BK EIA
A commercial EIA (Bachem; San Carlos, CA, USA) was used to measure immunoreactive,
rather than functional, BK in cell culture supernatants and pleural fluids, according to the
manufacturer’s instructions. Briefly, 50 µl standard or sample, 25 µl anti-BK antibody and
52
25 µl biotinylated BK tracer were loaded into each well of a 96-well microtitre plate and
incubated O/N at 4ºC or 2 hr at RT. Following this, the wells were washed five times with
300 µl PBS containing 0.05% (v/v) Tween 20 and incubated with 100 µl HRP-labeled
streptavidin diluted 1:200 for 1 hr at RT. The wash step was repeated and colour developed
following incubation with 100 µl tetramethylbenzidine (TMB) solution for 30 min at RT.
The reaction was terminated by the addition of 100 µl 2M HCl and the absorbances read at
450 nm. For each assay, EIA buffer in the absence of standard or sample was included to
account for background absorbance. Concentrations of immunoreactive BK were
determined by interpolation from the standard curve using purified BK provided in the kit.
3.9.2 IL-6 and IL-8 enzyme-linked immunosorbant assay (ELISA)
IL-6 and IL-8 release was determined using a specific ELISA, as described previously
(Asokananthan et al., 2002). Briefly, Maxisorp 96-well plates (Nunc; Rochester, NY, USA)
were coated with 100 μl/well of the primary antibody (0.5 μg/ml in alkaline carbonate
buffer, pH 9) and incubated O/N at 4C. The plates were washed with PBS containing
0.05% (v/v) Tween 20, and blocked with 200 μl PBS containing 0.05% (v/v) Tween 20 and
1% (w/v) BSA for 1 hr at RT. The wash step was repeated and 100 μl sample or standard
added. The plates were then incubated overnight at 4C, washed, and incubated with the
biotinylated secondary antibody (0.5 μg/ml) for 1 hr at RT. After washing, the plate was
incubated with 100 μl/well 1:4000 dilution of HSP-labeled streptavidin for 30 min at RT.
Following washing, the wells were incubated with 100 μl/well K-Blue ELISA Substrate
(Graphic Scientific; Brisbane, Australia). Reactions were terminated by the addition of 1 M
HCl and the absorbances at 450 nm measured. Cytokine concentrations were determined by
53
interpolation from the standard curve using purified recombinant IL-6 and IL-8 (BD
Pharmingen).
3.9.3 TNF- and monocyte chemotactic protein (MCP)-1 ELISA
TNF- and MCP-1 concentrations were measured using commercial ELISA kits
(eBioscience; San Diego, CA, USA) according to the manufacturer’s instructions. Briefly,
Maxisorp 96-well plates (Nunc) were coated with 100 μl/well of the capture antibody in 1 x
alkaline carbonate buffer (pH 9) and incubated O/N at 4C. The plates were washed with
PBS containing 0.05% (v/v) Tween 20, and blocked with 200 μl PBS containing 0.05%
(v/v) Tween 20 and 1% (w/v) BSA for 1 hr at RT. The wash step was repeated and 100 μl
sample or standard added. The plates were then incubated overnight at 4C, washed, and
incubated with the detection secondary antibody (0.5 μg/ml) for 1 hr at RT. After washing,
the plate was incubated with 100 μl/well HRP-labeled avidin for 30 min at RT. Following
washing, the wells were incubated with 100 μl/well of the substrate solution provided for
15 min at RT and reactions terminated by the addition of 1 M HCl. Cytokine concentrations
were determined by interpolation from the standard curve using the provided recombinant
TNF- and MCP-1.
54
3.10 Cell culture
3.10.1 Cell culture conditions
Unless otherwise stated, media were replaced every 48 hr until the cells were at least 80%
confluent. At all stages of culture, cells were maintained at 37ºC in 5% (v/v) CO2. The
origin of each cell line used and their growth requirements are described in Section 2.4.
3.10.2 Pre-coating of tissue culture plates with rat tail collagen and gelatin
MeT-5A and NCI-H2052 cells were cultured on plates coated with 0.1% (w/v) gelatin,
whereas C2C12 and MSTO-211H cells were cultured on rat tail collagen-coated plates.
Gelatin or rat tail collagen was added to each well (100 μl for 96-well plates or 500 μl for
24-well plates) and incubated for at least 30 min at 37ºC. Excess gelatin was aspirated, the
wells washed twice with PBS and then used immediately. For rat tail collagen coated
plates, wells were washed of excess collagen using PBS and sterilised under UV light for 1
hr.
3.10.3 Propagation of cell lines
At confluency, the cells were washed twice with PBS and incubated with 4 ml
trypsin/EDTA for approximately 5-10 min at 37ºC in 5% (v/v) CO2. The cells were
examined by light microscopy and the flasks gently tapped to dislodge the adherent cells.
The trypsinisation reaction was stopped by addition of 10 ml 10% (v/v) FCS in PBS. The
cell mixture was centrifuged at 1,400 rpm for 5 min at RT and the pellet resuspended in
55
complete media containing serum or additives. The concentration of resuspended cells was
determined (Section 3.9.6), and cells then seeded for experimentation or transferred to 75
cm2 cell culture flasks (Nunc) for propagation. C2C12 cells were maintained at
subconfluence to minimise widespread contact, which induces fusion and formation of
myotubes. For passage, U-937 and HCM-1 cells were collected by centrifugation and
seeded into 75 cm2 culture flasks (Nunc) at a density of 2 x 10
5 cells/ml, and maintained for
no more than 3-4 days.
3.10.4 Myotube differentiation of C2C12 cells
Sub-confluent C2C12 murine myoblasts were differentiated into myotubes by replacement
with low mitogenic medium containing DMEM supplemented with 2% (v/v) FCS and 4
mM L-glutamine for 7 days,. Medium was changed every 48 hr. Myotube formation was
confirmed by light microscopy as multinucleated cells.
3.10.5 Isolation of human and murine primary mesothelial cells
Isolation of primary murine peritoneal mesothelial (MPM) cells was kindly performed by
Dr Sally Maher and Ms. Ai Ling Tan (Lung Institute of Western Australia, University of
Western Australia, Perth, Australia). Mice were anaesthetised using methoxyflurane
(Medical Developments Australia; Springvale, Australia) and culled by cervical
dislocation. MPM cells were isolated from the omentum and fat pads of male C57BL/6
mice. Mesothelial cells were separated from their basement membrane by incubation in
0.25% (w/v) trypsin and 0.02% (w/v) EDTA in DMEM for 30 min at 37C under constant
agitation. Trypsin activity was inactivated following addition of 1 ml FCS and cells
56
harvested by centrifugation at 1200 rpm for 6 min at RT and plated into 25 cm2 cell culture
flasks (Nunc). All experiments were performed using cells at passage three. Isolation of
primary human pleural mesothelioma cells was kindly performed by Dr Bahareh Badrian
and Ms. Hui Min Cheah (Lung Institute of Western Australia, University of Western
Australia, Perth, Australia), using pleural effusions obtained by thoracoscopy from patients
diagnosed with malignant mesothelioma. Briefly, effusions were centrifuged at 1,200 rpm
for 15 min and plated in 75 cm2 cell culture flasks (Nunc). The medium was changed the
following day, and every two days thereafter. As they do not replicate under these
conditions, major contaminants of initial cultures were removed by cell passages. All
experiments were performed using cells at passage three. The work was approved by Sir
Charles Gairdner Hospital Research Ethics Committee.
3.10.6 Determination of cell count and viability
Cell count and viability were determined by trypan blue exclusion. After centrifugation, the
cells were resuspended in an appropriate volume of complete medium. The resuspended
cells (10 μl) were diluted 1:2 in trypan blue and loaded into the chamber of a Nuebauer
hemocytometer. Live cells (clear cytoplasm) were differentiated from dead cells (blue
cytoplasm) and the number of cells in the 1 x 1 x 0.1 mm grid was determined. The
concentration was calculated using the following equation:
Cells/ml = viable cell count x dilution factor x 104
57
3.10.7 Storage of cells by freezing
Following passage, the cells were resuspended in cell freezing medium (FCS containing
10% (v/v) DMSO) and transferred to a 1.8 ml CryoTube® vial (Nunc). The cells were
immediately stored at -80ºC in a “Mr Frosty” freezing container (Nalgene; Rochester, NY,
USA) containing isopropyl alcohol for controlled cooling. Following this, the cells were
transferred to liquid nitrogen for long-term storage.
3.11 Cell stimulation
3.11.1 Serum starvation and stimulation
Following passage, cells were seeded in culture plates in complete medium until confluent.
Following this, cells were washed twice in PBS and incubated in basal medium without
serum or additives for 24 hr. Cells were then incubated with stimuli diluted in basal
medium and culture supernatants collected for EIA (Section 3.8) or stored at -80°C. For all
experiments, cells stimulated with the vehicle alone or 200 ng/ml phorbol myristate acetate
(PMA) were included as negative and positive controls for cytokine release, respectively.
Following stimulation, cell viability was determined using an acid phosphatase assay
(Section 3.11.2).
3.11.2 Determination of cell viability
Cell viability was determined by measuring cytosolic acid phosphatase activity (Yang et
al., 1996). A the conclusion of an experiment, cells were rinsed in PBS to remove non-
58
adherent cells and then incubated with 250 µl acid phosphatase substrate solution for 2 hr in
the dark. The reaction was stopped by addition of 25 µl 1 M NaOH and the absorbances
read at 405 nm. For all experiments, acid phosphatase substrate solution in the absence of
cells was used to control for background absorbance.
3.12 Pleural fluid samples
3.12.1 Subjects
Paired pleural fluid and serum samples were kindly provided by Dr. Jenette Creaney
(National Research Centre for Asbestos Related Diseases, Western Australia Institute for
Medical Research, University of Western Australia, Perth, Western Australia) and were
originally obtained from a cohort of patients recruited from the respiratory clinics of Sir
Charles Gairdner Hospital or the Hollywood Specialist Centre (Perth, Western Australia) as
previously described (Creaney et al., 2008). This patient group is herein referred to as
Cohort 1 and includes patients diagnosed with malignant mesothelioma (MM) (n = 10),
lung cancer (n = 4), breast cancer (3), leukemia/lymphoma (n = 3), renal disease (n = 1),
benign transudative effusion (n = 1), and non-malignant exudative effusion (n = 18). The
final diagnosis was confirmed by pathology and included clinical follow-up of all cases
until death or to last citation in the Public Health database system (iSoft Clinical Manager)
to confirm that the clinical pattern matched the diagnosis. Additional pleural effusions were
also kindly provided by Professor Y C Gary Lee (Lung Institute of Western Australia,
University of Western Australia, Perth, Western Australia) and originally collected from
patients recruited from the Oxford Pleural Unit (Oxford Centre for Respiratory Medicine,
Oxford, UK), as previously described (Davies et al., 2009), and is referred to here as
59
Cohort 2. Cohort 2 includes patients diagnosed with MM (n = 3), adenocarcinoma (n = 5),
rheumatoid arthritis (n = 2), congestive heart failure (n = 4), hepatic hydrothorax (n = 1),
and non-malignant exudative effusion (n = 4). For each cohort, effusions were classified as
originating from either malignant or non-malignant disease on the basis of cytologic and
immunohistochemical features, and effusions from non-malignant patients were classified
as either transudates or exudates using Light’s criteria (Light et al., 1972). All malignancies
other than MM are referred here as non-MM malignancies. The studies during which the
above samples were obtained were approved by Sir Charles Gairdner Hospital, Hollywood
Hospital and the Mid and South Buckinghamshire and Central Oxford Research Ethic
Committees, respectively. All participants provided written consent. See Appendix I for
patient characteristics.
3.12.2 Pleural fluid collection
Pleural fluid samples were collected by aseptic technique in standard blood collection tubes
containing sodium citrate. The samples were centrifuged at 3,000 rpm for 10 min at 4°C
and the supernatants collected and stored at -80°C.
3.13 Statistical analysis
Data were presented as mean ± standard error of the mean and statistical significance of the
means determined using Student’s t-test. The correlation between two variables was
performed using Pearson’s correlation coefficient and the significance determined using a
critical value table. Differences between groups of patients were assessed using Wilcoxon
Rank Sum Test. Multiple comparisons were performed using ANOVA on-rank. A p value
60
< 0.05 was considered statistically significant. Bonferroni’s correction was used when
necessary and defined by the following equation: p value = 0.05/number of variables
CHAPTER 4
ACTIVATION OF THE PLASMA KALLIKREIN-KININ SYSTEM ON
RESPIRATORY EPITHELIAL CELLS
60
4.1 Introduction
At present, it is not clear whether plasma KKS activation occurs on respiratory epithelial
cells, but a variety of data indicate that it could. In this regard, several studies have
observed increased kinin formation in models of experimentally induced airway
inflammation. For example, Christiansen et al. induced kinin production in the airways of 5
atopic asthmatic patients following endobronchial allergen challenge (Christiansen et al.,
1992). Likewise, several reports demonstrated similar results following nasal provocation
of allergic subjects with allergen (Proud et al., 1983, Baumgarten et al., 1985, Baumgarten
et al., 1986) and chronic non-allergic rhinitis patients with methacholine (Baumgarten et
al., 1992). Furthermore, kinins are elevated in patients with naturally occurring airway
disease, including pneumonia, bronchitis (Baumgarten et al., 1992, Zhang et al., 1997),
asthma (Gawlik et al., 1995) and rhinovirus infection (Proud et al., 1990).
In such studies, the presence of both Lys-BK and BK indicates the involvement of several
enzymes in kinin formation, including PK. In support of this, Baumgarten et al.
demonstrated increased PPK influx and activation during the nasal response to allergen in a
cohort of allergic subjects (Baumgarten et al., 1986). Likewise, PK is elevated in the BAL
fluid of patients with acute pneumonia and chronic bronchitis (Zhang et al., 1997, Peng et
al., 1999). Furthermore, PPK mRNA has been detected in whole lung tissue (Hermann et
al., 1999, Neth et al., 2001) and PPK protein is expressed on respiratory epithelial cells and
lung carcinoma subtypes, including adenocarcinoma (Fink et al., 2007, Chee et al., 2008).
Therefore, such data are suggestive of localised participation of PK in plasma KKS
activation in the airways.
61
The work described in this Chapter was undertaken to determine whether human
respiratory epithelial cells supported the assembly and activation of the plasma KKS. These
studies were performed using a range of human respiratory epithelial cell lines including
A549 adenocarcinoma, BEAS-2B SV40-transformed and CFT-1 tracheal epithelial cells
containing the Δ508 mutation in the cystic fibrosis transmembrane regulator (CFTR). In
addition, limited studies were performed using primary NHBE cells to confirm the results
obtained using cell lines. Furthermore, studies were also performed using matrix, given
laminin is known to bind HK (Schousboe and Nystron, 2009) and that endothelial
extracellular matrix supports plasma KKS activation (Motta et al., 2001).
Initially, A549 and BEAS-2B cells were examined for the presence of HK receptor
associated proteins previously described on endothelium, and whether they co-localised.
Subsequently, functional assays using FITC-labeled HK were performed to determine the
involvement of these receptors in binding HK on A549 cells. As sulphated proteoglycans
are known to bind HK on endothelial cells (Renne et al., 2000), its involvement on
respiratory epithelium was also determined by culturing cells in the presence of sodium
chlorate, which has been shown to down-regulate intracellular sulphation reactions
(Safaiyan et al., 1999).
Following this, experiments were performed to determine whether respiratory epithelial cell
lines and primary cells could assemble PPK to generate PK and liberate BK. As a number
of partial or complete inhibitors of PRCP-mediated conversion of PPK to PK on endothelial
cells have been identified (Shariat-Madar et al., 2002), a panel of these inhibitors were used
to assess its potential role on respiratory epithelium. In addition, the potential role of
HSP90 in this system was investigated using a known inhibitor, novobiocin (Marcu et al.,
62
2000, Marcu et al., 2000). Lastly, the extent of this system was assessed by determining
whether additional cell types could support plasma KKS activation.
