Alterations of Polymorphonuclear Neutrophil (PMN)

Recruitment in a Murine Mode1 of Peritonitis and a

Secondary Injury

Daniel E. Swartz, M.D.

L.D. MacLean S urgical Research Laboratories

Division of General Surgery

Department of Surzery

McGill University, Montreal

March, 1999

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment

of the requirements for the degree of Master of Science in Ekperimental Szcrgery

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TABLE OF CONTENTS

Page

Abstract

Resumé

Acknowledgements

List of Abbreviations

Chapter 1 : Review of the Literature

1.1 Overview of the host response to uifection

1.1.1 SIRS and MODS

1.1.2 RoIe of the PMN

1.2 PMN recruitment

l.Zl Selectins

1 -2.2 Chernoattractants

1.2.3 P2 Integrins

1 -2.4 Kinetics of PMN-endothelial ce11 interactions

1.3 PMN transendothelial migration

1.3.1 Morphologie changes of activation, diapedesis and

vascular emigration

1 -3 -2 Chernotactic migration

2 -4 The PMN in host defense

1 -4.1 Phagocytosis of pathogens

vii

1.4.2 Release of degredative enzymes and toxic oxygen

Metabolites 32

1.4.3 Apoptosis 33

1.5 Consequences of altered PMN effector function 34

1 S. 1 Endo thelial ce11 destruction by PMNs 35

1.5.2 PMN recruitment to a remote site in the presence

of infiammation

1.6 Justification of a murine mode1 of secondary peritonitis

and a secondary injury

1.7 Hypotheses

1.8 Objectives

Chapter 2: Materials and Methods

2-1 Anirnals and housing

2.2 Reagants

2.3 Animal procedures

2.3.1 Cecal ligation and puncture

2.3.2 Polyvinyl sponge placement

2.3 -3 Optimal E. coli concentration

2.3 -4 Cremasteric dissection for intravital microscopy

2.4 Measurement of PMN-EC interactions using intravital

microscopy

2-5 Measurement of vesse1 kinetics

2.6

2.7

Chapter 3:

3.1

3 -2

3 -3

PMN isolation and counting

2.6-1 P M . pUnty

S tatistical anafysis

Results 53

Animal mortality 53

PMN regïonalization following cecal ligation and puncture 53

Circulating PMN counts followinp orchitis and cecal

Ligation and puncture 56

3.4 Intravital microscopy 57

3 -4. i Circulatory parameters 57

3-42 Kuietics of PMN and endothefial ce11 interactions 58

Chapter 4: Discussion 66

4.1 PMN defivery to polyvinyl sponge discs 67

4.2 Intravital microsocpic analysis of PMN fluxes, rolling and

firm adhesion 70

4.3 Future directions: a potential role for shed L-selectin 76

4.4 Conclusion 79

Chapter 5: Original Contributions to Knowledge

References

ABSTRACT

Secondary peritonitis is a significant cause of morbidity and mortality in the ICU

and ICU patients as a group have the highest rate of nosocomial infections. Once

recmited to the site of injury, the PMN interacts with endothelial cells (ECs) via rolling

adhesion, fm adhesion, and transendothelial migration. Using a murine cecal ligation

and puncture pentonitis mode1 and either skin or cremaster injury as the secondary site in

a two-front injury modei, we examined the role of injury severity on the triage of PMNs

to competing sites of injury. We demonstrated that a finite pool of PMNs was recruited to

tissues in numbers correlating to the severity major injury. Wiîh intravital microscopy we

demonstrated that numbers of PMNs invoived in rolling adhesion, rolling velocity and

stationary adhesion in the presence of one or more sites of injury were predictable,

consistent and likely mediated by changes in surface adhesion molecule expression.

La péritonite secondaire est une cause importante de morbidité et de mortalité

chez les patients à l'unité des soins intensifs, et le taux des infections nosocmiales est très

élévé chez ces patients. Une fois mobilisé au site de la blessure, le PMN interagit avec les

cellules endothéliales par l'adhérence roulant, l'adhérence ferme ainsi que la migration

transendothéliale. Dans le modèle du système de blessure à fionts chez la souris, le site de

lésion primaire consiste en une ligature caecale et en une péritonite perforée, tandis que le

site de lésion secondaire consiste en une lésion cutanée ou crémastérienne. À l'aide de ce

modèle nous avons évalué le r6le de la gravité de la blessure dans la mobilisation des

PMNs à des sites compétitifs. Nos travaux ont démontré que le nombre des PMNs

mobilisés est proportio~el à la gravité de la blessure. Nos résultats ont montré, à l'aide

de la microscopie intravitale, que le nombre des PMN impliqués dans l'adhérence

roulant, la vitesse roulante ainsi que dans l'adhérence fermement peut être prédit et qu'il

est uniforme en présence d'un ou de plusieurs sites de blessure. De plus, le nombre des

PMNs mobilisés est probablement lié à des changements de l'expression des molécules

adhésives à Ia- surface des cellules.

Many people have been instrumental in guiding me during the formulation of this

thesis which serves as the culmination of my experience in the Surgical Scientist

Program. 1 am particularly grateful to Dr. Nicolas Christou without whose support and

assistance none of this would have been possible. In addition to helping me create the

hypotheses and design the studies, Dr. Christou was there to make sense of results that 1

fomd nonsensical, to redirect me down the path when al1 roads hit a dead end, and to

refocus my thinking each tirne 1 contemp lated strangling the technician out of sheer

hstration.

Dr. Eleanor Minshall has been an endless fountain of encouragement without

whom 1 would still be revising this thesis for years to corne. Her editorial skills, artistic

illustrations and assistance with the many versions of the tables and charts were integral

in creating the polished product. Her patience and support dwing the many nights and

weekends 1 spent writing and rewriting were a tremendous sacrifice for which 1 will

always be gratefùl.

1 am gratefbl to Dr. Andrew Seely, who collaborated with me and assisted on

many of the experiments, for al1 his input and insight whether or not it was solicited.

Long hours and very late nights were spent in good Company operating on animals,

counting neutrophils, and engaging in fierce philosophical and metaphysical debates.

A special mention to some of the others whose involvement and assistance were

irreplaceable to this effort: Ms. Betty Giannias, Dr. Xuwu Chen, Ms. Mary Bouldadakis,

Dr. Felicia Huang and Dr. Teruo Sakamoto.

LIST OF ABBREVIATIONS

APACHE II

Dv

EC

EGF

ELAM- 1

£MLP

G-CSF

GlyCAM- 1

GM-CSF

GMP-140

E V

ICAM

ICU

1gSF

IL-1 p

LAD I

LAM- 1

LECAM

LTB3

MAdCAM- 1

MODS

Acute p hysiology and chronic health evaluation score II

Mean vesse1 diameter

Endo thelia1 cells

Epidermal growth factor @art of the se lech molecule)

Endothelial-leukocyte adhesion molecule-1

N-formyl-methionine-leucine-phenylalanine

Granulocyte colony-stimulahg factor

Glycosylation-dependent cellular adhesion molecule-i

Granulocyte-monocyte colony-stimulating factor

Granulocyte membrane protein- 140

High endothefial venules

Intercellular adhesion molecules

Intensive care unit

Immunoglobulin super family

Interleukin 1 p

Leucocyte adhesion deficiency type 1

Leucocyte adhesion molecule- 1

Leucocyte-endothelia1 ce11 adhesion rnolecule- 1

Leukotriene Bq

Mucosal addressin cellular adhesion molecule-1

Multiple organ dys fünction syndrome

vii

NK

PAF

PECAM

PMA

PMN

PSGL-Z

SCR

SIRS

TNF-a

VCAM-1

VBF

Vmean

Vrbc

VSR

VSS

Natural killer

Platelet activating factor

Platelet-endotheiial cell adhesion molecule

Phorbol myrktate acetate

Polymorphonuclear granulocyte

P-selectin glycoprotein ligand4

Short consensus repeats

Systemic inflarnrnatory response syndrome

Tumor necrosis factor cc

Vascular ce11 adhesion molecule- 1

Venular blood flow

Mean red blood ce11 velocity

Centerline red blood ce11 velocity

Venular shear rate

Venular shear stress

Chapter 1: REVZEW OF THE LITERATURE

1-1 Overview of the host response to infection

A successfid outcome of any injury, including infection, depends on the host's

ability to mount an appropriate immune response. The initial entry of infectious

pathogens into the host's tissues usually results fiom a failure of epithelial bamier

hc t i ons such as the gut mucosa (leading to peritonitis), lungs (leading to bronchitis and

pneumonia), and skin (leading to cutaneous and soft tissue uifections). Once entry into

host tissues has occurred, a complex interplay of cells and molecules of the immune

system attempts to contain the infection and recruit the necessary effector cells to

eradicate the rnicrobial invaders. Although the severity of infection, the type and

resistance of the pathogens and the anatomic location of the injury contribute to the

outcome, multivariate regression studies of patients with peritonitis 1 and animal studies

2 have dernonstrated that the predominant factor in detennining survival is the ability to

rnount an appropnzte immune response.