63
4.2 Results
4.2.1 Expression and co-localisation of the known HK receptor proteins on respiratory
epithelial cells
Figures 4.1 and 4.2 demonstrate the expression of uPAR, gC1qR and CK1 in permeabilised
and non-permeabilised A549 and BEAS-2B cells by immunocytochemistry and flow
cytometry, respectively, but not Mac-1. As only weak expression of this HK receptor
associated molecule was demonstrated on both cell lines, its role in binding HK was not
explored further. The degree of uPAR staining on the A549 and BEAS-2B cell lines was
similar, but gC1qR and CK1 was noticeably higher on the A549 cell line. Figure 4.3 shows
that uPAR, gC1qR and CK1 co-localised on both A549 and BEAS-2B cell lines. While
CK1 and uPAR, and gC1qR and CK1 co-localised on the majority of the cells examined,
co-localisation of uPAR and gC1qR was limited to a small proportion of the cells.
Immunofluorescence studies performed using commercially obtained tissue sections
indicated weak uPAR staining along the bronchial epithelium of normal human lung, in
contrast to relatively strong staining of gC1qR and CK1 (Figure 4.4). However, in contrast,
only moderate CK1 staining was observed on alveolar epithelium (Figure 4.5).
4.2.2 Binding of FITC-labeled HK to respiratory epithelial cells
Approximately one third of A549 cells examined bound FITC-labeled HK in the presence
of 50 µM ZnCl2, and binding was significantly inhibited by both EDTA and 50-fold molar
excess of unlabeled HK (p < 0.005) (Figure 4.6). Specific binding of FITC-HK was
observed at the earliest time-point measured (15 min), reached a plateau at 2 hr (Figure
4.7A), and was saturated at 8.3 nM FITC-HK (Figure 4.7B).
Figure 4.1 Immunohistochemical analysis of uPAR, gC1qR, CK1 and Mac-1
expression on respiratory epithelial cells
Immunohistochemical analysis of uPAR, gC1qR, CK1 and Mac-1 expression on A549 (A)
and BEAS-2B (B) cells. Expression on non-permeabilised (i) and permeabilised (ii) cells
was examined. Positive immunoreactivity is seen as brown precipitate. The figures are
representative of three independent experiments.
uPAR gC1qR CK1 Mac-1 IgG1 isotype
A
B i
ii
i
ii
Figure 4.2 Flow cytometric analysis of uPAR, gC1qR, CK1 and Mac-1
expression on respiratory epithelial cells
Flow cytometric analysis of uPAR, gC1qR, CK1 and Mac-1 on A549 (A) and BEAS-2B
(B) cells. Expression on non-permeabilised (i) and permeabilised (ii) cells was examined.
Representative figures are shown and the values associated represent mean SEM of three
independent experiments. Shaded: uPAR, CK1, gC1qR and Mac-1; Black line: isotype
control.
A
B
49 9 83
1.8
2.2
1.4
91
1.5
92
0.4 1.2 0.7
% M
ax
i
ii
i
ii
0.9
0.3 11
1.4 23 1
0.95
0.04
16 6
79
7 78
4 46 9
30 9
62 7
Fluorescence intensity
uPAR gC1qR CK1 Mac-1
Figure 4.3 Co-localisation of uPAR, gC1qR and CK1 on respiratory epithelial cells
Fluorescent microscopy analysis of co-localisation of uPAR, CK1 and gC1qR on A549 (A)
and BEAS-2B (B) cells. The figures are representative of three independent experiments.
Blue: nuclei.
gC1qR CK1 Merged
Merged
Merged CK1 uPAR
uPAR gC1qR
CK1 uPAR
gC1qR CK
1
uPAR gC1qR
A
B Merged
Merged
Merged
Figure 4.4 uPAR, gC1qR and CK1 expression on normal human bronchial
epithelium
Immunofluorescence of uPAR, gC1qR and CK1 expression on normal human bronchial
epithelium. The figures are representative of three independent experiments. Arrow,
bronchial epithelium.
Alex Fluor® 488 DAPI counterstain
IgG1
isotype
uPAR
gC1qR
CK1
Figure 4.5 uPAR, gC1qR and CK1 expression on normal human alveolar
epithelium
Immunofluorescence of uPAR, gC1qR and CK1 expression on normal human alveolar
epithelium. The figures are representative of three independent experiments. Arrow,
alveolar epithelium.
DAPI counterstain Alexa Fluor® 488
IgG1
isotype
uPAR
gC1qR
CK1
Figure 4.6 Binding of FITC-labeled HK to A549 cells
Binding of FITC-labeled HK to A549 cells in the presence of 50 μM ZnCl2 (A), 10 mM
EDTA (B) or 50-fold molar excess unlabeled HK (C). Representative figures are shown
and the values associated represent mean SEM of three independent experiments. *
Significantly greater than cell treated with FITC-labeled HK in the presence of 10 mM
EDTA or 50-fold molar excess unlabeled HK (p < 0.005).
4.2 ± 0.8
8.33 ± 1.3
30.5 ± 3.7*
Alexa Fluor® 488
A
B
C
% M
ax
101
102
103 10
4 10
5
100
80
60
40
20
0
0
20
40
60
80
0
20
40
60
80
100
100
Figure 4.7 Time course and dose response of FITC-labeled HK binding to A549
cells
Time course (A) and dose response (B) of FITC-labeled HK binding to A549 cells. Specific
binding () was calculated by subtraction of total binding () from non-specific binding
(). For the dose response experiment, cells were incubated with FITC-HK for 2 hr. Data
is presented mean SEM of three independent experiments.
15 30 60 120 180
0
5
10
15
20
25
30
% F
ITC
-HK
bin
din
g
A
1 2 5 10 8.3 16.6 41.5 83
FITC-HK concentration (nM)
0
B
10
20
30
40
50
60
Time (sec)
% F
ITC
-HK
bin
din
g
64
4.2.3 Inhibition of FITC-labeled HK binding to respiratory epithelial cells
When pre-treated with anti-uPAR, -gC1qR or -CK1 antibodies, HK binding to A549 cells
was inhibited by approximately 27%, 18% and 7%, respectively (p > 0.05). However, when
cells were treated with all these antibodies in combination, about 45% inhibition was
obtained (p = 0.02; Figure 4.8A). The culture of cells in sodium chlorate-supplemented
medium did not inhibit FITC-HK binding to A549 cells (Figure 4.8B).
4.2.4 PPK activation and liberation of BK
A549 and BEAS-2B cells incubated with the PK-selective substrate S-2302 after sequential
treatment with HK and PPK demonstrated an increase in absorbance over time due
formation of the chromogenic product (Figures 4.9A and B, respectively). These studies
were expanded to include primary NHBE cells and a cell line possessing the CFTR defect
(Figures 4.9C and D, respectively). In contrast to untreated cells or cells treated with HK
alone, modest PK activity was detected when cells were treated with PPK alone, with the
exception of BEAS-2B cells. Of the four cell types tested, the degree of PK activity
generated by BEAS-2B cells was the lowest and was approximately 3 to 4-fold lower than
that observed with either A549, CFT-1 or NHBE cells. Figure 4.10 shows that pre-
treatment of cells with fetal calf serum used in the tissue culture process had little effect on
PK activity generated by A549 cells. However, PK activity was enhanced in the presence of
serum when A549 cells were treated with PK alone (p < 0.0001). PK activity generated by
both serum treated and starved cells was significantly greater than that generated by
untreated cells and cells incubated with HK alone (p < 0.0001).
Figure 4.8 Inhibition of FITC-labeled HK binding to A549 cells
A549 cells were pre-treated with antibodies against uPAR (), gC1qR () and CK1 ()
alone or in combination () (A) or cultured in media supplemented with 5 mM (grey bars)
or 10 mM (black bars) sodium chlorate (B) and binding determined following 2 hr
incubation with FITC-HK. The data are present as the mean ± SEM of three independent
experiments.
1 5 10 20 EDT
A concentration 1 5 10 20 EDTA
0
20
40
60
80
100
A
% F
ITC
-HK
bin
din
g
1 2 4
Da
y
1 2 4
0
20
40
60
80
100
120
B
% F
ITC
-HK
bin
din
g
Days
Antibody concentration (μg/ml)
Figure 4.9 PPK activation on A549, BEAS-2B, CFT1 and NHBE cells
A549 (A), BEAS-2B (B), CFT1 (C) or NHBE (D) cells were sequentially treated with HK
and PPK () and PK activity monitored over time. Untreated cells () and cells treated
with HK () or PPK () alone were also included. A549 cells () treated with HK and
PPK were also included as a positive control for NHBE cells. The data are presented as
mean ± SEM from three independent experiments performed in triplicate.
0
0.5
1
1.5
2
2.5 A
Time (min)
0
0.5
1
1.5
2
2.5 D
0 10 20 30 40 50 60 70 80 90
Time (min)
0 10 20 30 40 50 60 70 80 90
Ab
sorb
an
ce (
40
5n
m)
C
0
0.5
1
1.5
2
2.5
B
0
0.5
1
1.5
2
2.5
Figure 4.10 The effect of serum on PPK activation using A549 cells
Cells cultured in serum (white bars) or starved of serum for 24 hr (black bars) were
sequentially treated with HK and PPK and PK activity determined after 90 min. The data
are presented as the mean ± SEM of three independent experiments performed in triplicate.
* Significantly greater than serum starved cells treated with PPK alone (p < 0.0001). **
Significantly greater than untreated serum starved cells or serum starved cells treated with
HK alone (p < 0.0001).
0
0.4
0.8
1.2
1.6
Untreated HK alone PPK alone HK + PPK
Ab
sorb
an
ce (
405
nm
)
*
**
**
65
Following incubation of A549 and NHBE cells with HK and PPK, modest, but significant,
release of BK was demonstrated when both cell types were treated with HK alone (A549, p
= 0.017; NHBE, p < 0.0001). BK release increased when cells were treated with PPK alone
(A549 and NHBE, p < 0.0001) or sequentially with HK and PPK (A549 and NHBE, p <
0.0001) (Figure 4.11). In addition, plasma KKS activation was also demonstrated when
A549 cell-free matrix and lysate, rather than whole, intact cells, were treated with HK and
PPK (Figures 4.12 and 4.13, respectively). Subsequent studies were performed to determine
whether A549 cells were responsive to BK. Calcium mobilisation was shown to be induced
in A549 cells in response to 1 μM BK (Figure 4.14). Similarly, stimulation with BK
resulted in the release of IL-6 and IL-8 (Figure 4.15A). Figure 4.15B shows that when cells
were treated with des-Arg9-BK IL-8 release was also observed, but only at concentrations
several orders of magnitude higher than when using BK.
4.2.5 Inhibition of PPK activation
In an attempt to determine whether PRCP might be involved in plasma KKS activation on
respiratory epithelium, several inhibitors known to inhibit PRCP-mediated conversion of
PPK to PK on endothelium were used (Figure 4.1). In addition, the role of HSP90 in this
process was also examined using a know HSP90 inhibitor, novobiocin.
4.2.5.1 Inhibition of PPK activation on respiratory epithelial cells
ANG II, the preferred substrate of PRCP (Shariat-Madar et al., 2002), inhibited PPK
activation by only 11%, and other known inhibitors such as antipain, leupeptin, 2-ME or
AEBSF had no noticeable inhibitory effect on activation. However, BK, another known
Figure 4.11 BK liberation from A549 and NHBE cells
Following sequential treatment of A549 (A) or NHBE (B) cells with HK and PPK, culture
supernatants were assayed for BK The data are presented as mean ± SEM from three
independent experiments performed in triplicate.
0
1
2
3
4
5
6
Untreated HK alone PPK alone HK + PPK
BK
con
cen
trati
on
(n
g/m
l)
p < 0.0001
p < 0.0001
p < 0.0001
B
0
1
2
3
4
p = 0.018
p < 0.0001
p = 0.002
p < 0.0001 A
Figure 4.12 PPK activation and BK liberation by A549 cell matrix
A549 cell matrix remaining in microtitre wells after cell removal was sequentially treated
with HK and PPK () and PK activity monitored over time. Untreated matrix () and
matrix treated with HK () or PPK () alone were also included (A). Following treatment
of A549 cell matrix with HK and PPK, culture supernatants were assayed for BK (B). The
data are presented as mean ± SEM from three independent experiments performed in
triplicate.
HK + PPK
0
0.5
1
1.5
2
2.5
3
5 10 20 30 40 50 60 70 80
Ab
sorb
an
ce (
405n
m)
Time (min)
90
0
1
2
3
4
5
6
7
Untreated HK alone PPK alone
BK
con
cen
trati
on
(n
g/m
l)
p = 0.0006
p < 0.0001
p = 0.0014
p < 0.0001
B
A
Figure 4.13 PPK activation by A549 cell lysate
HK, PPK and S-2302 were incubated with various amounts of A549 lysate protein in
microtitre plates (0.5 (), 1 (), 2.5 (), 5 () or 10 μg () protein) and PK activity
monitored over time (A). Lysate (2.5 μg) was incubated with HK, PPK and S-2302 and PK
activity monitored over time (). Untreated lysate () and lysate treated with HK () or
PPK () alone were also included (B). The data are presented as mean ± SEM from three
independent experiments performed in triplicate.
0
1
2
3
4
Ab
sorb
an
ce (
405n
m)
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50 60 70 80
Ab
sorb
an
ce (
405 n
m)
Time (min)
90
B
A
Figure 4.14 Spectrophotometric analysis of calcium mobilisation induced by BK in
A549 cells
Spectrophotometric analysis of calcium mobilisation induced by 1 μM BK. The arrow
indicates the addition of BK. The figure is representative of three independent experiments.
0.4
0.6
0.8
1
1.2
0 5 104
1 105
1.5 105
2 105
2.5 105
3 105
3.5 105
4 105
0 50 100 150 200 250 300 350 400
1.2
1
0.8
0.6
0.4
Flu
ore
scen
ce r
ati
o (
340/3
80 n
m)
Time (sec)
Figure 4.15 The effect of BK and des-Arg9-BK on IL-6 and IL-8 release from A549
cells
A549 cells were stimulated with BK (white bars) or des-Arg9-BK (black bars) for 24 hr and
IL-6 (A) and IL-8 (B) release determined by ELISA. The data are presented as the mean ±
SEM of three independent experiments performed in triplicate. * Denotes significantly
greater than vehicle control
0
50
100
150
200
250
300
IL-6
con
cen
trati
on
(p
g/m
l)
0
500
1000
1500
2000
Vehicle
control
0.1 1 10 100
IL-8
con
cen
trati
on
(p
g/m
l)
Kinin concentration (μM)
* * *
* *
*
*
* * * A
B
Table 4.1 Panel of inhibitors used for PPK activation assays and their
specificities
Inhibitor Specificity
Antipain Serine/cysteine proteases, trypsin-like serine proteases
Leupeptin Serine/cysteine proteases
EDTA Metalloproteases
Benzamidine Trypsin, tyrpsin-like enzymes, serine proteases
AEBSF Serine proteases
Plummer’s inhibitor Carboxypeptidase N
Phosphoramidon Neutral endopeptidase
Captopril Angiotensin converting enzyme
Apstatin aminopeptidase P
66
substrate inhibitor of PRCP (Shariat-Madar et al., 2002), inhibited activation by 98%, with
an IC50 of approximately 20 µM. Also, benzamidine and EDTA had little effect, while
cysteine inhibited activation by approximately 65% at 10 mM (Figure 4.16A and B). As
BK was a potent inhibitor of PPK activation on A549 cells, the effect of BK-derived
peptides on activation was examined. Incubation with 500 μM des-arg9-BK, BK 1-7 or BK
1-5 inhibited PPK activation on A549 cells by 6%, 20% and 4%, respectively (Figure
4.17A). Incubation with protamine sulphate also inhibited activation by 98% on A549 cells,
with an IC50 of approximately 4 μM (Figure 4.17B). As FXIIa is a known activator of PPK,
an antibody which blocks FXIIa activity was used to determine a role on respiratory
epithelium, but inhibition of PPK activation on A549 cells was not observed (Figure 4.18).
Additionally, the role of BK metabolising enzymes as PPK activators was examined using
known inhibitors. Figure 4.19A shows PPK activation on A549 cells was not significantly
inhibited by 1 mM Plummer’s inhibitor (CPN) (Sheikh and Kaplan, 1986a),
phosphoramidon (NEP) (Roques et al., 1993), captropril (ACE) (Sheikh and Kaplan,
1986b) or apstatin (aminopeptidase P) (Simmons and Orawski, 1992). Lastly, the HSP90
inhibitor novobiocin inhibited PPK activation by 95% at 5 mM, with an IC50 of
approximately 500 μM (Figure 4.19B). In this regard, immunocytochemistry studies
confirmed the presence of HSP90β expression on non-permeabilised A549 cells (Figure
4.20), indicating the chaperone is available on the extracellular surface to activate PPK.