1.1.1 SIRSandMODS

The systemic inflammatory response is a double-edged sword. While an

appropriate response is essential for a successful outcorne foliowing infectious injury, a

maladaptive and dysregulated response may incite more widespread and darnaging

systemic injury leading to vascular and other tissue injury, the systemic uinarnmatoiy

response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). The latter

entities have corne to light over recent decades as improvements in antimicrobial

treatments and care of cntically ill patients have demonstrated groups of patients who

survive their initial injury only to succomb weeks to months later of MODS.

Infection, SIRS and sepsis are distinct entities d e h e d by the 1992 Consensus

Conference of the American College of Chest Physicians and the Society of Critical Care

Medicine 3. Infection is defied as the documentation of microbial invasion of normally

sterile host tissues. SIRS, the host response to infection, is defined as two or more of the

followùig: temperature higher than 38°C or lower than 36°C; hem rate greater than 90

beats per minute; respiratory rate geater than 20 breaths per minute or artenal carbon

dioxide tension less than 32 mn Hg; and white blood ce11 count greater than 12,000 or

less than 4000 per mm' or greater than 10% bands on differential. This definition is valid

only in the absence of any other cause such as the immediate post-operative state. Sepsis

is defined as the presence of SIRS with docurnented infection.

A sustained immune response giving rise to a widespread inflammatory state

following a significant injury or insult can lead to dire systemic events such as MODS.

This syndrome is defined as a persistent maladaptive and dysregulated state of the

inflammatory and immune systems 4. As the acronym implies, multiple organs can be

involved and it is their progressive reIentIess destruction that is associated with

significant morbidity and morîality in K U patients. Multiple etiologic entities, both

infectious and non-infectious, employ the same mediators and effector moIecules in the

development of MODS. The pathophysio logy of this disease state involves both an

uncontrolled immunomediator response to injury as well as tissue hypoxia 4. Whether

MODS results purely f?om excessive or prolonged cytokinemia, uncontrolled

macrophage and PMN activation, gut epithelial breakdown with resident bacterial and

endotoxin translocation, microcircuiatory changes leading to tissue ischemia, or a

combination of one or more of these factors is still debated- Some authors favor a two-hit

phenomenon in which one of these events serves to "prime" the inflammatory system for

dramatic and excessive activation following a subsequent event 47 5.

1.1.2 Role of the PMN

The PMN is the predominant granulocyte in both the circulation as well as at the

site of acute infection in the initial 48 hours. PMNs are produced by the myeloid cell h e

in the bone rnarrow at a rate of about 101° cells per day, which may be increased by as

much as 10-fold in tirnes of injury 6 , and have a half-life of six to eight hours 7. Once

recruited to the site of injury, the PMN interacts with ECs via the specific regulation and

expression of surface receptors and ligands. The eventual goal is activation of the PMN to

become an effector ceIl capable of extravasating into the tissues and destroying the

foreign micro-organisms. Under normal circurnstances following the successfûl

elirnination of rnicrobial invaders, the PMN undergo es apop tosis, or prograrnmed ce11

death 8.

1.2 P M . recruitment

Initial interaction .s between ECs and PMNs involve dynarnic forces in which

erythrocytes moving rapidly in the center of the vesse1 exert a shear force on the larger

and less deformable PMNs pushing them outward toward the vesse1 walls 9. This

margination is thought to allow the PMN to keep in close proximity to the EC layer and

thus respond to the release of local factors. To maintain these interactions, shear forces in

the direction of blood flow must be sficiently low to permit cell-ce11 communication.

This effect can be reproduced in vitro where rolling adhesion between PMNs and ECs

varies inversely with perfusion pressure Io. The anatomic site where shear forces appear

to most favor PMN-EC interaction and transendothelial migration is the post-capillary

venule 1 1.

Under the appropriate circurnstances, specific receptors and their ligands direct a

sequence of events leading to extravasation by the P m : rolling adhesion, fim adhesion

and PMN activation. These events are mediated by a sequence of families of adhesion

molecules and chernoattractants: selectins, chernoattractants and integrins in a multi-step

mode1 as described by Springer 12. The specific leukocyte-endothelia1 interaction is

postulated to be determined by the combination of individual receptors corn each farnily

analagous to a telephone area code detemrined by the specific combination of three digits

(Figure 1.1).

1-21 Selectins

Rolling adhesion of the PMN along the vascular endothelium, first described by

Wagner in 1839 13, is defined as a low affuiity interaction between the leukocyte and EC

where the hydrodynarnic shear forces of the blood flow exerts a rotational motion of the

leucocyte. This event is mediated by the selectin family of adhesion molecules which

share a comrnon structure of an MIz-terminal calcium-dependent C-type lectin domairi,

an epidermal growth factor (EGF)-like region, several short conserisus

repeats (SCR) and a trammembrane region with a cytoplasmic tail 14. Multiple

molecules with this sequence homology have been identified (Table 1.1).

p Adhesion

Diapedesis

(integrin) Cliemokine

aD ICAM- 1

0 y

Figure 1.1. The sequence of receptor families involved in PMN-EC interactions:

selectins, chemokuies and integrins.

These include endothelid leukocyte adhesion molecule-l (ELAM-1), granulocyte

membrane protein-140 (GMP-140 or PADGEM) and lymphocyte homing receptor

(gp90Me1, Leukocyte Adhesion Molecule -1 (LAM-1) or leukocyte-endothelid ce11

adhesion molecule (LECAM)). As a result of the 1989 consensus conference these have

been reco*gnized as E, P and L-selectin, respectively. The role of the selectins in the acute

idammatory response is to mediate capture (or tethering) and rolling of PMNs to ECs.

Each selectin molecule is capable of recognizing specific carbohydrate ligands by the N-

temiinal lectin domain however the EGF and SCR regions may affect ligand binding 147

15. S tmctural differences between the selectin molecules include the number of SCR

uaits whereby L-selectin has 2, E-selectin has 6 and P-selectin has 10 (Figure 1.2).

Selectin Location PMN Predorninant Ligand Other Ligands

PMNs, most CD 34, PSGL-1 lymphocytes, al1 other leucocytes

ECs, phtelets PSGL- 1

ECs sLeX , unknown

E-selectin (via sLeS), GlyCPuM- 1, MAdCAM- 1

Table 1.1: Selectins involved in PMN-EC interactions (based on Springer 12).

L-selectin is constitutively expressed on al1 circulating leukocytes except a subset

of memory lymphocytes. Ct functions predominantly in interactions of PMNs and ECs in

acutely inflarned tissues as well as lymphocytes and their ligands on penpheral lymphatic

high endothelial venules (HEVs) 16. Upon PMN activation by chernoattractants, the

extracellular domains of L-selectin are rapidly shed from the ce11 surface by proteolytic

cleavage 17. AU three selectin molecules bind three types of carbohydrate moiteies via

the N-terminal lectin domain: sialylated tetrasaccharides (of which sialyl-Lewisx (sLeX)

and its steroisomer, sialyl-Lewisa (sLea) found on epithelial and cancerous cells, are the

protowes), phosphoqdated mono- and polysaccharides and sulfated polysaccharides and

lipids 14 18 19-

C Lectin domain

EGFdomain

8 Concensus repeat

rqY Mucin domain

Immunoglobulin-Iike domain

Figure 1.2. Structure and ce11 of expression of the selectins involved in PMN-EC

interactions.