Figure 4.16 Inhibition of PPK activation on A549 cells using a panel of protease and
substrate inhibitors
Inhibition of PPK activation on A549 cells using protease inhibitors (top panel), including
antipain (), leupeptin (), benzamidine (), 2-ME (), EDTA (), ABESF () and
cysteine () or substrate inhibitors (bottom panel), including BK (), BK 1-5 (), ANG
II () and ANG 1-7 ().The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.
0
20
40
60
80
100
120
% P
PK
acti
va
tio
n
0
20
40
60
80
100
10-5
10-4
0.001 0.01 0.1 1 10 100 1000 104
0.1 1 10 100 1000
% P
PK
acti
va
tio
n
Concentration (μM)
Figure 4.17 Inhibition of PPK activation on A549 cells using BK peptides and
protamine sulphate
Inhibition of PPK activation on A549 cells using BK (), des-Arg9-BK (), BK 1-7 ()
and BK 1-5 () (top panel) or protamine sulphate (bottom panel). The data are presented as
the mean ± SEM of three independent experiments performed in triplicate.
0
20
40
60
80
100
0.1 1 10 100 1000
Protamine sulphate concentration (μg/ml)
BK peptide concentration (μM)
% P
PK
act
ivati
on
0
20
40
60
80
100
0.1 1 10 100
% P
PK
act
ivati
on
Figure 4.18 Inhibition of PPK activation using A549 cells and a neutralising
antibody against FXII
A549 cells were treated with HK, followed by treatment with PPK in the presence of anti-
FXII antibody. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.
0
20
40
60
80
100
0.1 1 2 5 10 20
Antibody concentration (μg/ml)
% P
PK
act
ivati
on
Figure 4.19 Inhibition of PPK activation on A549 cells using inhibitors of BK
degrading enzymes and novobiocin
Inhibition of PPK activation on A549 cells using inhibitors of BK degrading enzymes,
including Plummer’s inhibitor (), captopril (), phosphoramidon () and apstatin ()
(top panel) or novobiocin (bottom panel). The data are presented as the mean ± SEM of
three independent experiments performed in triplicate.
0
20
40
60
80
100
0.1 1 10 100 1000
% P
PK
act
ivati
on
0
20
40
60
80
100
0.1 1 10 100 1000 104
% P
PK
act
ivati
on
Concentration (μM)
Figure 4.20 Immunohistochemical analysis of HSP90β expression on A549 cells
Immunohistochemical analysis of HSP90β expression on non-permeabilised (A) and
permeabilised (B) A549 cells. The isotype control is shown in the insert in the top left hand
corner. The figures are representative of three independent experiments.
B
A
67
4.2.5.2 Inhibition of PPK activation on A549 cell-free matrix and lysate
Similar to A549 cells, PPK activation on NHBE cells and A549 cell-free matrix was
strongly inhibited by 10 mM cysteine, 500 μM BK, 100 μg/ml protamine sulphate and 5
mM novobiocin (Figures 4.21 and 4.22, respectively). In contrast, however, PPK activation
on lysates was strongly inhibited by antipain, leupeptin and EDTA, and moderately
inhibited by 2-ME, benzamidine, AEBSF and ANG II (Figure 4.23). The effect of
novobiocin on HK-PPK complex activation by lysates was not examined as it formed a
precipitate in the presence of S-2302.
4.2.5.3 Inhibition of trypsin-activated PPK activity
As the inhibitors may also affect the proteolytic activity of the PK formed during HK-PPK
complex activation, the inhibition profile of trypsin-activated PPK was also examined
(Figure 4.24). Consistent with HK-PPK complex activation, BK inhibited the activity of
trypsin-activated PPK. In contrast, however, antipain, leupeptin and EDTA were strong
inhibitors, while 2-ME, AEBSF, cysteine and protamine sulphate had little effect (Figure
4.24B). The effect of novobiocin on trypsin-activated PPK was not examined as a
precipitate formed when S-2302 was added to the mixture.
4.2.6 Plasma KKS activation on epithelia derived from tissues other than human lung and
non-epithelial cells
Plasma KKS activation was also demonstrated on prostate-derived PC3 and colorectal HT-
29 epithelial cell lines following sequential treatment with HK, PPK and S-2302 (Figure
4.25). Cell lines of non-epithelial origin were also examined and, in this regard, MRC-5
Figure 4.21 Inhibition of PPK activation on NHBE cells
Inhibition of PPK activation on NHBE cells was investigated using a panel of protease
inhibitors, substrate inhibitors, inhibitors of BK degrading enzymes, protamine sulphate
and novobiocin. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.
0 20 40 60 80 100
500μM Antipain
500μM Leupeptin
1mM EDTA
1mM 2-ME
1mM Benzamidine
1mM AEBSF
10mM Cysteine
100μM BK
500μM ANG II
100μg/ml Protamine
1mM Phosphoramidon
1mM Captopril
1mM Apstatin
5mM Novobiocin
1mM Plummer’s inhibitor
% PPK activation
Figure 4.22 Inhibition of PPK activation using A549 cell-free matrix
Inhibition of PPK activation on A549 cell-free matrix using a panel of protease and
substrate inhibitors. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.
0 20 40 60 80 100 120
500 μM Antipain
500 μM Leupeptin
1 mM EDTA
1 mM 2-ME
1 mM Benzamidine
1 mM ABESF
10 mM Cysteine
500 μM ANG II
100 μM BK
100 μg/ml Protamine
5 mM Novobiocin
% PPK activation
Figure 4.23 Inhibition of PPK activation using A549 cell lysate
Inhibition of PPK activation on A549 cell lysates using a panel of protease and substrate
inhibitors. The data are presented as the mean ± SEM of three independent experiments
performed in triplicate.
0 20 40 60 80 100
500 μM Antipain
500 μM Leupeptin
1 mM EDTA
1 mM 2-ME
5 mM Benzamidine
1 mM ABESF
10 mM Cysteine
500 μM ANG II
500 μM BK
100 μg/ml Protamine
% PPK activation
Figure 4.24 Activity and inhibition of trypsin-activated PPK
Trypsin-activated PPK was incubated with S-2302 () and PK activity monitored over
time. Buffer alone (), trypsin with LBTI () and untreated PPK () were also included
(A). Inhibition of trypsin-activated PPK activation using protease inhibitors and substrate
inhibitors (B). The data are presented as the mean ± SEM of three independent experiments
performed in triplicate.
Ab
sorb
an
ce (
405n
m)
0
0.5
1
1.5
2
2.5
3
5 10 20 30 40 50 60 70 80 90
0 20 40 60 80 100
HEPES alone
Trypsin + LBTI
Untreated PPK 500 μM Antipain
500 μM Leupeptin
1 mM EDTA
1 mM 2-ME
1 mM ABESF
1 mM Benzamidine
10 mM Cysteine
500 μM ANG II
100 μM BK 100 μg/ml Protamine
% PPK activation
Time (min)
A
B
Try
psi
n-a
ctiv
ated
PP
K
Figure 4.25 PPK activation on additional epithelial cell lines
Human HT-29 colorectal (black bars) and PC3 prostate (diagonal lines) epithelial cells
were incubated sequentially with HK and PPK and PK activity determined after 90 min.
BEAS-2B cells (white bars) are included (Figure 4.9B). The data are presented as mean ±
SEM from three independent experiments performed in triplicate.
0
0.5
1
1.5
2
2.5
Untreated HK alone PK alone HK + PK
Ab
sorb
an
ce (
405n
m)
68
fibroblast, C2C12 myoblast, C2C12 myotube, HMC-1 mast cell and U-937 monocyte cell
lines also demonstrated PK activity (Figure 4.26). Of all the cell lines tested, A549, CFT1,
NHBE, MRC-5, C2C12 myoblasts and HMC-1 cells demonstrated the greatest amount of
PK activity.
Figure 4.26 PPK activation on cell lines of non-epithelial origin
MRC-5 human fibroblasts (white bars), C2C12 mouse myoblasts (grey bars), C2C12 mouse
myotubes (black bars), HMC-1 human mast cells (horizontal lines) and U-937 human
monocytes (diagonal lines) were incubated sequentially with HK and PPK and PK activity
determined after 90 min. The data are presented as mean ± SEM from three independent
experiments performed in triplicate.
0
0.5
1
1.5
2
2.5
3
Untreated HK alone PPK alone HK + PK
Ab
sorb
an
ce (
405n
m)
69
4.3 Discussion
On endothelium, uPAR, gC1qR and CK1 bind HK (Colman et al., 1997, Joseph et al.,
1999a) and, on neutrophils and platelets, Mac-1 may also be involved (Wachtfogel et al.,
1994, Barbasz et al., 2008). With the exception of Mac-1, all three proteins were detected
on both the transformed respiratory epithelial cells tested. However, the NHBE cells were
not tested due to time constraints. The intensity of uPAR was similar on both the A549 and
BEAS-2B cell lines, but the A549 cell line showed higher expression of gC1qR and CK1,
using either permeabilised and non-permeabilised cells. All three co-localised on A549
cells, although co-localisation of uPAR and gC1qR appeared limited to isolated groups of
cells, the reasons for which are unclear. However, it may reflect the presence of
subpopulations with differential expression profiles
(Croce et al., 1999) and/or the
relatively lower frequency of cell surface staining of uPAR compared to gC1qR. These
proteins were also noted on normal bronchial epithelium, but only CK1 was present on
alveolar epithelium.
A549 cells supported the Zn2+
-dependent binding of HK, consistent with endothelial cells
(Zhao et al., 2001), but it was only weakly inhibited by antibodies against uPAR, gC1qR
and CK1. Anti-gC1qR inhibited HK binding to endothelial cells by 72% (Joseph et al.,
1999a), but little inhibition was obtained with A549 cells. Likewise, anti-uPAR antibodies
completely abolished HK binding on endothelial cells (Mahdi et al., 2001), but our
antibody only weakly inhibited HK binding. Furthermore, anti-CK1 antibodies have been
shown to inhibit HK binding to endothelial cells (Shariat-Madar et al., 1999, Joseph et al.,
1999a) but, again, our CK1 antibody had negligible effect. Although these data may reflect
differing specificities of the antibodies used, this may not be the case for the gC1qR and
70
uPAR antibodies used here, as they demonstrate specificities similar to those previously
reported (Joseph et al., 1999a, Mahdi et al., 2001). When all three antibodies were used in
combination, approximately 45% inhibition was obtained, suggesting that although uPAR,
gC1qR and CK1 may be involved in binding HK on A549 cells, other components may
play a role, although not sulphated proteoglycans or Mac-1, given our findings.
Following sequential treatment with HK and PPK, PK formation was demonstrated on a
variety of respiratory epithelial cell types including A549 Type II pneumocytes, BEAS-2B
bronchial and primary NHBE cells. Additionally, plasma KKS activation was demonstrated
on the CFT-1 tracheal epithelial cell line, indicating the Δ508 mutation in the CFTR has no
effect on this system. Although BEAS-2B and CFT-1 cells were not tested, BK release was
demonstrated using A549 and NHBE cells following activation of the plasma KKS.
However, in future studies it would be of interest to examine the time course of BK
formation and the extent of BK metabolism during this process by epithelial-derived
peptidases. Consistent with previous reports (Koyama et al., 1998, Rodgers et al., 2002),
BK and des-Arg9-BK induced IL-6 and IL-8 release from A549 cells. Thus, plasma KKS
activation on respiratory epithelium may play role in initiating and/or propagating
inflammation within the lung.
Although no increase in PK activity was demonstrated when respiratory epithelial cells
were treated with HK alone, increased BK release was observed, suggesting BK formation
occurred independently of PK activation. Epithelial cells treated with PPK alone also
showed increases in both PK activity and BK release. One explanation for this finding is
the cell acquired HK due to pre-exposure to bovine serum, which was used to maintain the
cells in culture. However, cells deprived of serum still retained PK activity when treated
71
with PPK alone and serum did not significantly increase PK formation, except when cells
were treated with PPK alone. Alternatively, respiratory epithelial cells could activate PPK
independently of cell-bound HK, as previously described for endothelial cells (Motta et al.,
1998), but this would not explain the increase in BK release and, thus, an endogenous
source of kininogen is likely to be present. Additionally, PPK can liberate BK from HK
without conversion to PK (Joseph et al., 2009). Although the epithelial data described in
this Chapter clearly demonstrate conversion of PPK to PK, it is possible that BK is released
due to the combined actions of PPK and PK on HK.
Assembly of HK and PPK, and the formation of PK was also demonstrated on epithelial
cell lines derived from the prostate and gut. Thus, the results suggest plasma KKS
activation may be a universal feature of most, if not all, epithelia. Similarly, HK-PPK
activation was demonstrated on a variety of cell lines of non-epithelial origin, including
monocytes, as previously described (Barbasz and Kozik, 2009), but also myoblasts,
myotubes, fibroblasts and mast cells, indicating that this system is not restricted to
epithelial tissue. Despite this similarity, however, the extent of PK formation differed
significantly between cell types. For example, A549, NHBE, CFT-1, HT-29, MRC-5,
C2C12 myoblasts and HMC-1 cells generated similar levels of PK activity, but PK activity
generated by BEAS-2B, C2C12 myotubes and U-937 cells was approximately 3 to 4-fold
lower than that observed with A549 and NHBE cells. The reasons for this are unclear, but
may reflect the differential expression of HK binding proteins or activity of the HK-PPK
activating enzyme on the different cell lines. However, these possibilities were not explored
further.
72
Subsequent experiments determined that intact cells were not required for PPK activation.
For example, plasma KKS activation was demonstrated using A549 cell-free matrix, as
previously described for endothelium (Motta et al., 2001, Moreira et al., 2002), providing
further evidence that cellular membranes are not the sole physiologic surface capable of
plasma KKS assembly and activation. In this regard, laminin is known to bind HK
(Schousboe and Nystron, 2009) and, thus, may play a role in plasma KKS activation on
epithelial matrix. Similarly, A549 cell lysates were also shown to support PK formation,
indicating that plasma KKS activation is maintained following cellular disruption. This
observation may have relevance to cell necrosis, in which BK release from damaged cells
may provide danger signals for inflammatory cells (Aliberti et al., 2003). Either the
membrane or cytosolic fraction of the lysate may be responsible for HK-PPK complex
activation, as previously demonstrated (Joseph et al., 2002), but it is unclear whether the
same PPK activating enzymes are involved.
Following this, the identity of the enzyme involved in HK-PPK activation was investigated.
Activation of the HK-PPK complex on A549 and NHBE cells was inhibited by BK, but not
by ANG II, known substrate inhibitors of PRCP which is thought to activate the HK-PPK
complex on endothelial cells (Shariat-Madar et al., 2002). Complete inhibition by BK was
achieved at 100 μM, which contrasts with reported data for activation of the complex on
endothelial cells where significant, but not complete, inhibition was observed at a ten-fold
higher concentration (Shariat-Madar et al., 2002, Shariat-Madar et al., 2004). Additionally,
the limited effects of other known PRCP inhibitors including antipain and leupeptin suggest
a PRCP-independent activation mechanism on epithelium, but this possibility requires
further investigation. In this regard, parallel experiments should be performed using
73
endothelial cells to confirm that the presumed PRCP inhibitors used in this study do in fact
inhibit PRCP under these experimental conditions.
It was also shown that proteases which degrade BK were not involved in activation.
Interestingly, des-Arg9-BK, BK 1-7 and BK 1-5, which lack the C-terminal arginine in
addition to other residues, did not inhibit PPK activation, suggesting the activator could be
a carboxypeptidase with specificity towards C-terminal arginine. This possibility is
supported by data obtained showing inhibition of PPK activation by protamine sulphate,
which contains multiple arginine residues. In contrast, the inhibition profile of trypsin-
activated PPK differed significantly, suggesting the inhibitors used in the PPK activation
assay were acting on the PPK activator rather than the generated PK per se. For example,
cysteine and protamine sulphate were potent inhibitors of PPK activation on cells, but had
no effect on trypsin-activated PPK. Likewise, antipain, leupeptin and EDTA strongly
inhibited trypsin-activated PPK, but failed to produce a similar effect on PPK activation on
A549 and NHBE cells. In contrast, BK inhibited both trypsin-activated PPK and PPK
activation on cells, suggesting the inhibition of PPK activation by BK may be due to either
inhibition of the PPK activator or PK activity.