The ligands to which the selectin receptors bind have not been fully elucidated.

Ligands for lymphocyte L-selectin on HEVs are constitutively expressed 20 and include

CD34 2y 2l, the soluble ligand Glycosylation-dependent Cell-Adhesion Molecule- 1

(GlyCAM-1) 22 and Mucosal Addressin Cell Adhesion Molecule (MAdCAM-1) 23.

Ligands for PMN L-selectin appear not to be constitutively expressed but rather are

upregulated in cytokine-activated endothelium 24. These ligands include CD34 25, P-

selectin Glycoprotein Ligand-l (PSGL-l) on PMNs which allows binding between PMNs

15, and although not uniformly accepted, considerable evidence exists that L-selectin can

bind directly to E-selectin via sLeX 207 26727.

P-seiectin is expressed on both ECs where they are stored in intracellular Wiebel-

Palade bodies and on platelets where they are stored in alpha granules. Upon cellular

stimulation by leukotriene Bq , histamine 28, C5a 29, thrombin, bradykuun or fiee

oxygen radicals, these storage granules are transported to and rapidly fuse with the cell

surface leading to P-selectin expression within minutes. P-selectin expression appears to

be regulated by motifs within its intracytoplasmic tail which can cause rapid

intemalization with either lysosornal degredation or recycling into storage granules 30.

The principal ligand of P-selectin located on the PMN is PSGL- 1 3 1, a mucin-like

disulfide-linked homodimer which can also bind at lower affinity to L-selectin 1% 32 and

E-selectin 33. P-selectin can also bind to sLeX 34.

E-selectin, expressed only on ECs, requires de-novo synthesis following

stimulation of the endothelium with IL1 P, TNF-u and endotoxin. Monocional antibody

studies have deterrnined that it requires approximately three to six hours for E-selectin

expression following cytokine activation 9 3 3 5 3 3G and expression can be completely -

blocked by inhibitors of mRNA transcription (Actinomycin D) or translation

(cyclohexamide) 37. Expression of E-selectin is usually transient with a duration of 10-12

hours in cytokine-stimulated human umbiLical vein endothelid cells (HUVECs) in vitro

and longer in skin and oîher sites 35. Loss of expressed E-selectin occurs b y both

internalization and lysosomal degradation 35 as well as downregulation of DNA

transcription 39. Ligands for E-selectin include HECA-452 on T lymphocytes 14, L-

selectin presenting sLeX on PMNs 2 0 ~ 41 and PSGL-1 339 429 43-

As previously mentioned the role of the selectins with respect to PMN-EC

interactions in inflammation are in the capture and rolling of PMNs along the

endothelium; a prerequisite for stationary adhesion, activation and transendothelial

migration 4. Using soluble r-arbohydrate Ligands or monoclonal antibodies to the selectin

lectin domain, Tu and CO-authors were able to abolish rolling as weLl as finn adhesion of

PMNs on ECs 15. Furthemore, at physiologie shear stresses, PMNs attach and roll along

phospholipid bilayers expressing P-selectin or P-selectin with ICAM-1 (ligand for P2-

integrin, see below), but not ICAM-1 alone 45. Ail three selectins have been shown to

mediate rolling independently both i i z vivo using knockout mice and in vitro using genetic

transfectants or g l a s surfaces coated with a particular selectin or its ligand 9. Expression

of each selectin varies according to the type of injury (ischemia/reperfusion, infection,

trauma), the effector ce11 and organ involved and the temporal relationship to the time

since stimulus for its expression. These compIexities have hindered the development of a

clear understanding of selectin involvement in leukocyte trafficking 14.

In the acute inflammatory response, initia1 tethering and rolling of PMNs is

mediated predominantly through P-selectin while L-selectin-dependent rolling is present

after 30 minutes and E-selectin is not present until after two to four hours. Studies

perforrned with P-selectin knockout mÏce undergoing intravitai microscopy of mesenteric

venules exhibited a marked 1eucocytosis with complete ablation of rolling initially that

retunied to normal levels after 2-4 hours; a duration that the authors postulated to

coincide with E-selectin expression 46. This data was corroborated with monoclonal

antibodies againçt P-selectin in dogs 47 and in mice 28. The role of L-selectin in PMN

recruitment to inflamed endothclium was demonstrated by Ley and CO-authors who

compared r o l h g adhesion of PMNs (L-selectin (+), sLex (+)) with HL-60 promyelocytes

which express sLex but no t L-selectin (L-selectin (-), sLeX (+)) and 3 00.19 cells

transfected with L-selectin (L-selectin (+), sLex (-)) in rat mesenteric venules under

intravital microscopy. The 300.19 c e h showed almost no rolling initially but increased

markedly after 20-30 minutes while the HL-60 cells demonstrated moderate rolling

initially but this was reduced to practicaily zero after 20 to 30 minutes. The wild-type

PMNs revealed consistently greater rolling t h a n the other ceIls throughout the

experiment. This data supports that early rolling at least in part involves sLex, probably

binding to P-selectin, while L-selectin-dependent rolling does not occur until after 20 to

30 minutes 48. In an ex-vivo murine study, L-selectin knockout mice whose cremaster

muscles were exteriorized and examined under intravital microscopy were compared to

normal control mice. The authors demonstrated normal rolling initially followed by up to

a 65% reduction in rolling PMNs after 30 minutes 4? E-selectin does not appear to play a

role in PMN recruitment until2-4 hours after cytokine stimulation and

monoclonal antibodies directed against E-selectin, such as EL-246, had no effect on PMN

tethering and rolling for the fkst three hours 52.

The kinetics of selectin-rnediated PMN rolling is hypothesized by Lawrence and

Splinger to involve labile bond formations between the selectins and their ligands. Upon

dissociation of one bond, the PMN is propelled downstream by the hydrodynamic forces

of the blood in a rotational motion until the ce11 is tethered by the most upstream bond.

The velocity of the rolling PMN is determined by the rates of bond association and

dissociation which are unique to each selectin-ligand pair. Applying pharmacokinetic rate

constants where b, refers to bond association and bff refers to dissociation, the

equilibrium constant, k, kn/ kr), must remain constant in order for rolling to continue

45- Puri and colleagues measured the PMN tethering (capture), rolling velocity and

rolling adhesion strength of al1 three selectins in a laminar flow in vitro assay with either

P-selectin, E-selectin or CD34 (L-selectin ligand) adsorbed to glass slides. Tethering at

physiologie shear stresses (1.5 - 2 dyn/crn2) correlated with receptor site density and was

sirnilar for al1 three selectins but slightly higher on L-selectin followed by P-selectin and

somewhat fewer numbers of PMNs tethered on E-selectin. Rolling velocity at these shear

stresses was eight-fold greater on L-selectin than E- or P-selectin. By incremeotally

increasing the shear stress, rolling adhesion strength was calculated by recording the

shear stress at which 50% of the PMNs remained bound. The rolling velocity was almost

twice as great for L-selectin as E- or P-selectin 53. While both L-selectin and PSGL-1 are

concentrated on the microvilli tips of the PMN plasma membrane 54, L-selectin is

present in two to three-fold greater concentration on the PMN ce11 surface 55. These

hd ings support an increase in L-selectin exposure to its ligand on the EC and may

account for its greater rolling adherence and velocity.

It is unclear what significance is attributed to the faster rolling velocity of L-

selectin-mediated P M ' rolling as opposed to P- or E-selectin, however it is likely that

slower velocities permit greater exPosud of the PMN to chemoattractants on the

endothelial surface leading to increased interactions with the integrin receptors 53.

Because L-selectin is expressed consititutiveiy whereas P- and E-selectin require some

fonn of stimulation for expression, L-selectin may be relegated to a role of systemic

endothelial surface sampling in the non-inflamed state characterized by greater tethering

(ability to sarnple) and faster rolling velocity (greater endothelial suiface area samp led) .

Other studies are needed to fürther delineate the individual roles of the selectins,

1.2.2 Chemoattractants

The second group of rnolecules involved in PMN recmitment in in£laxmnation are

the chemoattractants. They serve as the second of the three-digit area code analogy

advanced by Spnnger 12 (see Figure 1 -2). Chernoattractants refer to a diverse group of

rnolecules that serve several fûrictions: they can activate and alter integrin adhesiveness;

stimulate PMN changes in morphology, actin polymerization and r e sp i r a to~ burst; and

they direct migration of the leukocyte across the endotheiial basement membrane to the

site of injury. Activated leukocytes migrate along a concentration graaient of soluble

chemoattractant molecules which dif ise from their point of production. PMNs have been

demonstrated to respond to concentration differences across 1% of their diameter and

migrate progessively in the direction of higher chemoattractant concentration 56.

Chemotactic stimuli have to date been classified into two groups: classical

chernoattractants and chemokines (chemoattractant cytokines). Classical chemoattractants

have counter-receptors on monocytes and PMNs and include N-formyl peptides, C5a,

leukotriene Bq (LTB4) and platelet-activating factor (PAF), The chernokines have been

recently subdivided into four families: C-X-C, C-C, C and C-X3-C. C-X-C (a)

chemokines, which rnap to chromosome 4, and C-C (P) chemokines, which map to

chromosome 17, were the first two families identified and were described with respect to

whether or not an arnÏno acid, cX", separates the f i t two cysteine residues 12. Very

recently two fiirther goups have been added: C (y) chemokines which map to

chromosome 1 and contain a single cysteine residue 57 and CX3C (6) chemokines which

map to chromosome 16 58 (Table 1.2). C-X-C chemokines act primarily on PMNs as

well as non-hematopoietic cells involved in wound healing and include IL-8, CTAP-III,

gro/MGSA and ENA-78. C-C chemokines interact with monocytes, eosinophils and

lymphocytes and include MCP- 1, MIP-1 a, M W I P, RANTES and 1-3 09. Only the

classical chemoattractants and C-X-C chemokines pertain to PMN recruitrnent in acute

inflammation and hence the other groups will not be considered herein.

Chemoattractant Ce11 or Process of Origin Target CeiI

CIassical chemoattractants N-formyl peptides CSa L m 4 PAF

C-X-C chemokines IL-8

C-C chemokines MCP- 1

Lymp htoac tin

Bacterial protein processing Complement activation Arachidonate metaboIism Phosphatidylcholine rnetabolism

T cell, monocyte, EC, fïbroblast, kentinocyte, chondrocyte, rnesothelial ceil Platelets Fibroblast, melanomas, EC, monocyte Epithelïum

T cell, monocyte, fibroblast, EC, smooth muscle Monocyte, T cell, basophil T ceIl, platelets T cell, rnast ceii

Cytotoxic T celIs, thymocytes

Granulocyte Granuiocyte PMN, monocyte Granulocyte

PIvfN, basophil

PMN, basophil, Fibroblast PMN, melanoma, fibroblast

PMN

Monocyte, basophil

GranuIocyte, T ce11 Monocyte, eosinophil, T ceiI monocyte

T cell, monocyte

Table 1.2: chernoattractants @ased on Springer l2 and Ward 59).

Chemokines, produced by virtually al1 cells involved in inflammation, consist of

approxirnately 7-8 kd proteins which diffuse across the endothelium. Over forty distinct

chemokines have been charactenzed to date 59. Once in the bloodstream, it is thought

that chemoattractants are rapidly diluted and can no longer effect PMN-EC binding.

Administration of IL-8 intradermally in rabbits led to marked extravasation of PiMNs

whereas this did not occur in intravenous administration 60. It is generally accepted that

PMN tethering and rolling enhances ce11 exposure to chemoattractants. Chemokines bind

to a G-protein-coupled receptor on the PMN which transduces the chemokine signal into

integrin adhesiveness 61.

The presence of chemokines have been demonstrated in most inflamed tissues

including the skin, brain, joints, lungs, vessels, kidney and gut as well as virtually al1 cells

involved in the inflammatory response. Stimuli for chemokine secretion are IL-1 P, TNF-

a, IFN-y, interleukin 4 (IL-4) as well as bacterial products such as lipopolysaccharide 62.

The specific chemokine involved is determined by the particular tissue or cell involved,

the type of infiammatory infiltrate and the nature of the invading organisrn. When the EC

is stimulated by pro-idiammatory cytokines, such as TNF-a and IL-10, it produces IL-8

and other chemoattractants which become bound to the luminal surface of the activated

cell.

The role of chemokines in PMN attachent and emigration has been borne out in

many in vitro and in vivo investigations. Using transwell chambers separated by a human

umbilical vein endothelial cell (HUVEC)-lined filter (a common endothelial ce11

monolayer culture grown in vitro), cytokine-stirnulated ECs synthesized and secreted IL-

8 into the basal chamber. PMNs added to the apical charnber were subsequently

identified in the basal charnber. IL-8 added to the apical charnber inhibited PMN

emigration 63. In a rabbit mode1 of ischemia-reperfusion injury to the lung, monocIona1

antibody against IL-8 inhibited PMN emigration into the lung and markedly reduced

tissue injury 64. Oda and CO-authors topically adrninistered the chemoattractants N-

formyl-Met-Leu-Phe ( W P ) and LTB4 to hamster cheek pouch post-capillary venules

and examined the effect on PMN recruitment using intravital microscopy. They noted a

marked increase in adherence peaking within 10 minutes followed by the disappearance

of PMNs fi-om the bloodstream and a reappearance 30 minutes later in the interstitiun 64.

1.2.3 p21ntegrins

PMN rolling on endothelium is characterized by low-affinity selectin-receptor

binding. It is a necessary prerequisite to firm adhesion under conditions of flow as well as

activation and sub s equent emigration uito extravascular tissues 9. Extensive information

on the role of integrins in PMN recruitment has been obtained fkom studying patients

with the congenitally acquired syndrome of Leucocyte Adhesion Deficiency type 1 (LAD

I), where the affected individual is devoid of P2 integrins. Examination of the PMN-EC

interactions in these patients reveals a chronic neutrophilia, normal rolling but absence of

firm adhesion, a marked reduction in PMN emigration and an increased susceptibility to

severe infections despite high circulating PMN counts 65.

The integrin family of receptors and their Iigands mediate stationary, or "çm"

adhesion of the PMN to the EC. Integrins are leukocyte-associated heterodimeric

molecules composed of an a and a P subunit of which the susbset involved in PMN-EC

binding utilize the P2 subuaits and are known as "P2 integrins". There are three P2

integrins: Leucocyte Function-associated Antigen- l (LFA- 1) (aLP2, CD 1 1 dCD la),

Mac-1 (ccMP2, CD 1 WCD 18) and p l5OY% (aXB2, CD 1 WCD 18). CD 1 1 aKD18 is

expressed on lymphocytes, monocytes and PMNs wlule CD 1 1 b/CD 18 and CD I 1 c/CD 1 8

are expressed only on monocytes and PMNs 12 (Table 1.3). LFA-1 and Mac4 are

constitutively expressed on PMNs (and other leucocytes) and when activated by

infiammatory mediators, undergo a rapid conformational change to a high avidity state

allowing stationary (b) adhesion 9.

In tegrins Names Location Ligands

P2 integtrins d B 2 LFA-1, CD 1 1aICD 18 PlMN, monocyte, B and

T cells

CM32 Mac- 1, CD 1 1 b/ CD 18 PMN, monocyte

P150,95, CD 1 lc/CD 18 PMN, monocyte

VLA-4, CD49dCD29 B and T celis, monocyte, neural crest cells, fibroblast, muscle

LPAM-1, CD49dfCD- B and T ceiis

ICAM-1, ICAM-2, ICAM-3 ICAM-1, iC3b, fibrinogen, factor X iC3b, fibrinogen

VCAM- 1, fibronectin

MAdCAM- 1, VCAM- 1, fibronectîn

Table 1.3: Integrins in Leukocyte-Endothelia1 Interactions (fiom Springer 12).