Formation of PK was also inhibited by novobiocin, an antibiotic known to interfere with
eukaryotic HSP90 function (Marcu et al., 2000, Marcu et al., 2000). HSP90 was reported to
catalyse the activation of the HK-PPK complex (Joseph et al., 2002) and the data present
here, although indirect, suggest it is also involved in some way on respiratory epithelial
cells. Immunocytochemistry studies showed that HSP90β (HSP90 was not tested) was
shown to be expressed on the surface of A549 cells. However, as HSP90 is non-proteolytic,
it has been proposed that it may induce enzymatic activity in HK or expose an active site
74
within PPK before cleavage (Joseph et al., 2002). Alternatively, HSP90 may have an
accessory role in HK-PPK complex activation, akin to the maturation of extracellular
matrix metalloproteinase 2 by HSP90 (Eustace et al., 2004).
The inhibition profile of HK-PPK complex activation on A549 cell-free matrix was similar
to that observed on A549 and NHBE cells, indicating deposition of the respiratory
epithelial cell HK-PPK activator on extracellular matrix. In this regard, PRCP is
responsible for plasma KKS activation on endothelial extracellular matrix (Moreira et al.,
2002). As such, the inhibition by novobiocin indicates association of HSP90 with epithelial
cell extracellular matrix, although this was not directly confirmed. In contrast, however, the
inhibition profile obtained using A549 cell lysate differed significantly. For example, PK
formation was strongly inhibited by antipain, leupeptin and EDTA, but moderately
inhibited by benzamidine and ANG II. Thus, this result suggests the presence of additional
activators within the cytosol of respiratory epithelial cells capable of activating the HK-
PPK complex. Inhibition by antipain, leupeptin and AEBSF suggests the activator could be
a serine protease, which may possess trypsin-like activity given the effect of benzamidine
on activation. In addition, inhibition by EDTA may indicate the involvement of a
metalloprotease, and inhibition by 2-ME and cysteine suggest the protease involved possess
critical disulphide bonds in their structure.
Within the lung, hK1 is thought to be the major kininogenase (Christiansen et al., 1987,
Schenkels et al., 1995) but recently, PPK has been demonstrated in extrahepatic sites,
including the lung (Hermann et al., 1999, Fink et al., 2007). These observations, combined
with the known leakage of plasma proteins into the mucosa of individuals with
inflammatory lung disease (Persson et al., 1995, Persson et al., 1998, Khor et al., 2009,
75
Persson and Uller, 2009), indicate PK may participate in local kinin formation. In addition,
the influx of HK and PPK has been described in the upper airways following allergen
challenge and was accompanied by the activation of PPK and generation of kinins
(Baumgarten et al., 1985, Baumgarten et al., 1986). As BK is formed following the
assembly of HK and PPK, plasma KKS activation along the respiratory epithelium, alone
or in combination with hK1 and FXIIa, might contribute to the development of chronic
inflammation within the lung (Sato et al., 1996, Koyama et al., 1998, Koyama et al., 2000,
Bertram et al., 2007). As proposed for endothelial cells (Schmaier, 2000), activation of the
plasma KKS on the respiratory epithelium may occur independent of FXIIa, but FXII auto-
activation (Reddigari et al., 1993b) or conversion by PK (Rojkjaer et al., 1998) would
augment this process by activating PPK directly. Although the data demonstrate activation
of the plasma KKS by respiratory epithelial cells per se, host-derived (Imamura et al.,
1996, Kozik et al., 1998, Stuardo et al., 2004) or microbial (Molla et al., 1989, Imamura et
al., 1994) proteases present at inflammatory foci may also activate HK or PPK bound to the
epithelium.
In conclusion, the data show that A549 cells bind HK in a Zn+-dependent manner, which
partially involves uPAR, gC1qR and CK1, but not Mac-1. In the presence of HK, primary
respiratory epithelial cells, A549 cells and extracellular matrix and lysate bind PPK and
catalyse its conversion to PK in a process dependent on HSP90, although not necessarily on
PRCP. Additionally, assembly of HK and PPK resulted in the generation of BK from HK.
Furthermore, the data obtained also suggests plasma KKS activation may be common
feature of most cell types. Although, the data presented were obtained primarily using
transformed cell lines, data obtained using NHBE cells and normal human tissue suggest
that plasma KKS may operate on normal lung epithelium. Finally, the data obtained suggest
76
that enzymes other than PRCP and FXIIa may be involved in the conversion of PPK to PK.
77
4.4 Summary
A549 and BEAS-2B cells expressed the known HK binding proteins, uPAR, gC1qR
and CK1, but not Mac-1.
A549 cells specifically bound FITC-labeled HK, which was only partially
dependent on uPAR, gC1qR and CK1. However, sulphated proteoglycans did not
appear to be involved.
Sequential treatment of A549, BEAS-2B, CFT-1 and NHBE cells with HK and PPK
induced PK formation and release of BK from HK. Similarly, plasma KKS
activation was demonstrated on A549 cell-free matrix and lysate.
HK-PPK complex activation on A549 and NHBE cells was weakly inhibited by
several known partial or complete inhibitors of PRCP-mediated HK-PPK activation.
HSP90 inhibition strongly inhibited PK formation on A549 and NHBE cells and
A549 cells expressed HSP90 on the cell surface.
Plasma KKS activation was demonstrated on additional epithelia derived from the
prostate and gut, and cells of non-epithelial origin.
CHAPTER 5
ACTIVATION OF THE PLASMA KALLIKREIN-KININ SYSTEM ON PLEURAL
MESOTHELIAL CELLS
77
5.1 Introduction
In the previous Chapter, it was shown that the plasma KKS may operate not only on
respiratory epithelium, but also on epithelia from other tissues, as well as non-epithelial
cells. In the context of the lung, it was shown that type II pneumocytes, bronchial
epithelium and tracheal epithelial cells possessing the Δ508 mutation in the CFTR could
activate this system. In this Chapter, the possibility that other cell types originating from
the lung could activate the plasma KKS was investigated, with particular focus on pleural
mesothelial cells.
The primary function of mesothelial cells is to provide a non-adhesive barrier to apposing
organs, but they may also contribute to fluid balance, healing, repair and inflammation
(Mutsaers and Wilkosz, 2007). With regard to fluid balance, studies indicate kinins such as
BK not only play a role, but that plasma KKS activation may occur on mesothelial cells.
For example, Uchida et al. demonstrated that intra-pleural administration of carrageenin
into rats induced activation of PPK and HK, but not LK, in pleural exudates, indicating
activation of the plasma kinin-forming pathway (Uchida et al., 1983). In support, Ruud and
co-workers showed increased PK activity, in parallel with a reduction in PPK levels, in
peritoneal effusions obtained from animals in models of experimental pancreatitis (Ruud et
al., 1982, Ruud et al., 1984, Ruud et al., 1985). Further studies also showed the PK
localised to the pleural mesothelium and Chee et al. demonstrated expression of PK by the
epithelioid and sarcomatoid components of pleural tissue isolated from patients with
biphasic mesothelioma (Chee et al., 2007). Therefore, given these results, it is highly likely
PK contributes to local kinin formation on the pleural mesothelium and, thus, plays a role
in inflammation.
78
In this Chapter, studies were initially performed to determine whether pleural fluids
obtained from patients with benign and malignant effusions contained BK. Pleural fluids
were already collected from two multicentre clinical trials (Creaney et al., 2008, Davies et
al., 2009) and comprised patients with a variety of pleural, pulmonary and extra-pulmonary
conditions (Appendix I). Subsequently, plasma KKS activation resulting in BK release was
examined on a variety of pleural mesothelial cell lines, primary human mesothelioma cells
and primary murine mesothelial cells. The cell lines tested included MeT-5A, MSTO-
211H, NCI-H2052 and NCI-H28 and are common cell types used in various studies
regarding the pleural mesothelium (Schmitter et al., 1992, Tsao et al., 2007, Tsuji et al.,
2010). In addition, the inhibitors used in the previous chapter were also employed here to
characterise the protease responsible for HK-PPK complex activation. Furthermore, the
responsiveness of mesothelial cells to kinins, with respect to calcium mobilisation and pro-
inflammatory mediator release, was investigated.
79
5.2 Results
5.2.1 BK in pleural effusions
Initially, BK concentrations in paired pleural effusion and serum samples from Cohort 1
comprising patients with a variety of clinical conditions were determined to assess the
likelihood of local BK formation in the pleural space. BK was detected in all patient
samples examined (median: serum, 45 ng/ml; pleural effusion, 40 ng/ml) and
concentrations were shown to be greater in pleural fluids than in the matched serum
samples in 18 of the 40 patients (Figure 5.1). For one patient, pleural and serum BK
concentrations were approximately the same. For Cohort 1 and 2 patients, significant
differences in pleural fluid BK were not detected when benign with malignant effusions
(Cohort 1, p = 0.9; Cohort 2, p = 0.272) (Figure 5.2A) or benign with MM and non-MM
malignant effusions (Cohort 1, p = 0.9; Cohort 2, p = 0.5) (Figure 5.2B) were compared.
Similarly, differences in serum BK concentrations (Cohort 1 samples) were not observed
when benign with malignant (p = 0.53) (Figure 5.3A) or benign with MM and non-MM
malignant samples (p = 0.46) (Figure 5.3B) were compared. For Cohort 2, BK
concentrations in pleural fluids were significantly greater in exudates than transudates
(median, 62.1 versus 24.2 ng/ml; p = 0.025) (Figure 5.4). BK concentrations in exudates
and transudates from Cohort 1 were not compared due to the limited number of
transudative effusions.
Figure 5.1 BK concentrations in paired serum and pleural effusion samples from
Cohort 1 patients
Paired serum and pleural effusion samples were analysed for the presence of BK using a
competitive EIA. Dashed line: median BK concentration in sera; solid line: median BK
concentration in pleural effusions.
Serum Pleural effusion
1
10
100
1000
10000
Log B
K c
on
cen
trati
on
(n
g/m
l)
Figure 5.2 BK concentrations in pleural effusions from patients with non-
malignant and malignant disease
Comparison of the BK concentrations in pleural effusions from patients with non-malignant
and malignant disease (A). A comparison of the BK concentrations in pleural effusions
from patients with non-malignant disease, MM and non-MM malignancies (B). BK
concentrations were determined using a competitive EIA. Dashed lines: median BK
concentration; black dots: Cohort 1 patients; white dots: Cohort 2 patients.
Benign Malignant Benign Malignant
1
10
100
1000 A
Benign MM Non-MM Benign MM Non-MM
1
10
100
1000
Log B
K c
on
cen
trati
on
(n
g/m
l)
B
Log B
K c
on
cen
trati
on
(n
g/m
l)
Figure 5.3 Comparison of serum BK concentration from Cohort 1 patients
with non-malignant and malignant disease
Comparison of the BK concentrations in serum from patients with non-malignant and
malignant disease (A). A comparison of the BK concentrations in serum from patients with
non-malignant disease, MM and non-MM malignancies (B). BK concentrations were
determined using a competitive EIA Dashed lines: median BK concentration.
Benign Malignant
1
10
100
1000
10000
Log B
K c
on
cen
trati
on
(n
g/m
l)
Benign MM Non-MM
1
1
0
100
1000
1000
0
Log B
K c
on
cen
trati
on
(n
g/m
l)
B
A
Figure 5.4 BK concentrations in transudative and exudative pleural effusions from
Cohort 2 patients
BK concentrations were determined using a competitive EIA. Dashed lines: median BK
concentrations.
Transudates Exudates
1
10
100
1000 p = 0.025
Log B
K c
on
cen
trati
on
(ng/m
l)
80
5.2.2 PPK activation on mesothelial cells
To determine whether mesothelial cells could be involved in local BK formation within the
pleural space, a variety of mesothelial cell types were incubated with HK and PPK, and
kinin liberation then determined, as described for the respiratory epithelium studies
(Chapter 4). Following sequential treatment with HK and PPK, all human- and non-human-
derived cell types tested supported activation. Similar results were also obtained using cell-
free matrix from MeT-5A (Figure 5.5). Modest PK activity was observed when cells were
treated with PPK alone, in contrast to that observed with untreated cells or cells treated with
HK alone. The rank order of activation was MeT-5A ≈ MeT-5A matrix > primary
mesothelioma cells > NCI-H28 > MPM > NCI-H2052 > MSTO-211H.
Following incubation, significant BK release was observed with cells treated with HK
alone (p < 0.0001), which increased upon treatment with PPK alone or HK and PPK
combined (p < 0.0001) (Figure 5.6). The rank order of BK release was MeT-5A matrix >
MeT-5A > primary mesothelioma cells > MPM > NCI-H28 > NCI-H2052 > MSTO-211H.
A positive correlation between PK activity and BK release was observed (p > 0.01) (Figure
5.7).
Following this, MeT-5A cells were incubated with pleural effusions from Cohort 2 to
determine whether PK activity could be generated due to the presence of any of the plasma
KKS components. Following incubation, PK amidolytic activity was demonstrated using
MeT-5A cells, but this was significantly increased after cells were treated with malignant
pleural effusions (median, 0.3 versus 0.23; p = 0.04) (Figure 5.8). A dose response of
amidolytic activity using purified PK revealed a linear relationship over the concentration
Figure 5.5 PPK activation on mesothelial cells and mesothelial cell-free matrix
Mesothelial cells and cell-free matrix were sequentially treated with HK and PPK and PK
activity determined following incubated with S-2302 for 90 min. The data are presented as
mean ± SEM from three independent experiments performed in triplicate.
0
0.5
1
1.5
2
2.5
3
Ab
sorb
an
ce (
405n
m)
MeT-5A
NCI-H28
NCI-H2052
MSTO-211H
Murine peritoneal
mesothelial cells
Human primary
mesothelioma cells
MeT-5A
matrix
Untreated HK alone PPK alone HK + PPK
Figure 5.6 BK liberation from mesothelial cells and mesothelial cell-free matrix
Following sequential treatment of mesothelial cells and mesothelial cell-free matrix with
HK and PPK, culture supernatants were analysed for BK using a competitive EIA. The data
presented are means ± SEM from three independent experiments performed in triplicate. *
Denotes significantly greater than untreated control (p < 0.0005). ** Denotes significantly
greater than HK alone and untreated controls (excluding NCI-H28 cells) (p < 0.005). ***
Denotes significantly greater than all other treatments (p < 0.005).
0
2
4
6
8
10
12
Untreated HK alone PPK alone HK + PPK
BK
con
cen
trati
on
(n
g/m
l)
***
** *
MeT-5A
NCI-H28
NCI-H2052
MSTO-211H
Human primary
mesothelioma cells
MeT-5A
matrix
Murine peritoneal
mesothelial cells
Figure 5.7 Relationship between PK activity and BK release from mesothelial cells
and matrix
PK activity is plotted against BK release for MeT-5A (), NCI-H28 (), NCI-H2052 (),
MSTO-211H (), MPM (), primary mesothelioma cells () and MeT-5A matrix ().
0.1
1
1 10
y = 2.2667 + 0.6407log(x)
R= 0.87394
Lo
g a
bso
rb
an
ce (
40
5 n
m)
Log BK concentration (ng/ml)
Figure 5.8 PK activity generated following incubation of MeT-5A cells with pleural
effusions from Cohort 2 patients
Confluent MeT-5A cells were incubated with pleural effusions for 1 hr and PK activity
determined by the addition of S-2302 for 4 hr. Comparison of PK activity generated by
effusions obtained from patients with non-malignant and malignant disease (A). A
comparison of PK activity generated by effusions obtained from patients with non-
malignant disease, MM and non-MM malignancies (B). Dashed line: median absorbance at
405 nm.
Benign Malignant
0
0.1
0.2
0.3
0.4
0.5
0.6
Ab
sorb
an
ce (
405 m
n)
Benign MM Non-MM
0
0.1
0.2
0.3
0.4
0.5
0.6
Ab
sorb
an
ce (
405 n
m)
p = 0.04
A
B
81
range of 0.01-0.1 nM (p > 0.05) and all absorbance values obtained following incubation of
MeT-5A cells with effusions fell within this curve (Figure 5.9).