Adhesiveness of integrin molecules is activated and altered by chemoattractants.

With respect to PMN-EC interactions in the inflarnmatory response, fMLP and IL-8

moderate the P2 integrins LFA-1 and Mac-l 66. Yuan and CO-authors demonstrated

increased PMN adhesiveness using intravital microsocpy of porcine coronary venules

under flow conditions by pretreatment of the PMNs with CSa without effect on PMN

rolling 10.

PMN activation by chemoattractants induces shedding of L-selectin and rapid up-

regulation of Mac4 expression and adhesiveness 67. The increased adhesiveness is likely

due to the concentrated clustering of P2 integrins on the PMN surface as demonstrated in

reçponse to phorbol ester stimulation 68. However chernoattractants in the absence or

inhibition of the P2 integrins are insufîïcient to promote firm adherence and emigration of

PMNs. Arfors and CO-authors administered murine monoclonal antibody directed against

CD18 with intradermal injection of fMLP, LTB4, CSa and histamine to rabbits and

examined the tenuissimus muscle under intravitai microscopy. They noted that rolling

was not altered however PMN adherence and emigration in post-capillary venules was

abolished 69.

The ligands for the B2 integrins, located on ECs, belong to the Immwoglobulin

Superfamily (IgSF) of adhesion molecules composed of an immunogiobulin domain of

90- 100 amino acids. They include intercellular adhesion molecule- 1 (ICAM- 1), ICAM-2,

and ICAM-3. Other IgSF members which bind to a4 integrins are involved in mainiy

lymphocyte adhesion and include vascular ce11 adhesion moIecule-1 (VCAM-1) and

mucosal addressin ce11 adhesion molecule (MAdCAM-1). MAdCAM-1 is unique in

lymphocyte binding properties in that it has homology to immunoglobu1in (and thus

binds to integrins) as well as a carbohydrate domain (to bind to L-selectin) 23. While

ICAM-1, ICAM-2 and ICAM-3 can bind to LFA- 1, only ICAM-1 can bkd to Mac-1.

Thus in order to inhibit LFA-1 interactions with the endothelium, ail three ICAMs need

to be bIocked 70.

1.2.4 Kinetics of PMN-endothefial cell interncdions

Ln surnrnary, the three step mode1 for PMN recnritment prior to transendothelial

migration involves the adhesion molecu1e:ligand sequence of selectin-chemoattractant-

integrin as advanced by Springer 12. The selectins (L on the PMN and P and E present on

the endothefium) with their ligands mediate PMN capture (tethering) and rolling along

the endothehm via association and dissociation of l ow-awty bonds. Frorn a

physiological viewpoint this is thought to reduce the shear forces present in venular blood

flow as it passes the lruninal wall to permit adhesion and exposure to chemoattractants.

Chemoattractants, produced and released by the EC in response to IL4 and TNF-cc,

induce activation of P M . with shedding of L-selectin and up-regulation of high-avidity

P2 integrins such as LFA-1 and Mac-1. Leucocyte integrin binding to their respective

cellular adhesion molecule counter-receptors on the EC lead to stationary, or £km,

adhesion of the PMN. Now in its activated form, the neutrophil undergoes ernigration

from the vascular space between endothelid junctions and across the basement

membrane where it will migrate along a chernotactic gradient to the site of rnicrobial

invasion,

1.3 PMN transendothelial mi.gration

Transendothelial migration of the PMN generally requires al1 of the

aforementioned recruitment steps. The evidence for the requirement of selectins has been

demonstrated by studies in which anti-l-seiectin monoclonal antibody 717 72 and

knockout mice deficient in both E and P-selectin 73 are associated with a significant

reduction in ernigration. Likewise the importance of the R2 integrins in transendothelial

migration are supported by its ablation in the presence of anti-CD 18 monoclonal antibody

T4 and anti-ICAM-1 monoclonal antibody 7 5 9 76. Fuaher evidence for the requirernent of

P1 integrins in transendothelial migration cornes ~ o m studies of patients with leucocyte

adhesion deficiency syndrome (LAD) type 1 who are deficient in PMN expression of

LFA-1 and Mac-1. PMNs fiom these patients demonstrate a virtual ablation of vascular

emigration 65. The impairment of emigration following the administration of Pertussis

toxin, which irreversibly inhibits the G-pro tein-coupled receptor, to marnmalian

leukocytes in vitro demonstrates a necessary role for chemoattractants 77.

In vitro studies have also demonstrated that the presence of a chernotactic gradient

involving IL-8 63, PAF 783 79, £MLP or LTB4 807 gL can induce transendothelid

migration. Migration involves dynamic changes of PMN receptor expression. From

studies in which PMN receptor expression was examined following PMN migration in a

skin window preparation 82 or fiom pustules 83, it has been shown that PMN exudation

is associated with up-regdation of fMLP and C3bi receptors as well as £MLP-induced

chernotaxis, hydrogen peroxide and fiee oxygen radical production 87. In addition PMN

transmigration across TNF-a-stimulated endothelium in vitro is dependent on IL-8- TNF-

a-stimulated HUVEC monolayers was associated with three-fold increase in PMN

transmigration and high concentrations of IL-8 in the supernatant compared with

unstirnulated endothelium and CO-incubation with anti-IL-8 monoclonal antibody or

actinomycin D-inhibition of protein synthesis almost completely inhibited transmigration

84- Studies in vitro have demonstrated that LPS from E. coli up-regulates DL-8R

expression on PMNs to peak around 30 minutes before down-regulation to baseline

within 2 hours 85. Changes in IL-8 receptor (IL-8R) expression correlated with

intracellular Ca++ levels and low Ca* levels prevented IL-8R down-regdation 86.

A recent in vivo human study usin% skin windows compared IL-SR and CSaR

expression on PMNs before and after transmigration in ICU patients with SIRS and

healthy controls. The process of transmigration correlated with a reduction in IL,-8R but

not CSaR in both patients and controls. Furtherrnore patients with SIRS had reduced

CSaR but not IL-8R in their circulation. TNF-a levels were increased in the exudate

environment over the circulation and in vitro incubation of PMNs with TNF-a was

associated with a reduction in IL-SR expression suggesting that TNF-a may contribute to

akered PMN IL-8R expression before and after transendothelial migration. This study

provides fiuther evidence for the dynarnic effect of PMN ce11 surface receptor expression

by the process of transendothelial migration. This data also suggests a potential for

different roles for specific chemoattractants such that sorne, such as IL-8, may contribute

to vascular emigration while others, such as C5a, may mediate post-migration chernotaxis

87

1.3.1 MovhoZogic changes of activation. diapedesis and vascular ernigration

The rnorphology of PMN emigration hüs been documented via elecîron

rnicroscopic studies. Initial contact of the EC by the PMN involved rnicrovilli-like

extensions where L-selectin and PSGL-1 are concentrated 54 while the nucleus,

organelles and majority of the cytoplasm rernained in a relatively spherical cellular

envelope. Once contact is made, the PMN acquire a flattened appearance which increases

the apposition between the cells. The mechanisms to account for these changes have not

been well-defhed but are likely mediated by chemoattractants. Pseudopodia are then

extended by the PMN between two adjacent ECs and the cytoplasm and granules

followed by the organelles and finally the nucleus flow into the pseudopodia 88. The

PMN having migrated rapidly across the EC then remains superficial to the basement

membrane in close association with the EC 88 for up to 30 minutes before migration

across this layer is observed 81.