5.2.3 Inhibition of PPK activation on mesothelial cell lines
As with respiratory epithelium (Chapter 4), the possible identity of the HK-PPK complex
activator was assessed using a panel of inhibitors. Table 5.1 shows that PPK activation on
MeT-5A, NCI-H2052, NCI-H28 and MPM cells was strongly inhibited by 10 mM cysteine,
100 μM BK, 100 μg/ml protamine sulphate and 5 mM novobiocin. All cell types tested
were moderately inhibited by 2-ME and AEBSF. Antipain and leupeptin, at 500 μM,
inhibited PPK activation on MeT-5A, NCI-H2052, and MPMC by approximately 30-50%,
but they neither had any effect on NCI-H28 cells. EDTA, benzamidine and ANG II had
negligible effect on PPK activation on any of the cell lines tested.
5.2.4 Mesothelial cells express HSP90, but not PRCP or FXII
Given the inhibition data, immunocytochemistry experiments were then performed to
determine the expression profile of PPK activators on mesothelial cells. MeT-5A, NCI-H28
and NCI-H2052 cells were subject to immunocytochemistry to determine the expression
profile of possible cell surface HK-PPK activators. While extracellular HSP90β was
expressed on all cells tested, weak HSP90 staining was evident only on MeT-5A cells. In
contrast, cell surface expression of either PRCP or FXII was not detected on mesothelial
cells by immunocytochemistry (Figure 5.10).
Figure 5.9 Dose response of amidolytic activity using purified PK
S-2302 was incubated with increasing concentrations of PK and absorbances at 405 nm
determined after 4 hr. The data presented are the mean ± standard deviation of one
experiment performed in triplicate.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.02 0.04 0.06 0.08 0.1
y = 0.067944 + 6.2285x
R= 0.99966
Ab
sorb
an
ce (
405 n
m)
Concentration (nM)
Figure 5.10 Immunohistochemical analysis of surface expressed HSP90,
HSP90β, PRCP and FXII on mesothelial cells
Immunohistochemistry of surface expressed HSP90, HSP90β, PRCP and FXIIa on MeT-
5A (A), NCI-H28 (B) and NCI-2052 (C) cells. Positive immunoreactivity is seen as brown
precipitate. The non-immune control antibody are shown in the inserts. The figures are
representative of three independent experiments.
HSP90 HSP90β PRCP FXII
A
B
C
Table 5.1 Inhibition of PPK activation on MeT-5A, NCI-H28, NCI-H2052
and MPM cells
% PPK activation
Inhibitor MeT-5A NCI-H228 NCI-H2052 MPM
10 mM Cysteine 1.5 ± 0.5 1.7 ± 0.8 0 ± 0.1 0.01 ± 0.2
100 μM BK 5.2 ± 0.7 23.2 ± 2.7 10.8 ± 1.1 9.2 ± 3.3
100 μg/ml Protamine 2.3 ± 0.5 1.9 ± 1 2.6 ± 1.3 0.9 ± 0.5
5 mM Novobiocin 8.9 ± 0.2 8.8 ± 1.1 8 ± 0.9 17.7 ± 4.9
500 μM Antipain 59.4 ± 8.3 97.7 ± 1.4 43.4 ± 3 70 ± 6.9
500 μM Leupeptin 58.6 ± 9.8 101.1 ± 1.9 43.4 ± 4.8 73 ± 7.3
1 mM EDTA 99.1 ± 2.4 101.5 ± 1.7 101.4 ± 0.9 106.4 ± 2
1 mM 2-ME 46.8 ± 9.7 59.5 ± 1.3 47.7 ± 0.8 63.6 ± 3.8
1 mM Benzamidine 99.5 ± 1.9 108 ± 1.4 102.4 ± 1.8 107 ± 1.9
1 mM ABESF 75.2 ± 7.5 69.6 ± 0.6 57.6 ± 0.9 88.7 ± 1.7
500 μM ANG II 100.3 ± 1.2 102.3 ± 1.5 99.7 ± 2.2 103 ± 0.4
Cells were treated with HK, followed by incubation with PPK in the presence or
absence of inhibitors. PK activity was determined following addition of S-2302 for
90 min and compared against data obtained in the absence of inhibitors, which was
defined as 100% activation. The data present are the means ± SEM of three
independent experiments performed in triplicate.
82
5.2.5 The effect of BK and des-Arg9-BK on calcium mobilisation, and cytokine and
chemokine release from mesothelial cell lines
To determine whether mesothelial cells responded to kinins, calcium mobilisation and pro-
inflammatory mediator release were assessed. BK (1 μM) stimulated calcium mobilisation
in MeT-5A and NCI-H2052 cells, with approximately 20-25% of cells responding, in
contrast to that observed with NCI-H28 cells (Figures 5.11A-C). However, 1 μM des-Arg9-
BK had little effect on any of the mesothelial cell lines tested (Figures 5.10D-G).
Stimulation of MeT-5A, NCI-H2052 and NCI-H28 cells with either BK or des-Arg9-BK
had little effect on IL-6 and IL-8 release (Figures 5.12 and 5.13), in contrast to the results
obtained using A549 cells (Chapter 4). All cell lines tested did, however, produce IL-6 and
IL-8 when stimulated with PMA (Figure 5.14). BK did not induce MPC-1 or TNF-
release from MeT-5A cells (Figure 5.15).
Figure 5.11 Flow cytometric analysis of calcium mobilisation in mesothelial
cells induced by BK and des-Arg9-BK
Flow cytometric analysis of calcium mobilisation in MeT-5A (A,D), NCI-H2052 (B,E) and
NCI-H28 (C,F) cells in response to 1 μM BK (A-C) or des-Aarg9-BK (D-F). The figures
are representative of three independent experiments. Solid arrows: addition of BK or des-
Arg9-BK; dashed arrows: addition of 1 μM ionomycin.
0
10
20
30
40
0 10
0
20
0
30
0
0
1
0
2
0
3
0
4
0
0 100 200 300
0
20
40
60
80
0 10
0
20
0
300
0
10
20
30
0 10
0
20
0
300
% r
esp
on
din
g c
ells
0
2
0
4
0
6
0
0 100 200 300
0
1
0
2
0
3
0
0 10
0
20
0
300
A
B
C
D
E
F
Time (sec) Time (sec)
Figure 5.12 The effect of BK on IL-6 and IL-8 release from mesothelial cells
MeT-5A (diagonal lines), NCI-H2052 (horizontal lines), NCI-H28 (white bars) and A549
(black bars) were stimulated with BK for 24 hr and IL-6 (A) and IL-8 (B) release
determined by ELISA. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.* Denotes significantly greater than vehicle control (p <
0.0001).
0
200
400
600
800
1000
1200
IL
-6 c
on
cen
tra
tio
n (
pg
/ml)
0
500
1000
1500
2000
2500
Vehicle
control
0.1 1 10 100
IL-8
con
cen
trati
on
(p
g/m
l)
BK concentration (μM)
A
B
* * * *
* * *
*
Figure 5.13 The effect of des-Arg9-BK on IL-6 and IL-8 release from
mesothelial cells
MeT-5A (diagonal lines), NCI-H2052 (horizontal lines), NCI-H28 (white bars) and A549
(black bars) were stimulated with des-Arg9-BK for 24 hr and IL-6 (A) and IL-8 (B) release
determined by ELISA. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.* Denotes significantly greater than all other treatments
(p < 0.05).
0
200
400
600
800
1000
1200
1400
0
500
1000
1500
2000
2500
3000
Vehicle
control
0.
1 1 10 100
Des-arg9-BK concentration (μM)
A
B
*
*
IL-8
con
cen
trati
on
(p
g/m
l)
IL
-6 c
on
cen
tra
tio
n (
pg
/ml)
Figure 5.14 The effect of PMA on IL-6 and IL-8 release from mesothelial cells
MeT-5A, NCI-H2052 and NCI-H28 were stimulated with vehicle (white bars) or PMA
(black bars) for 24 hr and IL-6 (A) and IL-8 (B) release determined by ELISA. * Denotes
significantly greater than vehicle control (p < 0.05). The data are presented as the mean ±
SEM of three independent experiments performed in triplicate. ND: not detected.
10
100
1000
10 4
10 5
IL-6
con
cen
trati
on
(p
g/m
l)
1
0
100
1000
10 4
10 5
MeT-5A NCI-H2052 NCI-H28
IL-8
con
cen
trati
on
(p
g/m
l)
ND ND
A
B
*
*
*
*
* *
Figure 5.15 The effect of BK on MCP-1 and TNF- release from MeT-5A cells
MeT-5A cells were stimulated with BK for 24 hr and MCP-1 (A) and TNF- (B) release
determined by ELISA. The data are presented as the mean ± SEM of three independent
experiments performed in triplicate.
0
500
1000
1500
2000
2500
3000
MC
P-1
con
cen
trati
on
(p
g/m
l)
0
20
40
60
80
100
Vehicle
control
0.1 1 10 100
TN
F-α
con
cen
tra
tion
(p
g/m
l)
BK concentration (μM)
A
B
83
5.3 Discussion
The present study demonstrated that pleural mesothelial cell lines, primary human
mesothelioma cells, primary murine mesothelial cells and human mesothelial cell
extracellular matrix support the assembly and activation of the plasma KKS and, thus,
could be a site of local BK formation within the pleural space. Following sequential
treatment with HK and PPK, malignant and benign mesothelial cells of benign and
malignant origin catalysed the conversion of PPK to PK, liberating BK.
Previous studies demonstrated the consumption of HK and PPK, and the formation of PK
in pleural and peritoneal fluids (Uchida et al., 1983, Ruud et al., 1985, Waldner et al.,
1993), indirectly demonstrating a role for local plasma KKS activation in serosal tissues.
However, the underlying mechanism of activation has not been described in any detail.
Thus, the results of this study are consistent with previous findings, but also provide
information regarding the possible activation process per se. For example, in a large
proportion of patient samples tested in this study, BK concentrations in pleural fluids were
greater than those in corresponding serum samples, suggesting that kinin may be produced
locally within the pleural space, in concert with contributions from the systemic circulation.
BK was detectable in all samples regardless of disease diagnosis, although differences in
pleural fluid BK concentrations were not observed between disease groups. However, BK
concentrations were significantly elevated in patients with exudative effusions, suggesting
increased kinin formation in response to local inflammation of the pleura. Furthermore,
mesothelial cells incubated with pleural effusions generated PK, indicating the presence of
plasma KKS components in pleural fluid. Interestingly, the PK activity generated was
84
significantly higher from cells treated with malignant pleural effusions, suggesting the
presence of elevated levels of KKS components in such disease states, although the
confirmation of specific components were not undertaken.
Although the samples were immediately frozen following collection, the lack of kininase
inhibitor treatment of the pleural fluids represents a limitation to the study since, in vivo,
BK has a short half-life (Saameli and Eskes, 1962, Ferreira and Vane, 1967) due to its rapid
degradation by kininases (Erdos and Sloane, 1962, Sheikh and Kaplan, 1986b, Roques et
al., 1993). Thus, the accurate measurement of BK concentrations in biological samples is
difficult and, in the pleural cavity, it is rapidly metabolised following induction of pleurisy
(Hori et al., 1988, Majima et al., 1992). Similarly, its detection in effusion samples is
largely dependent on treatment of samples with peptidase inhibitors at collection (Tissot et
al., 1985). Additionally, the presence of large amounts of kininogen and kinin-forming,
and/or kinin-degrading proteases could influence the determination of BK concentrations
upon collection of effusions. In addition, the nature of the sample per se may influence the
presence of such components. Further, given that the bradykinin was not extracted by
treated of the samples by ethanol precipitation, as described for similar biological samples
(Malavazi-Piza et al., 2004), it is possible that inflammation-induced proteases in the
samples may have influenced the data obtained. It would be appropriate to repeat these
assays after such treatment in future studies.
The use of specific kininase inhibitors (Tissot et al., 1985, Hori et al., 1988) or EDTA
(Proud et al., 1983, Zhang et al., 1997) limits the extent of BK degradation and could be
included in future studies. Alternatively, the identification of stable metabolites, such as BK
1-5 or des-Phe8-Arg
9-BK, could provide a more accurate indication of in vivo BK release
85
(Majima et al., 1992, Shima et al., 1992, Majima et al., 1993, Majima et al., 1996,
Murphey et al., 2000). Despite these caveats, BK was still detected in human pleural
effusions from patients with a variety of disease diagnoses, suggesting kinins may be
important within the pleural space.
Recurrent pleural effusions cause significant morbidity, and occur in a variety of pleural,
pulmonary and extra-pulmonary diseases. Pleural effusion is associated with increased
permeability (Medford and Maskell, 2005, Jantz and Antony, 2008), which supports the
passage of high molecular weight proteins through the pleura (Asseo and Tracopoulos,
1981, Alexandrakis et al., 2000). This leakage may contribute to the HK and PPK pool
within the pleural cavity, thereby providing the necessary components for BK formation
along the mesothelium. Moreover, extravasation of FXII may augment this process by
activating PPK directly following its activation by PK (Rojkjaer et al., 1998) or auto-
activation (Reddigari et al., 1993b) on mesothelial cells. Overall, BK may exacerbate
effusion development, given observations showing enhanced plasma exudation by kinins in
models of pleurisy (Katori et al., 1978, Uchida et al., 1983, Hayashi et al., 2002).
As demonstrated with respiratory epithelium (Chapter 4), an increase in BK release was
demonstrated with mesothelial cells treated with HK alone although no significant PK
formation was observed, indicating that BK may form independently of PK on
mesothelium. Likewise, mesothelial cells treated with PPK alone showed increases in both
PK activity and BK release. Although not demonstrated for mesothelium, serum starved
respiratory epithelial cells treated with PPK alone still generate PK activity (Chapter 4),
suggesting acquisition of HK from bovine serum is not responsible. Thus, it is likely that an
86
endogenous source of kininogen is present on the cell surface, which assembles PPK and
supports its conversion to PK and liberation of BK.
The inhibition profile of HK-PPK complex activation does not allow a clear delineation of
the activating enzyme responsible. The moderate inhibition by antipain and leupeptin on
MeT-5A, NCI-H2052 and MPM cells indicate the HK-PPK activator could be a serine
protease. Similarly, antipain and leupeptin inhibit PRCP-mediated activation of the HK-
PPK complex on endothelial cells. However, in this study, a five-fold greater concentration
of antipain and leupeptin was required to achieve approximately half the inhibition
observed using endothelial cells (Motta et al., 1998, Joseph et al., 2002, Shariat-Madar et
al., 2002, Shariat-Madar et al., 2004). Additionally, antipain and leupeptin had little effect
on NCI-H28 cells, although AEBSF inhibited activation by approximately 30%. Activation
was also inhibited by BK, a substrate inhibitor of PRCP (Shariat-Madar et al., 2002).
However, consistent with the previous data using respiratory epithelium (Chapter 4), a lack
of inhibition by ANG II, another PRCP substrate (Shariat-Madar et al., 2002), was
demonstrated. Combined with the absence of PRCP immunoreactivity on mesothelial cells,
our data suggest a PRCP-independent mechanism of activation. The inhibition by 2-ME
and cysteine suggests a protease susceptible to reduction of disulphide bonds may be
involved, as seen with respiratory epithelium (Chapter 4). Additionally, as previously
shown for respiratory epithelium (Chapter 4), protamine sulphate was a potent inhibitor of
HK-PPK activation on all cells tested, suggesting a carboxypeptidase with specificity
towards C-terminal arginine residues is responsible.
Mesothelial cell lines expressed HSP90, and PK formation was strongly inhibited by
novobiocin, which is known to interfere with eukaryotic HSP90 function (Marcu et al.,
87
2000, Marcu et al., 2000), consistent with previous reports using endothelium and
epithelium (Joseph et al., 2002); Chapter 4). HSP90β could represent the dominant isoform
responsible for HK-PPK activation on mesothelial cells, given our immunocytochemistry
data, as both and β isoforms catalyse the conversion of PPK to PK (Joseph et al., 2002).
However, the exact contribution of HSP90 to HK-PPK complex activation in not known
since HSP90 is not a protease per se, but rather a chaperone. In this regard, it may support
maturation of a membrane-associated protease capable of activating the HK-PPK complex,
as demonstrated with other cell membrane-associated proteases (Eustace et al., 2004).
At a concentration sufficient to induce BK receptor activation (MacNeil et al., 1997, Andre
et al., 1998), the mesothelial cell lines tested were heterogeneous with regard to their
responsiveness to BK, as judged by calcium mobilisation studies. For example, MeT-5A
and NCI-H2052 cells, but not NCI-H28 cells, were active, suggesting a differential
expression of the B2R or the impairment of a functional receptor. In addition, given that in
all cell lines tested, significant calcium responses were not induced by des-Arg9-BK, a B1R
agonist, the results also suggest functional B1R is not constitutively expressed on
mesothelial cells, although both B1R and B2R expression has been demonstrated on
mesothelioma cells (Chee et al., 2007). Notwithstanding these considerations, the results
obtained suggest B2R is the dominant functional receptor subtype on mesothelial cells.
BK has been shown to induce cytokine and chemokine release from various cell types
(Tiffany and Burch, 1989, Koyama et al., 1998, Wiernas et al., 1998, Koyama et al., 2000)
and mesothelial cells are known to synthesise mediators in response to inflammatory
stimuli (Lanfrancone et al., 1992, Topley et al., 1993). However, neither BK nor des-Arg9-
88
BK appeared to induce or up-regulate IL-6, IL-8, TNF- or MCP-1 release from
mesothelial cells. These findings contrast with those obtained using A549 cells where IL-6
and IL-8 was clearly released after exposure to both agonists (Chapter 4).
The failure to observe BK-induced cytokine and chemokine release could suggest the cell
lines used in this study were not representative of primary mesothelial cells, which are
known to release IL-6 and IL-8 in response to inflammatory stimuli (Lanfrancone et al.,
1992, Topley et al., 1993, Topley et al., 1993, Arici et al., 1996, Witowski et al., 1996).
Similarly, pleural mesothelioma cell lines release these mediators constitutively or when
stimulated (Demetri et al., 1989, Schmitter et al., 1992, Galffy et al., 1999). Consistent
with these findings, all cell lines tested constitutively produced IL-8, albeit to varying
degrees, and MeT-5A constitutively produced MCP-1. However, only unstimulated NCI-
H2052 cells released IL-6. Given this, the high constitutive expression of these cytokines
by some of the cell lines tested may have masked any response induced by BK or des-Arg9-
BK. Alternatively, BK liberated from the mesothelium could provoke paracrine responses
in other cell types present in the pleural space, including macrophages (Tiffany and Burch,
1989, Sato et al., 1996) and fibroblasts (Koyama et al., 2000). These cells, in turn, could
coordinate inflammation via cytokine cross-talk with mesothelial cells (Cailhier et al.,
2006).
In summary, the data detailed in this Chapter show that mesothelial cells assemble and
activate HK and PPK to liberate BK in a process dependent on HSP90, but independent of
PRCP. This study demonstrates that mesothelial cells can activate the plasma KKS. Given
the data demonstrating plasma KKS activation on murine peritoneal mesothelial cells, the
presence of kinins and plasma KKS components in peritoneal effusions (Ruud et al., 1985,
89
Maeda et al., 1988, Waldner et al., 1993, Cugno et al., 2001) and the structural and
functional similarity of mesothelial cells of different anatomical origin (Raftery, 1973,
Whitaker et al., 1980, Mutsaers, 2002), this study also suggests a role for the plasma KKS
on other mesothelia.
90
5.4 Summary
BK concentrations were detected in human pleural fluids and were elevated in
patients with exudative effusions.
In a large proportion of patients, pleural fluid BK concentrations were higher than
the corresponding serum samples, suggesting local BK formation was involved.
Treatment of MeT-5A cells with pleural effusions generated PK activity.
Benign and transformed mesothelial cell lines, primary human mesothelioma cells,
primary murine peritoneal mesothelial cells and MeT-5A cell-free matrix assembled
HK and PPK to generate PK, and release BK.
Similar to respiratory epithelium, HK-PPK complex activation appeared
independent of PRCP, but dependent on HSP90.
B2R represented the dominant functional BK receptor subtype in mesothelial cells.
BK and des-Arg9-BK did not induce pro-inflammatory mediator release from
mesothelial cells.
CHAPTER 6
THE ROLE OF THE B2R AND PROTEASE-ACTIVATED RECEPTORS IN
KALLIKREIN SIGNALING IN MESOTHELIAL CELLS
90
6.1 Introduction
In the previous Chapter, it was shown that assembly of HK and PPK on pleural
mesothelium resulted in PK formation and liberation of BK. Although kinin release
represents the major functional outcome of tissue and plasma KKS activation, hK1 and PK
demonstrate additional activities independent of kininogen. In this regard, hK1 and PK
directly activate the B2R and it has been proposed that this process involves a proteolytic
mechanism of activation similar to that observed during proteolytic activation of PARs
(Hecquet et al., 2000, Biyashev et al., 2006). In addition, kallikreins per se also activate
PARs on a variety of cell types (Oikonomopoulou et al., 2006, Mize et al., 2008,
Stefansson et al., 2008, Vandell et al., 2008, Gratio et al., 2010). With this background, it
was decided to determine whether PK and hK1 can activate the B2R or PARs on
mesothelial cells.
PARs are members of the GPCR superfamily, of which four have been described: PAR1
(Vu et al., 1991), PAR2 (Nystedt et al., 1994), PAR3 (Ishihara et al., 1997) and PAR4 (Xu
et al., 1998). Thrombin is the archetypal activator of PAR1, PAR3 and PAR4, while trypsin
and tryptase activate PAR2 and PAR4. PARs are activated following cleavage at specific
amino acid residues within the N-terminal domain of the receptor. Following this, the
newly exposed “tethered” ligand interacts with downstream regions of the same molecule
to initiate G protein-coupled signal transduction pathways (Ossovskaya and Bunnett, 2004)
(Table 6.1). PARs and their activators have been studied on a variety of cell types such as
respiratory epithelium (Asokananthan et al., 2002), but limited studies have been performed
on mesothelium. However, it has previously been shown that PAR2 activation in
mesothelial cells induces pleural inflammation (Lee et al., 2004), and thrombin stimulates
Table 6.1 Sites of cleavage and tethered ligands of PARs
PAR Activating proteases Cleavage site
PAR1 Thrombin LDPR41
↓ S42
FLLRN
PAR2 Trypsin, tryptase SKGR34
↓ S35
LIGKV
PAR3 Thrombin LPIK38
↓ T39
FRGAP
PAR4 Thrombin, trypsin PAPR47
↓ G48
YPGQV
The arrow denotes the cleavage site. The tethered ligand sequence is shown in bold.
91
mesothelial cell proliferation, chemotaxis (Hott et al., 1992), and mediator synthesis (Hott
et al., 1994, Mandl-Weber et al., 2002). Additionally, thrombin is known to potentiate AA
release from endothelial cells induced by hK1 and PK activation of B2R (Hecquet et al.,
2006). At present, however, data demonstrating the presence and relevance of all four
known PARs on mesothelial cells is lacking. Similarly, elucidation of the types of proteases
expressed by mesothelial cells or present in the pleural cavity capable of activating PARs,
or the B2R, is required.
In this Chapter, human PK, trypsin-activated PPK and the hK1 ortholog, porcine pancreatic
kallikrein, were studied for their ability to induce calcium mobilisation in the MeT-5A
pleural mesothelial cell line. MeT-5A cells were chosen for this study as they are
commonly used as an immortalised model of normal mesothelial cells (Schmitter et al.,
1992). In addition, the receptors involved in mediating kallikrein-induced signals were
investigated, with particular focus on PARs and B2R. In so doing, the expression and
functionality of these receptors on MeT-5A cells was examined.
92
6.2 Results
6.2.1 The effect of tissue and plasma KKS associated enzymes on calcium mobilisation in
MeT-5A cells
Initial studies aimed to determine whether mesothelial cells could be activated by proteases
associated with the tissue and plasma KKS. Calcium mobilisation was induced in MeT-5A
cells by 1 μM porcine kallikrein (Figure 6.1), as it was in A549 cells (Figure 6.2).
However, further studies using A549 cells were not performed due to time constraints. In
contrast, human PK and trypsin activated PPK had no significant effect on calcium
mobilisation in MeT-5A cells (Figures 6.3A and 6.4, respectively), although both
demonstrated amidolytic activity against the PK-selective substrate, S-2302 (Figures 6.3B
and 4.24A, respectively). As such, a role for PK and trypsin-activated PPK on MeT-5A
cells was not examined further.
6.2.2 Expression and functionality of PARs and B2R on MeT-5A cells
Figure 6.5 shows that all four PARs were expressed by MeT-5A cells as determined by
immunocytochemistry (Figure 6.5). Staining was observed on all cells and the intensity
appeared similar between all four PARs. Thrombin and trypsin induced calcium
mobilisation in these cells (Figure 6.6) and , at a concentration shown to activate PARs on
respiratory epithelial cells (Asokananthan et al., 2002), calcium mobilisation was induced
by PAR1 and PAR2 APs, but not by APs of PAR3 or PAR4 nor any of the four PAR CPs
(Figure 6.7). Calcium mobilisation in Met5A cells was also induced by BK, which was
inhibited by pre-treatment with the selective B2R antagonist, Hoe 140 (Figure 6.8),
indicating BK was acting through B2R.
Figure 6.1 Calcium mobilisation induced by porcine kallikrein in MeT-5A
cells
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by 1 μM
porcine kallikrein (A). The arrows indicate addition the addition of porcine kallikrein or
ionomycin. The figure is representative of three independent experiments. The amidolytic
activity of 0.1 (), 0.5 () and 1 μM () porcine kallikrein over 60 min using S-2266 (B).
Untreated S-2266 is included (). The data are presented as the mean ± standard deviation
of one experiment performed in triplicate.
0
5
10
15
20
25
30
0 50 100 150 200 250 300
% R
esp
on
din
g c
ells
Time (sec)
Ionomycin A
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50
Ab
sorb
an
ce (
405 n
m)
Time (min)
60
B
Figure 6.2 Calcium mobilisation induced by porcine kallikrein in A549 cells
Flow cytometric analysis of calcium mobilisation in A549 cells induced by 1 μM porcine
kallikrein. The arrows indicate addition the addition of porcine kallikrein or ionomycin.
The figure is representative of three independent experiments.
0.4
0.6
0.8
1
1.2
0 1 105
2 105
3 105
4 105
5 105
6 105
7 105
Flu
ore
scen
ce r
ati
o (
340/3
80 n
m)
0 100 200 300 400 500 600 700
Time (sec)
1.2
1
0.8
0.6
0.4
Ionomycin
Figure 6.3 Calcium mobilisation induced by human PK in MeT-5A cells
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by 10 (green),
20 (blue), 50 (red) or 100 nM (black) human PK, or 1 μM porcine kallikrein (dotted line),
included as a positive control (A). The arrows indicate the addition of PK, porcine
kallikrein or ionomycin. The figures are representative of three independent experiments.
The amidolytic activity of 10 (), 20 (), 50 () and 100 nM () human PK over 60 min
using S-2302 (B). Untreated S-2302 () is included. The data are presented as the mean ±
standard deviation of one experiment performed in triplicate.
0
1
2
3
4
0 10 20 30 40 50
Ab
sorb
an
ce (
405 n
m)
Time
(min)
60
0
1
0
2
0
3
0
4
0
5
0
6
0
0 5
0
10
0
15
0 200 25
0
30
0
35
0
% R
esp
on
din
g c
ells
Time
(sec)
Ionomycin A
B
Figure 6.4 Calcium mobilisation induced by trypsin-activated PPK in MeT-5A
cells
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by trypsin-
activated PPK (black). Ionomycin and 400 μM PAR1 AP (red) were included as positive
controls. The arrows indicate the addition of trypsin-activated PPK, PAR1 AP or
ionomycin. The figure is representative of three independent experiments.
0
10
20
30
40
0 50 100 150 200 250 300
% R
esp
on
din
g c
ells
Time (sec)
Ionomycin
Figure 6.5 Immunohistochemical analysis of surface expressed PARs on MeT-5A
cells
Immunohistochemical analysis of extracellular PARs on MeT-5A cells. The isotype control
for each PAR is shown in the insert on the top left hand corner. The figures are
representative of three independent experiments.
PAR1 PAR2
PAR3 PAR4
Figure 6.6 Calcium mobilisation induced by thrombin and trypsin
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by 1 U/ml
thrombin (A) and 200 ng/ml trypsin (B). The arrows indicate the addition of the protease or
ionomycin. The figures are representative of three independent experiments.
0
5
10
15
20
25
30
35
% R
esp
on
din
g c
ells
0
5
10
15
20
25
0 100 200 300
Time (sec)
B
A
Ionomycin
Ionomycin
% R
esp
on
din
g c
ells
Figure 6.7 Calcium mobilisation induced by PAR APs and CPs in MeT-5A cells
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by 400 μM
PAR-APs (black line) and -CPs (red line). The arrows indicate addition of the peptide or
ionomycin. The figures are representative of three independent experiments.
0
5
10
15
20
25
30
35
0 100 200 300
Time (sec)
0
5
10
15
20
25
30
35
40
% R
esp
on
din
g c
ells
0
5
10
15
20
25
0
5
10
15
20
25
30
35
0 100 200 300
Time (sec)
PAR1 PAR2
PAR3 PAR4
Ionomycin
Ionomycin Ionomycin
Ionomycin
% R
esp
on
din
g c
ells
Figure 6.8 Calcium mobilisation induced by BK and inhibition by antagonism of
B2R
Flow cytometric analysis of calcium mobilisation in MeT-5A induced by 10 μM BK (A)
and inhibition by pre-treatment with 1 μM Hoe 140, the B2R antagonist (B). The arrows
indicate the addition of BK, Hoe 140 or ionomycin. The figures are representative of three
independent experiments.
0
5
10
15
20
25
0 100 200 300
% R
esp
on
din
g c
ells
0
5
10
15
20
25
0 100 200 300 400
Time (sec)
BK
BK
Hoe 140
B
A
Ionomycin
Ionomycin
% R
esp
on
din
g c
ells
93
6.2.3 Specificity of PAR1 and PAR2 on mesothelial cells
Subsequent studies were required to determine the specificity of PARs on MeT-5A cells for
trypsin, thrombin and PAR APs. Treatment of cells with thrombin resulted in
desensitisation of subsequent responses to PAR1 AP, PAR2 AP, and trypsin. However,
exposure of cells to trypsin did not result in desensitisation of PAR1 AP, PAR2 AP or
thrombin responses (Figure 6.9). PAR1 AP desensitised the response induced by PAR2 AP,
but not vice versa. Likewise, exposure of cells to PAR1 AP desensitised responses to
thrombin and trypsin. However, treatment with PAR2 AP had no effect on the subsequent
thrombin response, and did not completely desensitise the response to trypsin (Figure 6.10).
6.2.4 The effect of porcine kallikrein on B2R and PARs
Pre-treatment of MeT-5A cells with 1μM Hoe 140, a B2R antagonist, had no effect on the
response induced by porcine kallikrein. Likewise, no significant cross desensitisation was
observed between BK and porcine kallikrein (Figure 6.11). The calcium response induced
by porcine kallikrein was desensitised by previous exposure to trypsin, thrombin and APs
of PAR1 and PAR2. However, prior exposure of cells to porcine kallikrein desensitised the
response to trypsin and PAR2 AP, but not thrombin or PAR1 AP (Figures 6.12 and 6.13).
Figure 6.9 Desensitisation of calcium mobilisation in MeT-5A cells by treatment
with thrombin and trypsin
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by PAR1-AP,
PAR2-AP, trypsin or thrombin following pre-treatment with trypsin or thrombin. The
arrows indicate the addition of protease, peptide or ionomycin. The figures are
representative of three independent experiments.
0
5
10
15
20
25
30
35
% R
esp
on
din
g c
ells
Thrombin
AP1
0
5
10
15
20
25 Thrombin
AP2
0
5
10
15
20
25
30
0 100 200 300 400
Thrombin
Trypsin
% R
esp
on
din
g c
ells
%
Res
pon
din
g c
ells
0
5
10
15
20
25
Trypsin
AP1
0
5
10
15
20
25
30
Trypsin AP2
0
5
10
15
20
25
30
0 100 200 300 400
Time (sec)
Trypsin
Thrombin
Time (sec)
Ionomycin Ionomycin
Ionomycin Ionomycin
Ionomycin Ionomycin
Figure 6.10 Desensitisation of calcium mobilisation in MeT-5A cells by treatment
with PAR APs
Flow cytometric analysis of calcium mobilisation in MeT-5A cells induced by PAR1-AP,
PAR2-AP, trypsin or thrombin following pre-treatment with PAR1-AP or PAR2-AP. The
arrows indicate the addition of protease, peptide or ionomycin. The figures are
representative of three independent experiments.