Burns and CO-authors using astrocyte-conditioned HLTVEC monolayers (which

create interendothelid tight junctions as found in the in vivo endothelium 89)

demonstrated that PMN emigration occurs around ti&t junctions at tricellular comers of

ECs 90- Platelet-endothelid cellular adhesion molecule (PECAM, CD3 l), a member of

the irnmmoglobulin superfamily (IgSF) located on monocytes, granulocytes, natural

killer (NK) cells, some T cells and concentrated at tricellular comers of ECs, is required

for emigration across EC junctions- Monoclonal anti-PECAM antibodies administered to

cytokine-stimulated HUVECs prevented Ieukocyte migration across ECs but not PMN

binding to EC junctions gl . PECAM appears to be the sole adhesion molecule to mediate

transendothelial migration and has no other known fûnction and thus, in contrat to the

redundancy of the other adhesion molecules, appears to serve as the final common

mediator of leukocyte recruitment to extravascular tissue 92. This factor has Ied to

theoretically attractive anti-idammatory therapy directed at PECAM 937 94.

1-32 Chernotactic migration

Neutrophil cytokinesis, the process of ce11 migration, has not been fully elucidated

however it is clear that migration is directly irnplicated in the capacity of the PMN to

undergo respiratory burst and oxygen-fiee radical formation 95. The cytoskeleton,

particularly F-actin and microtubules, are intunately involved in this activity 96. 97.

PMNs were previously thought to migrate by cyclical endocytosis, a process in which

cells such as fibroblasts and lymphocytes maneuver. Here, coated pits at various areas of

the plasma membrane containing specific receptors and other proteins, but no lipids, are

internalized by endocytosis and are returned to the membrane at the front of the ce11 98.

This creates a rearward flow of membrane Iipids toward the back of the ce11 and which

directs movement in an anterograde fashion. Lee and CO-authors elegantly refiited this

theory by photobleaching a line ont0 membrane lipids perpendlcular to the axis of ce11

movement and dernonstrating that PMN membrane lipids actually move toward the fiont

of the ce11 during anterograde motion 99.

It is likely that PMNs migate by F-actin filament rearrangement, similar to the

process of neuronal growth, such that portions of the plasma membrane in the front of the

ce11 undergo contraction and internalization. This induces gradients of tension which pull

the ce11 forwards while the internalized portion of membrane are passed to the back of the

ce11 where they fuse with the membrane 96.

1.4 The PMN in host defense

1.4.1 Phagocytosis of pathogens

Following recruitment of the PMN to sites of injury and inflammation, the PMNs

ingest and destro y microbial invaders. P hagocytosis, the process of intracy-top lasmic

ingestion, involves surrounding opsonized microbial pathogens with pseudopodia that

fuse to create the enclosed intracytoplasmic vesicle known as a phagosome 100.

Phagocytosis is mediated by two classes of receptors: immunoglobulin (Fcy) and

cornplement (C3b/C3bi) receptors. The immunoglobulin Fcy receptors, namely FcyRI,

FcyRII and FcyRm, recognize the Fc domain of imrnunoglobulin G (1gG)-opsonized

particles. Cytokine-stimulated and unstimulated PMNs have different mediators of

phagocytosis: stimulated PMNs require protein kinase C activation while unstimulated

PMNs require phospholipase D activation - These are not the only signalling

pathways involving Fcy receptors since fMLP-stimulated PMNs, for instance, use neither

protein kinase C nor phospholipase D activation but rather involve increased

intracytoplasmic calcium ions and iiiositol L,4,5-trisphosphate 102- Complement

receptors for C3bK3bi are alternately involved in the signalling pathway of phagocytosis

and have been previously demonstrated to be up-regulated following transendothelial

migration 82. These interactions induce a signal transduction event involving the PMN

membrane and cytoskeleton which is required for phagocytosis. A variety of molecular

stimuli at infiammatory sites can induce pha~ocytosis which is locally-confined thus

preventing widespread proinflammatory and tissue-destructive processes 103-

1.4.2 ReZeme of degredative en,ymes and toxic O-xygen nzetubolites

Once enplfed by the PMN, the foreign material remains bound within

phagosomes where lysosomal enzymes contained in cytoplasmic granules fuse and

" degranulate" to destroy the pathogens O3 - Primary (azurophilic) granules contain

rnyeolperoxidase and lysosomal enzymes such as cathepsin G and elastase Io4. Many of

these proteases are positvely-charged and thus bind with more aanity to negatively-

charged ce11 membranes and extracellular rnatrix proteins enhancing their destructive

capacity 105. Secondary (specific or peroxidase-negative) granules contain lactoferrin,

lysozyme and other enzymes without myeloperoxidase 106-

The PMN membrane-associated NADPH-induced respiratory burst pathway

represents the other primary mechanism by which PMNs kill bacteria. Activation of this

NADPH oxidase system is associated wîth a respiratory burst in which oxygen

consumption is increased and superoxide anions are generated. Although hydrogen

peroxide is produced by the respiratory burst, rnyeloperoxidase released by azurophilic

granules catalyzes a reaction between hydrogen peroxide and chloride radicals to yield

hypochlorous acid, a powerful oxidant that is considered the predominant agent of PMN

oxygen-fiee radical injury 107-

Althou& the oxidative and non-oxidative mechanisms of microbial killing may

function independently, the destruction of many pathogens rquire both systems

functioning together. Gram-negative bacteria have been show to be resistant to lysozyme

alone but are killed when exposed to both lysozyme and oxidants 108. Turnor ce11 lysis in

vitro has also been demonstrated to require both defensin and oxygen species 109. The

serine protease, elastase, also acts synergistically with oxygen-fiee radicals. Among the

PMN degradative enzymes, elastase is most commonly associated with tissue injury 110

given its ability to hydro lyze many extracellular matrix (elastin, fibronectin and CO llagen

types DI and IV) and plasma proteins (complement proteins and clotting factors) 1 11.

Tissue destruction by elastase is compounded by either the inactivation of plasma anti-

proteinases, such as ai-proteinase inhibitor, or by the presence of hypochlorous acid 1 12.

1.4.3 Apoptosis

Apoptosis, or prograrnmed ce11 death, is the standard by which PMNs are cleared

fkom the site of inflammation. The activated PMN having engulfed and destroyed the

microbial pathogen in normal circurnstaiices will undergo apoptosis and the rernnants

phagoc ytosed by surrounding mononuclear phagocytes 13. The process of apoptosis is a

tightly regulated process in which the ce11 undergoes shrinkage, fragmentation

(karyorrhexis), dissolution (karyolysis), internucleosomal DNA cleavage with

partitioning into membrane-bound apoptotic bodies L4. Apoptosis rates have been

enhanced upon PMN ingestion in vitro of E- coli in an oxygen-dependent rnanner 1 15.

Mediators of the in£larnmatory process, such as C5a, fM3LP, LPS and GM-CSF have been

demonstrated to delay apoptosis rates of PMNs 87 l3 . Seely and CO-authors

demonstrated 50% of transmigrated PMNs remained present d e r 8 hours and were

unresponsive to TNF-a-induced apoptosis when compared to PMNs in circulation l6.

Teleologically the point in time at which apoptosis occurs during the recruitment

and effector activities of the PMN determines which side of the "double-edged sword" is

at work This paradox of PMN function refers to both their essential role in host

defense to eradicate rnicrobial invasion and ensuing infection as well as their capacity to

effectuate host tissue injury, a maladaptive systernic inflamrnatory response and MODS.

Resolution of an inffammatory process relies on the inactivation and clearance of the

extravasated PMNs and their products; a process performed by inflammatory and

monocyte-derived macrophages 1 18 within a period of minutes 19.

1.5 Consequences of altered PMN effector fùnction

The paradox of PMN recruitment lies in its necessity for an individual's suMval

in the face of injury as well as its capacity to eIicit inflamrnatory injury in the host leading

to cellular, tissue and organ destruction (or MODS), the leading cause of death in the K U

120. Chronic uinammatory conditions such as rheurnatoid arthritis 121 and ischemia-

reperfusion injury 1073 122y 123 are also mediated prirnarily by PMNs. There are several

theones regarding the meçhaïiisms of maladaptive PMN responses. Some microbes (e-g.