0
5
10
15
20
25
30
% R
esp
on
din
g c
ells
AP1
Thrombin
0
5
10
15
20
25
30
AP1
Trypsin
0
5
10
15
20
25
0 100 200 300 400
AP1
AP2
% R
esp
on
din
g c
ells
%
Res
pon
din
g c
ells
0
5
10
15
20
25
Thrombin
AP2
0
5
10
15
20
25
AP2
Trypsin
0
5
10
15
20
25
0 100 200 300 400
Time (sec)
AP2
AP1
Time (sec)
Ionomycin Ionomycin
Ionomycin Ionomycin
Ionomycin Ionomycin
Figure 6.11 The effect of Hoe 140 on porcine kallikrein-induced calcium
mobilisation in MeT-5A cells and cross-desensitisation with BK
Calcium mobilisation induced by 1 μM porcine kallikrein alone (A) or following pre-
treatment with 1 μM Hoe 140 for 2 min (B). Desensitisation of 1 μM porcine kallikrein
response following pre-treatment with 10 μM BK (C) and 10 μM BK response following
pre-treatment with 1 μM porcine kallikrein (D). The figures are representative of three
independent experiments.
0
10
20
30
40
50
0 100 200 300
% R
esp
on
din
g c
ells
0
10
20
30
40
50
0 100 200 300
0
10
20
30
40
50
60
70
0 100 200 300 400
% R
esp
on
din
g c
ells
Time (sec)
0
5
10
15
20
25
30
35
40
0 100 200 300 400
Time (sec)
Porcine
kallikrein
Porcine
kallikrein
Porcine
kallikrein
Porcine
kallikrein
BK BK
A B
C D
Ionomycin Ionomycin
Ionomycin Ionomycin
Figure 6.12 Cross-desensitisation between porcine kallikrein and thrombin or
trypsin in MeT-5A cells
Desensitisation of 1 μM porcine kallikrein response following pre-treatment with 1 U/ml
thrombin (A) or 200 ng/ml trypsin (B). Desensitisation of response induced by 1 U/ml
thrombin (C) or 200 ng/ml trypsin (D) following pre-treatment with 1 μM porcine
kallikrein. The figures are representative of three independent experiments.
0
5
10
15
20
25
30
35
40
0 100 200 300 400
% R
esp
on
din
g c
ells
0
5
10
15
20
25
30
35
40
0 100 200 300 400
0
10
20
30
40
50
0
10
20
30
40
50
60
% R
esp
on
din
g c
ells
Porcine
kallikrein
Porcine
kallikrein
Porcine
kallikrein
Porcine
kallikrein
2
Porcine
kallikrein Porcine
kallikrein
Trypsin
Trypsin
Thrombin
Thrombin
Thrombin
Trypsin
Ionomycin Ionomycin
Ionomycin Ionomycin
A B
C D
Figure 6.13 Cross-desensitisation between porcine kallikrein and PAR APs in MeT-
5A cells
Desensitisation of 1 μM porcine kallikrein response following pre-treatment with 400 μM
PAR1-AP (A) or 400 μM PAR2-AP (B). Desensitisation of response induced by 400 μM
PAR1-AP (C) or 400 μM PAR2-AP (D) following pre-treatment with 1 μM porcine
kallikrein. The figures are representative of three independent experiments.
0
5
10
15
20
25
30
35
% R
esp
on
din
g c
ells
0
5
10
15
20
25
30
0
5
10
15
20
25
30
0 100 200 300 400 500
% R
esp
on
din
g c
ells
Time (sec)
0
5
10
15
20
25
30
35
0 100 200 300 400 500
Time (sec)
AP1
AP1
Porcine
kallikrein
AP2
AP2 Porcine
kallikrein
Porcine
kallikrein Porcine
kallikrein
AP1 Porcine
kallikrein
Porcine
kallikrein AP2
A B
C D
Ionomycin Ionomycin
Ionomycin Ionomycin
94
6.3 Discussion
Previous studies indicate that KLKs are capable of activating PARs (Table 6.2). For
example, hK4 (Mize et al., 2008, Wang et al., 2010) and hK14 (Oikonomopoulou et al.,
2006) activate PAR1, whereas PAR2 is amenable to activation by hK2 (Mize et al., 2008),
hK5 (Oikonomopoulou et al., 2006) and hK6 (Angelo et al., 2006). Similar to PARs, B2R
is a GPCR susceptible to pharmacological activation by porcine kallikrein, hK1 and PK
(Hecquet et al., 2000) and, thus, some of the effects attributed to kinins may have been due
to direct actions of kallikreins on B2R. This, therefore, provides an alternative pathway of
receptor activation in the absence of kinin formation. As such, this study aimed to
determine the involvement of the B2R and PARs in kallikrein-induced cell signaling.
To exclude contaminant activation by kinins liberated from trace amounts of adherent
kininogen, cells were washed several times in zinc-free buffers prior to porcine kallikrein
stimulation. Likewise, cells were harvested in media containing EDTA, which would
sequester cell-bound zinc. Although this study demonstrates the presence of functional B2R
on MeT-5A cells, Hoe 140 pretreatment had little effect on porcine kallikrein-induced
calcium mobilisation, suggesting a lack of involvement for this receptor. Additionally, no
significant cross-desensitisation of porcine kallikrein with BK was observed indicating that
the B2R is not activated in a similar manner to PARs. Given these findings, the results are
consistent with data from previous reports describing direct receptor proteolysis as a non-
preferred mechanism of B2R activation by kallikreins (Houle et al., 2003, Marceau and
Houle, 2003), at least on mesothelial cells.
Table 6.2a PAR-activating kallikreins
KLK PAR activated Method Reference
hK2 PAR2 Analysis of ERK1/2 signaling Mize, Wang and
in PAR1 knockout fibroblast Takayama, 2008
cell line
expressing PAR2
hK4 PAR1/2 Analysis of ERK1/2 signaling Mize, Wang and
in PAR1 knockout fibroblast Takayama, 2008
cell line
expressing PAR1/2
PAR1 Loss of biotinylated tag Wang et al.,
on PAR1 expressed by 2010
CHO cells
PAR1 Measurements of calcium Gratio et al.,
mobilisation in colorectal 2010
epithelial cell line
PAR2 Analysis of ERK1/2 signaling Ramsay et al.,
and measurements of calcium 2008
mobilisaiton in prostate
epithelial cell line
hK5 PAR2 Immunofluoresence analysis Stefansson et al.,
and measurements in calcium 2008
mobilisation in rat kidney
epithelial cells expressing
PAR2
hK5 PAR2 HPLC-mass spectral analysis
Oikonomopoulou
of PAR peptide cleavage et al., 2006a
products, measurements of
calcium mobilisation and
platelet aggregation assay
hK6 PAR1/2 Measurements of calcium Vandell et al.,
mobilisation in NSC34 neuron 2008
and Neu7 astrocyte cell lines
expressing PAR1 and PAR2
PAR2 FRET peptides spanning Angelo et al.,
cleavage site of PARs 2006
Table 6.2b PAR-activating kallikreins
KLK PAR activated Method Reference
hK6 PAR2 HPLC-mass spectral analysis
Oikonomopoulou
of PAR peptide cleavage et al., 2006a
products, measurements of
calcium mobilisation and
platelet aggregation assay
hK14 PAR1/2/4 HPLC-mass spectral analysis
Oikonomopoulou
of PAR peptide cleavage et al., 2006a
products, measurements of
calcium mobilisation and
platelet aggregation assay
PAR2 Immunofluoresence analysis Stefansson et al.,
and measurements in calcium 2008
mobilisation in rat kidney
epithelial cells
CHO: Chinese hamster ovary; FRET: fluorescence resonance energy transfer;
HPLC: high pressure liquid chromatography.
95
Given the above results, the role of PARs in porcine kallikrein-induced responses in
mesothelial cells was examined. Although mesothelial cells are known to respond to trypsin
(Aronson et al., 1976) and thrombin (Hott et al., 1992, Hott et al., 1994, Mandl-Weber et
al., 2002), PAR2 is the only PAR subtype that has been comprehensively described on this
cell type (Lee et al., 2004). Therefore, studies were performed to determine whether pleural
mesothelial cells expressed all four PARs. In this regard, all four PARs were detected on
MeT-5A cells by immunoytochemistry and the intensity of staining of each PAR appeared
similar. In addition, calcium mobilisation occurred in response to thrombin, trypsin, PAR1
and PAR2 APs, but not PAR3 or PAR4 APs. Despite the observed expression of PAR3 and
PAR4 on MeT-5A cells, the inability to elicit a calcium response using these APs might
suggest impairment of receptor function, but PAR3 (Bar-Shavit et al., 2002, Wang et al.,
2002, Bretschneider et al., 2003) and PAR4 (Asokananthan et al., 2002, Wang et al., 2002)
APs are known to induce responses in a variety of cells types. However, the insensitivity of
MeT-5A cells to the PAR3 AP, TFRGAP, is consistent with previous functional data
obtained with respiratory epithelium (Asokananthan et al., 2002).
Exposure of MeT-5A cells to PAR2 AP did not completely desensitise the response to
trypsin. Likewise, trypsin failed to abolish responses to PAR2 AP. Thus, additional
receptors on mesothelial cells may be subject to trypsin activation, such as PAR1 (Ubl et
al., 1998, Grishina et al., 2005, Wang et al., 2006), PAR4 (Xu et al., 1998) or the B2R
(Hecquet et al., 2000). However it should be noted that calcium mobilisation in response to
trypsin may have desensitised if treatment was repeated or used at a higher concentration.
This would need to be addressed in subsequent studies. Furthermore, thrombin desensitised
MeT-5A cells to subsequent treatment to trypsin or PAR2 AP, suggesting direct activation
of PAR2 by thrombin or transactivation of PAR2 by thrombin-activated PAR1, as described
96
for endothelial cells (O'Brien et al., 2000). Interestingly, the PAR1 AP, TFLLRN, also
abolished responses to trypsin and PAR2 AP on MeT-5A cells, but not vice versa. Ligand
cross-reactivity with PAR2 may explain this finding, although the frog peptide TFLLRN is
highly specific for PAR1 (Asokananthan et al., 2002, Jeng et al., 2006).
Cross-desensitisation of porcine kallikrein was observed with trypsin and PAR2 AP,
suggesting the porcine kallikrein may activate PAR2. The desensitisation of porcine
kallikrein by prior exposure to thrombin or PAR1 AP may be explained by the ability of
each to desensitise PAR2. Similarly, the ability of porcine kallikrein to partially inhibit
thrombin responses is likely due to desensitisation of PAR2, rather than direct activation of
PAR1 by porcine kallikrein. Alternatively, porcine kallikrein may disarm PAR1 by
disrupting the tethered ligand sequence and would explain the attenuated response to
thrombin, but not PAR1 AP. Nonetheless, consistent with previous studies with other TKs
(Angelo et al., 2006, Mize et al., 2008, Ramsay et al., 2008, Stefansson et al., 2008),
porcine kallikrein activates PAR2 on mesothelial cells and is a likely result given the well
established affinity of PAR2 for trypsin-like serine proteases (Nystedt et al., 1994).
However, additional experiments should be performed to confirm that porcine kallikrein
cleaves PAR2. For example, whether treatment with porcine kallikrein induces
internalisation and redistribution of PAR2 could be examined. Also, it should be noted the
high concentration of porcine kallikrein used for this study, which exceeds concentrations
commonly used for kallikreins, and other proteases, in similar reports (Hecquet et al.,
2000). Subsequent studies are required to address this issue, as the employed concentration
of porcine kallikrein likely has little physiological relevance.
97
As porcine kallikrein demonstrates substrate specificity towards Phe-(or Leu)-Arg-X bonds
(Fielder, 1987), the protease may cleave the same Arg34
-Ser35
bond utilised by trypsin
(Nystedt et al., 1994) and tryptase (Molino et al., 1997) to activate the receptor. As such,
kallikreins have been shown to preferentially cleave synthetic N-terminal PAR2 peptides to
expose the tethered ligand sequence (Angelo et al., 2006, Oikonomopoulou et al., 2006,
Oikonomopoulou et al., 2006). In addition, porcine kallikrein may cleave other sites in
PAR2. In this regard, hK5 and hK14 cleave the Arg29
-Ser30
bond of PAR2, whereas hK6
cleaves within the Lys39
-Val40
and Lys49
-Gly50
bonds (Oikonomopoulou et al., 2006). In
principle, if porcine kallikrein were to produce such cleavages in PAR2 it may disrupt
formation of the tethered ligand. Nevertheless, cleavage by porcine kallikrein is likely
restricted to the Arg34
-Ser35
bond given PAR2 was activated on mesothelial cells.
Porcine kallikrein is often used as an hK1-like TK (Mikolajczyk et al., 1998, Hecquet et al.,
2000, Biyashev et al., 2006) as it demonstrates biological and functional similarities to the
human ortholog (Clements, 1989). In this regard, porcine kallikrein activates human B2R
(Hecquet et al., 2000, Hecquet et al., 2002, Biyashev et al., 2006, Hecquet et al., 2006) and
liberates Lys-BK from human LK (Figarella et al., 1978, Muller-Esterl et al., 1985). Thus,
it is tempting to extrapolate the present findings to include hK1. Given this, hK1 may
possess a dual activating role within the pleural space by directly activating mesothelial
PAR2, while concomitantly liberating Lys-BK from LK to activate B2R. In this regard,
hK1, B2R (Chee et al., 2007) and PAR2 (Lee et al., 2004) are all expressed by the pleural
mesothelium. Additionally, hK1 may disrupt B2R signaling by displacing BK from its
receptor, but this likely occurs at significantly lower concentrations than those used in this
study (Hecquet et al., 2000, Hecquet et al., 2002). Furthermore, pleural mesothelial cells
express additional KLKs, including hK4 (Davidson et al., 2006) and hK6 (Petraki et al.,
98
2001), which are known to activate PAR2 (Oikonomopoulou et al., 2006, Oikonomopoulou
et al., 2006, Wang et al., 2010). Therefore, within the pleural space, a variety of KLKs may
be available to activate PAR2, with the overall effect dependent on the spectrum of KLKs
expressed.
Although several reports indicate a role for TKs as PAR2 activators, the downstream effects
of this activation process requires elaboration. Various in vivo studies are consistent with an
inflammatory role for PAR2 (Vergnolle, 1999, Steinhoff et al., 2000, Su et al., 2005, Hyun
et al., 2008) and, as such, receptor activation by TKs may support pleural inflammation. In
this regard, PAR2 agonists are potent cytokine secretagogues for various cell types
(Asokananthan et al., 2002, Ikeda et al., 2006, Ramachandran et al., 2006, Niu et al.,
2008), including mesothelial cells, and intra-pleural administration of PAR2 AP induces
infiltration of inflammatory cells into the pleura (Lee et al., 2004). hK1 may induce similar
responses from pleural mesothelial cells and this notion is supported by recent studies
reporting a role for the PAR2-activating KLKs, hK4 (Wang et al., 2010) and hK5 (Briot et
al., 2009), in cytokine and chemokine release.
In contrast to TKs, PK may not cleave PARs (Molino et al., 1997, Molino et al., 1997) and
studies describing its ability to activate B2R are conflicting (Hecquet et al., 2000, Houle et
al., 2003). In this regard, PK had no significant effect on calcium mobilisation in
mesothelial cells, although it was used at concentrations reported to induce AA release
(Hecquet et al., 2000, Hecquet et al., 2002, Hecquet et al., 2006). Additionally, similar
results were also observed with trypsin-activated PPK. Thus, any effects attributed to PK
on mesothelial cells are likely due to indirect activation of B2R by BK following
proteolysis of cell-bound HK. Alternatively, rather than causing activation, PK may cleave
99
receptors at residues distal from the usual cleavage site, thereby inhibiting proteolytic
activation, such is the case for PAR1 or PAR2 (Molino et al., 1997, Molino et al., 1997).
In conclusion, this study demonstrates that porcine kallikrein, the porcine ortholog of hK1,
stimulated calcium mobilisation in pleural mesothelial cells. Although reports implicate
kallikreins in the activation of B2R, this study suggests that this receptor may not be
involved. Rather, porcine kallikrein was shown to activate PAR2 and, thus, supports a role
for TKs as PAR activators. As an indirect result, this study also provides several novel
findings regarding the expression, functional significance and specificity of PARs on
pleural mesothelium. Thus, all four PARs were detected on MeT-5A cells, and thrombin,
trypsin and APs of PAR1 and PAR2 were shown to induce calcium mobilisation. The
activation of PARs by kallikrein on pleural mesothelial cells is likely limited to TKs, given
the negative findings obtained using PK and trypsin-activated PPK. Within the pleural
space, activation of PARs by TKs would likely contribute to inflammatory processes
common to various pleural diseases, including cellular infiltration and effusion
development. Furthermore, a similar setting may exist within other serosal compartments
given the similarities of pleural mesothelium with peritoneal and pericardial mesothelia
(Mutsaers, 2002), and the widespread distribution of KLKs within these tissues (Petraki et
al., 2001, Petraki et al., 2002, Davidson et al., 2005, Shih et al., 2007). Lastly, the limited
data obtained using A549 cells suggest respiratory epithelial cells may be subject to direct
activation by kallikreins.
100
6.4 Summary
Porcine kallikrein, an ortholog of hK1, but not human PK or trypsin-activated PPK,
induced calcium mobilisation in MeT-5A and A549 cells.
MeT-5A cells expressed all four PARs and calcium mobilisation was induced in
response to thrombin, trypsin, PAR1 AP and PAR2 AP, but not APs of PAR3 or
PAR4.
Calcium mobilisation in MeT-5A cells was induced by BK, which was inhibited by
the B2R antagonist, Hoe 140.
Calcium mobilisation induced by porcine kallikrein was not blocked by Hoe 140
and no cross-desensitisation was observed with BK.
Calcium mobilisation induced by porcine kallikrein was desensitised by previous
exposure of MeT-5A cells to thrombin, trypsin, PAR1 AP or PAR2 AP.
Porcine kallikrein desensitised calcium mobilisation induced by trypsin and PAR2
AP, but did not completely abolish the response to thrombin or PAR1 AP, indicating
porcine kallikrein is acting through PAR2.
CHAPTER 7
GENERAL DISCUSSION AND FUTURE PERSPECTIVES
101
7.1 General discussion and future perspectives
A body of data has been generated indicating that kinin-forming pathways may contribute
to pro-inflammatory responses. For example, airway provocation with kinins stimulates
bronchoconstriction (Fuller et al., 1987, Polosa and Holgate, 1990, Polosa et al., 1994,
Polosa et al., 1997) and symptoms of rhinitis (Proud et al., 1988, Brunnee et al., 1991,
Rajakulasingam et al., 1991) in asthmatic and allergic patients, respectively. Likewise,
intra-thoracic administration of kinins induces plasma extravasation and inflammatory cell
influx in animal models of pleurisy (Saleh et al., 1997, Vianna and Calixto, 1998). In
addition, the potential importance of kinins in lung disease is supported by data showing
the attenuation and augmentation of pulmonary symptoms by kinin receptor antagonists
(Austin and Foreman, 1994, Akbary et al., 1996, Pruneau et al., 1999, Turner et al., 2001,
Hirayama et al., 2003) and kininase inhibitors (Lotvall et al., 1991, Klitzman et al., 1994,
Schilero et al., 1994, Schilero et al., 1996, Yuhki et al., 2004), respectively.
The tissue KKS is the most studied kinin-forming system in the lung, and hK1 has been
described as the major kininogenase in this tissue (Christiansen et al., 1987, Christiansen et
al., 1992, Zhang et al., 1997, Christiansen et al., 2008). In addition, the potential
importance of the tissue KKS in lung disease is suggested by the experimental efficacy of
hK1 inhibitors in preventing kinin-induced responses in animal models of allergic airway
disease (Szelke et al., 1994, Evans et al., 1996, Forteza et al., 1996, Sexton et al., 2009).
However, PK is also expressed within the lung (Christiansen et al., 1987, Zhang et al.,
1997, Chee et al., 2007, Chee et al., 2008) and may represent the dominate kallikrein in
various airway (Baumgarten et al., 1986, Zhang et al., 1997) and pleural diseases (Uchida
et al., 1983, Fujie et al., 1993, Costa et al., 2002, Malavazi-Piza et al., 2004). Despite these
102
observations, a role for the plasma KKS within these tissues has not been described.
However, the data described in this thesis indicate the assembly and activation of the
plasma KKS on respiratory epithelial and pleural mesothelial cells, suggesting it may be
important in lung disease.
Although the results of this thesis describe the novel activation of the plasma KKS on
respiratory epithelium, previous studies have clearly demonstrated the presence of such a
system on other epithelia. In this regard, ECV304 cells have been used to demonstrate HK-
PPK assembly and activation on endothelium (Motta et al., 2001). However, given that
ECV304 cells were recently identified as being derived from the bladder carcinoma cell
line, T24/83 (Dirks et al., 1999, Brown et al., 2000), these results inadvertently describe a
role for the plasma KKS on epithelium. In this regard, the data described in this thesis
indicate plasma KKS activation may be a common feature of epithelia per se. Likewise, the
results suggest this system is active on different types of mesothelia. Within a given tissue,
activation of the plasma KKS on epithelia and mesothelia is unlikely to significantly
contribute to the fluid phase kinin pool. Instead, this role is assumed by the tissue KKS.
Rather, activation of the plasma KKS represents a mechanism in which to rapidly and
effectively deliver kinins to receptors on the same, or nearby, cell (Barbasz and Kozik,
2009).
Kinins are known to act on a diverse range of epithelial cell types, including endometrial
(Matthews et al., 1993), vas deferens (Pierucci-Alves and Schultz, 2008), epididymal
(Cuthbert and Wong, 1986, Cheuk and Wong, 2002), intestinal (Cuthbert et al., 1985, Baird
et al., 2008), breast (Greco et al., 2004) and airway epithelium (Leikauf et al., 1985, Proud
et al., 1993). Similarly, BK is known to induce responses in mesothelial cells, including
103
calcium mobilisation (Andre et al., 1998) and prostaglandin release (van de Veld et al.,
1986, Satoh and Prescott, 1987). However, these studies fail to consider the involvement of
local plasma KKS activation on epithelial and mesothelial cells as the source of BK.
Rather, the effects of BK on epithelia and mesothelia are attributed to local hK1 activity
(Pierucci-Alves and Schultz, 2008) or the delivery of BK from proximal cells (Andre et al.,
1998), or following fluid passage from distal sites (Matthews et al., 1993). Thus,
identifying the plasma KKS as a feature of epithelia and mesothelia may impact on our
understanding of physiological and pathological processes in which kinins are important.
Although the conclusions drawn from this thesis specifically relate to epithelia and
mesothelia, plasma KKS activation may be a common feature of most, if not all, cell types.
Currently, cells which are thought to possess this system include endothelial cells (Zhao et
al., 2001), platelets (Gustafson et al., 1986), neutrophils (Gustafson et al., 1989),
macrophages (Barbasz and Kozik, 2009), astrocytes (Fernando et al., 2003) and smooth
muscle cells (Fernando et al., 2005), and this thesis supports and extends these findings to
include additional cell types. Therefore, within the lung plasma KKS activation may not be
restricted to the epithelium and mesothelium. As such, the ubiquity of this system may
account for the observed plasma KKS activation accompanying various inflammatory
conditions (Aasen et al., 1980, Carvalho et al., 1988, Herrera et al., 1989, Stadnicki et al.,
1998). Given HK and PPK are expressed within the lung (Kleniewski and Bogumil-
Oczkowska, 1980, Fink et al., 2007), plasma KKS activation may be autonomous of
systemic contribution of these proteins during physiological settings. However,
extravasation of bulk plasma proteins during inflammation (Persson and Uller, 2009) would
significantly enhance this process, contributing to widespread kinin liberation.
104
Moreover, functional overlap between the tissue and plasma KKS is expected within the
lung. For example, HK binding to the epithelium and mesothelium would not only facilitate
the assembly and activation of PPK, but allow cleavage of HK by hK1 approaching the cell
surface from the fluid phase. As such, this phenomenon has previously been described for
cell surface-absorbed HK on U-937 macrophages (Barbasz and Kozik, 2009). This process
would preferentially liberate Lys-BK from HK (Kaplan et al., 2002) and, similar to plasma
KKS activation, represents a mechanism to localise this kinin to the cell surface.
Concomitantly, hK1 likely activates cell surface expressed receptors on epithelium or
mesothelium, such as PARs, given the results with porcine kallikrein (Figure 7.1). Overall,
these processes would act in parallel to elicit secondary responses involved in initiating
and/or propagating inflammation.
The results obtained with respiratory epithelium and pleural mesothelium described in this
thesis demonstrate some key differences from plasma KKS on endothelium. First, binding
of HK to respiratory epithelium was only partially dependent on the endothelial HK
binding proteins, uPAR, gC1qR and CK1, and did not involve sulphated proteoglycans.
Second, the results suggest the endothelial HK-PPK activator, PRCP, is not involved in
plasma KKS activation on respiratory epithelium or pleural mesothelium. The most
suggestive data supporting this conclusion are the unique inhibitory profiles obtained using
partial and complete inhibitors of HK-PPK complex activation on endothelial cells
(summarised in Tables 7.1-3). Although not described in this thesis, co-
immunoprecipitation experiments were performed to isolate HK binding proteins on
epithelial cells, but time constraints prevented further progress. Similarly, experiments to
isolate and identify the epithelial and mesothelial HK-PPK activator were halted due to
time constraints. Affinity chromatograph using protamine sulphate or BK as a ligand may
Figure 7.1 Schematic of the role of the KKS within the lung
HK and PPK, expressed within the lung or acquired via the systemic circulation, assemble
on the surface of respiratory epithelium or pleural mesothelium. PPK is converted to PK
and liberates BK from HK to activate B2R on the same or nearby cell (1). hK1 present in
the fluid phase liberates Lys-BK from cell-absorbed HK (2) and directly activates cell
surface expressed receptors, including PARs (3). Plasma KKS activation on additional cell
types, such as stromal, vascular and infiltrating cells, contributes to the total kinin pool
within the lung (4).
HK
PPK
HK
PK
BK
HK Lys
-BK
hK1 1
2 3
Endothelial cells
Platelets
Monocytes
Macrophages
Neutrophils
Mast cells
Fibroblasts
Smooth muscle cells
4
HK PPK Blood
vessel
Epithelium/
Mesothelium
Table 7.1 Inhibitory profiles of HK-PPK complex activation on
endothelium and respiratory epithelium
% Inhibition
Inhibitor Endothelium * A549 NHBE
500 μM Antipain (100 μM) 100 31 29
500 μM Leupeptin (100 μM) 100 33 24
10 mM EDTA (20 mM) 85 3 4
1 mM 2-ME (5%) 98 19 35
1 mM Benzamidine 0 0 0
10 mM Cysteine 99 63 99
500 μM ANG II (100 μM) 100 8 8
100 μM BK 50-75 95 99
* Values for endothelium were originally reported by Motta et al. (1998), Shariat-
Madar et al. (2002), and Joseph et al. (2002). Values in parentheses indicate the
concentration used for experiments performed with endothelial cells.
Table 7.2 Inhibitory profiles of HK-PPK complex activation on
endothelium and pleural mesothelium
% Inhibition
Inhibitor Endothelium* MeT-5A NCI-H2052 NCI-
H28
500 μM Antipain (100 μM) 100 41 57 2
500 μM Leupeptin (100 μM) 100 41 57 0
10 mM EDTA (20 mM) 85 0.1 0 0
1 mM 2-ME (5%) 98 55 52 41
1 mM Benzamidine 0 0.6 0 0
10 mM Cysteine 99 99 100 98
500 μM ANG II (100 μM) 100 0 0.3 0
100 μM BK 50-75 94 89 77
* Values for endothelium were originally reported by Motta et al. (1998), Shariat-
Madar et al. (2002), and Joseph et al. (2002). Values in parentheses indicate the
concentration used for experiments performed with endothelial cells.
Table 7.3 Inhibitory profiles of HK-PPK complex activation on
endothelium and respiratory epithelial cell, matrix and lysate
% Inhibition
Inhibitor Endothelium * A549 cells A549 matrix A549 lysate
500 μM Antipain (100 μM) 100 31 50 99
500 μM Leupeptin (100 μM) 100 33 47 100
10 mM EDTA (20 mM) 85 3 27 95
1 mM 2-ME (5%) 98 19 35 63
1 mM Benzamidine 0 0 0 73
10 mM Cysteine 99 63 95 90
500 μM ANG II (100 μM) 100 8 5 37
100 μM BK 50-75 95 94 56
* Values for endothelium were originally reported by Motta et al. (1998), Shariat-
Madar et al. (2002), and Joseph et al. (2002). Values in parentheses indicate the
concentration used for experiments performed with endothelial cells.
105
prove useful given their role as substrate inhibitors of HK-PPK complex activation.
Although identification of the activator is yet to be determined, the results obtained from
this thesis suggest that a carboxypeptidase with specificity towards C-terminal arginine
residues may be responsible.
Considering the observed specificity of the activator, the location of a probable activation
site in PPK is problematic. In this regard, PPK activation typically involves cleavage of the
internal Arg371
-Ser372
bond (Hooley et al., 2007). Additionally, PPK does not contain a C-
terminal arginine residue (Chung et al., 1986), suggesting the protease may not be acting as
a carboxypeptidase or demonstrate specificity towards this residue on PPK. Western blot
analysis of PPK activation on epithelium and mesothelium would provide insight into the
specificity of the activator by revealing the pattern of PPK proteolysis (Motta et al., 1998).
In such experiments, FXIIa- and PRCP-activated PPK could be included as a comparison to
determine whether similar regions of PPK are cleaved.
The results obtained showing novobiocin inhibited PK formation on epithelium and
mesothelium support the involvement of HSP90 in plasma KKS activation (Joseph et al.,
2002), but, in that regard, its function is currently unclear. Although HSP90 lacks
proteolytic activity, previous data have demonstrated an affinity towards CTI. In addition,
purified HSP90 catalysed the conversion of PPK to PK in a cell-free system (Joseph et al.,
2002), suggesting the protein directly mediates activation. However, given the well
established chaperone function of HSP90, an indirect role in activation is as likely. For
example, HSP90 on the cell surface may function to stabilise the activation of a membrane-
bound protease capable of activating PPK. In this regard, HSP90 was found to mediate
activation of pro-MMP-2 on the cell surface of HT-1080 fibrosarcoma cells (Eustace et al.,
106
2004). Although HSP90 may function autonomously (Joseph et al., 2002), it typically acts
in concert with a variety of other chaperones or co-chaperone molecules, including HSP70,
p23 and hop (Caplan, 2003, Terasawa et al., 2005). Thus, a scenario can be envisaged, in
which association with a HSP90-co-chaperone complex is required for proper activation of
the HK-PPK complex activator. As such, the co-chaperones p23 and hop (Eustace and Jay,
2004), and the chaperone HSP70 (Shin et al., 2003) are extracellularly expressed by tumour
cells, however, their precise role in this setting remains unclear.
Given the role of kinins in inflammation, this thesis highlights the potential implications of
the plasma KKS as a potential therapeutic target in respiratory and pleural disease. In this
regard, HSP90 inhibition may be suitable considering the availability of inhibitors currently
in use in clinical trials (Neckers, 2003). As such, a compartmentalised approach using cell-
impermeable HSP90 inhibitors may be efficacious given the locality of the plasma KKS
and the necessity to maintain intact intracellular HSP90 activity (Eustace et al., 2004).
Recently, HSP90 inhibition has been shown to impact on various animal models of
experimentally-induced inflammatory disease (Chatterjee et al., 2007, Rice et al., 2008,
Dimitropoulou et al., 2010), and these findings may be explained, in part, by inhibition of
the plasma KKS. For example, a study by Dimitropoulou et al. indicates HSP90 inhibition
could be used to manage asthma severity as ovalbumin-sensitised mice treated with the
HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, demonstrate less
inflammatory changes in the lung including neutrophil infiltration and mucus
hypersecretion. A combinatorial approach may be applied to these studies, in which HSP90
inhibitors are used in concert with B2R antagonists and/or inhibitors of PK or hK1.
107
In summary, the data described in this thesis highlight the importance of the plasma KKS in
the airway and pleural space. Future work should focus of the identification of the HK-PPK
complex activator on epithelial and mesothelial cells. Similarly, the role of HSP90 in this
system requires further examination. Furthermore, the identity of the proteins involved in
binding HK on these cell types should be determined. Finally, the work conducted
throughout this thesis could be extended to include further cell types and applied to animal
models to determine its in vivo significance.
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108
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