Candida) rnay be too large to be fully engdfed by the PMN such that phagocytosis rnay

be partial or absent with release of Iysosomal enzymes and oxygen-fi-ee radicals into the

irnmediate vicinity 124- Alternatively, extensive release or backflow of inflammatory

mediators into the circulation rnay induce premature PMN activation and release of

cytotoxic agents by the PMN overwhelm host antioxidant protease inhibitors which lead

to EC injury and destruction 125. Oxidant injury rnay trigger a vicious cycle since

superoxide production also induces fùrther PMN recruitment to the site of inflammation

126-

The host defense mechanisrns against PMN-induced injury include the

antiproteases and antioxidants. When deficient, such as in ai-antitrypsin deficiency in

pulmonary ernphysema 12', or ovenvhelrned, as occurs in a dysregulated inflammatory

response 1 17, hydrolytic tissue injury c m occur. Despite adequate concentrations in the

circulation of most individuals, antiproteases are effectively excluded fiom the sites of

PMN-EC interaction by the dense adherence of these two ceIl types. Antiproteases are

also excluded fiom sites of interstitial inflammation by the high oxidative stress which

destroys the molecules by binding to exposed thiol groups 125. Sorne antiproteases, such

as ai-protease inhibitor and a2-macroglobulin, are inactivated by hydrogen peroxide and

chlorinated oxidants 2 17.

PMN activation and oxidative burst rnay be triggered by the sarne mediator at

different concentrations or two mediators which induce activation rnay act synergistically

to cause premature burst. N-formyl peptides, such as fMLP, induce chernotaxis and

migration when present in the nanomolar range and respiratory burst in the micromolar

range 128- Phorbol esters, such as phorbol myristate acetate (PMA), and fMLP at levels

which induce activation independently may act together to trigger oxidative burst and

release of toxic oxygen species 129.

1.5.1 Endothelial cell destruction by PMNs

Sepsis in ICU patients is invariably associated with a loss of vascular integrity

proportional to the degree of the inflamrnatory response manifesthg as interstitial edema

and hypotension despite extensive volume resuscitation. This endo thelial destruction has

been attributed to Pm-dependent mechanisms since Sacks and CO-authors demonstrated

that activated PMNs in vitro, when incubated with cultured ECs, induced EC lysis which

was inhibited by the addition of catalase and superoxide dismutase l30- Other studies

have supported that proteolytic enzymes induce EC detachment 2 132 while reactive

oxygen species are responsible for EC destruction l33-l35- Agonists of PMN activation

irnplicated in Pm-dependent EC injwy and detachrnent include PIVIA, W P , CSa, PAF

136. Phospholipase C, p hospholipase A2 and streptolysin S are membrane-active agents

which have been demonstrated to enhance susceptibility of ECs to injury by hydrogen

peroxide 137. Finally unstimulated PMNs incubated with cytokine-activated endothelium

can also induce EC detachrnent in vitro, an effect which can be inhibited with the

addition of IL-8 136. This latter effect rnay be due to the stimulatory effect of vascular

emigration by IL-8 which would lessen the duration of contact between PMNs and the

endo theliurn.

Endothelial damage in ischemia-reperfision injury aIso implicates PMN

involvement since hypoxic ECs demonstrate an increased PMN adherence 138 and

activation 139- Anti-CD18 monoclonal antibodies administered to a rabbit hemorrhagic

shock mode1 resulted in impaired PMN-EC adherence, enhanced survival and marked

reduction in end-organ injury 140- PMN activation results in cytoskeletal alterations that

render the celIs less deformable and can lead to microvascular plugging during penods of

ischemia with enhanced exposure to ECs and thus increasing the risk for damage to the

endothelium 107-

Most in vitro studies of PMN-induced EC injury taise place in plasma-fi-ee

systems using a variety of inert balanced salt solutions 135-1389 141. When human

plasma was used for the medium in which EC cytotoxicity was assayed, there was almost

a complete ablation of endothelial cytolysis suggesting the presence of an unidentified

protective factor in plasma 142. Little is known about this hypothesized factor, other than

that it is likely a protein since its effect is decreased following pronase digestion of

denaturation of plasma proteins 143, and fùrther investigations are required to elucidate

its structure and mechanism.

1.5.2 PMN recrzritment to a remote site in thepresence of inflammation

Although granulocytopoiesis is a well-recognized compensatory event in

inflammation, significantly fewer PMNs are delivered to remote sites when an active

inflammatory response to an injury elsewhere is present. Using a skin window technique

82 where plasma-filled polyethylene chambers are applied over denuded skui for eighteen

to twenty hours, Ahmed and CO-authors demonstrated a 72% reduction in PMN delivery

to ICU patients as compared to healthy controls 144. Although it is unclear why

neutrophilic states, such as a significant infection, are associated with reduced PMN

delivery to an idammatory focus, the consequences of this reduced delivery are

apparent,

As fewer PMNs are available at sites remote fkom the point of principal injury,

individuals with one insult may be at greater nsk for subsequent infections and death.

Critically il1 patients with intra-abdominal infections are more susceptible than healthy

controls or non-critically il1 hospital patients to infectious complications such as wound

infections, pneumonia, urinary tract and vascular-catheter-related infections l. In a recent

multi-center evaluation of over 10,000 critically iII patients, there was a 45% rate of ICU-

acquired infections and risk factors for rnortality were identified as pneumonia,

bacteremia and sepsis 145. These cntically il1 patients with sepsis also demonstrate an

increased rate of non-uifectious complications, such as acalculous cholecystitis,

pancreatitis and ARDS, which are recognized as predisposing factors for the development

of MODS 4. The hi& rnortality (50-80% of ICU deaths 4) and lack of an effective

treatment for MODS serves as the impetus for investigating t5e mechanisms of PMN

recruitment in sepsis; how PMNs are recruited to one site over another.

1.6 Justification of a murine mode1 of peritonitis and a secondary injury

Secondary peritonitis is an injury to the pentoneal space, often uifectious in origin

secondary to breakdown of the gut epithelial lining with release of enteric bactena.

Mortality rates in the literature Vary from O to 70% with a recent senes of 239 patients

with severe peritonitis (APACHE II scores greater than 10) demonstrating a 32%

rnortality 1. In animal models, pentonitis has been most reproducible and clinically

analagous following cecal ligation and puncture first reported by Chaudry l46 and

exhaustively used elsewhere 147-150 as well as in studies pertaining to this thesis. In this

procedure, animals are subjected to laparatomy, ligation of the cecum without causing

proximal bowel obstruction, and one or more punctures of the cecum with a needIe.

In the following experiments, we used a murine cecal ligation and puncture

peritonitis mode1 \ . th intra-abdominal infection as the primary site of injury and either

skin or cremaster injury as the secondary site.

1.7 Hypotheses

This aim of this thesis is to investigate the PMN-endothelial interactions in sepsis

with respect to differences of a primary and second site of injury. The following

hypoîheses will be tested.

Hypothesis 1. Prior to a cornpensatory response of increased neutrophil

production by the marrow, a regionalization phenomenon exists such that PMN delivery

to a pnmary site is proportional to the d e p e of injury with fewer cells available to sites

of secondary injury.

Hypothesis 2. Following an event such as secondary peritonitis, the PMN

activation results in decreased PMN adherence to sites remote fiom the prirnary injury.

Hypothesis 3. Following an event such as secondary peritonitis, shedding of L-

selectin results in decreased rolling and adhesion of PMNs at remote sites.

1.8 Objectives

The soal of this thesis is to both quanti@ and gain insight into the mechansims of

PMN delivery to secondary sites of inflammation. In the first part, polyvinyl sponge discs

placed in the abdomen and/or the dorsal subcutaneous tissue of mice served as both

irritants (recruïters ofPMNs) as well as a vehicle for collecting the ceils. In the second

part, intravital microscopy of the cremaster muscle permitted ex vivo analysis of numbers

of rolling and stationary PMNs as well as rolling velocity. Using the cremaster muscIe as

a point of reference, three scenarios of PMN-EC interactions were studied: a site remote

fiom a prirnary injury (pentonitis), a local primary injury (orchitis) and a local secondary

injury in the presence of a primary injury (orchitis plus peritonitis). Unlike previous

studies which use in vitro conditions with transfected cells, monoclonal antibodies and

flow chambers of adhesion molecule-coated polyrners, the methodo l o g in this thesis

parailels clinical conditions of peritonitis and secondary infections which involve

multiple adhesion molecules and idammatory mediators in an unrnodified fashion.

Chapter 2: MATERIALS AND METHODS

2.1 m a l s and housing

The protocols for these studies were approved by the McGill University Animal

Care Cornmittee. CD1 male mice (25-35 gram; Charles River, St. Constant, Quebec)

were used &er a 3-5 day acclimitization period to the Royal Victoria Hospital Animal

Facility. Al1 procedures, care and housing took pIace in this facility and conformed to the

Canadian Coucil on Animal Care.

2.2 Reagants

Dulbecco's Phosphate Buffered Saline

Ficoll-Paque (research grade)

Heparin (Heapalean;

1000 USP unitshl)

Iso fluorane

Ketamine (Ketaset)

Phorbol 12-Myrisatate 13-Acetate

Turk's stain

Xylazine (Rompurn)

Gibco, Grand Island, NY

Pharmacia, Uppsala, Sweden

Organon Teknika, Toronto, ON

Ayerst Laboratories, Montreal, QC

Sigma Chernicals, Oakville, ON

O.Olg Gentian violet, 3% Glacial acetic acid

Bayer hc., Etobicoke, ON

Bicarbonate-buffered saline was prepared as follows: NaCI, 13 1.9 rnmol/L; NaHC03, 20

mmoI/L; KC1,1.7 mmoYL; MgC12, 1.2 mmol/L.

2.3 Animal procedures

2.3-1 Cecal ligation and puncttrre

Anesthesia was administered by isoflouorane induction and nose-cone ventilation

with 2-4% isofluorane titrated to optimal anesthesia and analgesia. The abdomen was

shaved with electric clippers and the skin prepped with 1% proviodine solution. A 1 cm

rnidline Iaparotomy incision was made and the cecum was carefully delivered into the

operative field. Peritonitis was created in CLP mice according to the technique of

Chaudry et al 146 in which stool was gently milked into the cecum prior to Ligahon with

3-0 silk proximal to the iliocecal valve so as not to cause intestinal obstruction. Two

punctures with a 21 guage needle were made I cm apart and manual compression

extnided feces fkom the puncture sites. The cecum was replaced into the peritoneum and

the abdomen was closed in two layers withr 3-0 Dermalon suture: a continuous layer for

the abdominal fascia and intempted for the skin. The anirnals were allowed to awake

under heating lamps and received buprenorphene analgesia (0.05-0.1 mgkg

subcutaneously) immediately post-operatively and then every eight to tweIve hours as

needed.

2.3.2 Polyvinyl sponge placement

Polyvinyl sponge discs (Ni-PACT, Eudora, Kansas) 5rnm in diameter and 3mrn

thick were prepared according to the technique of Brozna and Ward 151. After washing

in tap water for 90 minutes, they were soaked in distilled water at room temperature for

30 minutes and then boiled in distilled water for an additional 30 minutes. The sponges

were stored in sterile distilfed water until use.

Four groups of 20 mice were assigned as follows: CLP (cecal ligation and

puncture), CM (cecal manipulation), SP (sponge placement) and CON (control) (Table

2.1).

Table 2.1: PMN Regionalization Study Protocol

Group CLP CM SP CON

L-O laparotomy L- CLP X

t=6h laparotomy and abdominal sponge insertion X

t=6h back incision and sponge insertion X

t=3Oh sacrifice and sponge removal X

Table 2.1: Animal were assigned to the following groups: CLP (cecal Sgation and

puncture), CM (cecal manipulation or, sham laparotomy), SP (sponge placement only),

CON (control with sponges placed in back oniy).

At time = O, CLP mice undenvent cecal ligation and puncture as previously

descrïbed. CM mice had their cecums replaced into the abdomen without ligatioo and

puncture and the laparotomy closed in two layers with 3-0 Dermalon suture. Peritonitis

was created in CLP mice according to the technique of Chaudry et aï 146 in which stool

was gently rnilked into the cecum prior to Iigation with 3-0 s i k proximal tu the iliocecal

valve so as not to cause intestinal obstruction- Two punctures with a 21 guage needle

were made 1 c m apart and manual compression extnided feces f?om the puncture sites.

The cecum was replaced into the peritoneum and the abdomen was closed as previousIy

mentioned. The animds were allowed to awake under heating Iamps and received

buprenorphene analgesia (0.05-0- 1 mgkg subcutaneo us ly) imrnediately post-operatively

and then every eight to twelve hours as needed.

Six hours foiIowing the initial laparotomy, ( t h e = 6h) the CLP and CM groups

were re-anesthetized and prepped in the same fashion and their laparotomy wounds

reopened. The SP group also undenvent laparotomy at this time. The CLP mice had their

necrotic cecums resected and al1 three groups had their peritoneal cavities imgated with

30cc of w m e d 0.9% saline. One polyvinyl sponge was placed in each of the four

abdominal quadrants in each group and the Iaparotomy wounds were closed in the same

manner. AI1 anirnals were then placed in a prone position, the rnidline dorsum was shaved

and prepped and a 1-5 cm transverse incision was made through the skin and dennis- Four

subcutaneous pockets were created in each animal by blunt dissection. One sponge was

placed in each of the four pockets. The dorsal incision was closed as a single layer with

3-0 nylon intermpted suture. Each animal received 3.5 ml of warm 0.9% saline and

buprenorphene 0.05 mg/kg subcutaneously, and gentamycin 3mgkg and metronidazole

7.5 mgkg intrarnuscularly. The CON group underwent placement of dorsal sponges only.

Twenty-four hours after sponge placement, the animals were sacrificed using a

CO2 chamber. The sponges were carefully rernoved, placed in 5 ml syringes and the fluid

was squeezed into pre-weighed test tubes. The tubes were weighed with the fluÏd and

then 2 ml of s tede 0.9% saline were drawn through the syringe bearing sponges and the

fluid was expressed into the test-tube. This was repeated twice for a total of 6 cc. Each

tube was centrifuged for 8 minutes at 1500 rpm. The supernatant was discarded and the

pellet reconstituted with 300 pl of PBS of which 50 pl were added to 450 pl of Turks

stain. PMNs were counted with a hemocytometer and light microscope.

23 .3 Optimal E-coli concentration

To determine the optimal concentration of E. coli to create a local inflarnmatory

response in the cremaster muscle sheath for intravital microscopy, serial dilutions o f 1 o',

10~, 1 o7 and 10%rganisms in l5Opl of sterile 0.9% saline were generated using E. coli

NTCBOO 1. These organisms were graciously provided by the Department of

Microbiology and Immunology at McGill University and the material was handled using

aseptic technique and standard laboratory precautions. Each concentration was

administered to three mice by intracremasteric injection and the rnice were observed for

18 hours. Animals receiving 1 o8 and 1 o9 oorganisms becarne rnarkedly systernically toxic

and succumbed upon induction with anesthesia. Anirnals receiwig 107 and 10' organisms

appeared moderately il1 but tolerated the anesthesia well. Upon cremasteric dissection,

anirnals which had received 107 organisms demonstrated both gross and microscopie

inflarnmatory changes while those with 106 organisms had no visible evidence of such

changes. Thus 1 o7 organisms in 150 pl 0.9% saline was selected as the optimal

concentration for use in this study.

2.3.4 Cremastenc dissection for intravital rnicrascopy

Animals were subjected initially to intraperitoneal anesthesia with ketamine (200

mgkg) and xylazine (10 mgkg). Under a heat lamp, a longitudinal midline neck incision

was made and the right interna1 jugula vein was cannulated, During the course of the

expebent , ketamine and xylazine were administered intravenously in 50-1 00 pl boluses

titrated to anesthesia. Cremasteric muscle dissection for intravital microscopy was

perfomied according to the technique of Granger 9. In the supine position with the

assistance of a dissection light microscope (Nikon SMZ-IB Stereoscopic Dissecting

Microscope, Nikon Canada Inc., Montreal, QC), a 3 mm transverse skin snip of the

scrotum was made and the cremasteric sheath was exteriorized and cleared of its fascia1

attachments. The muscle was splayed open by an anterior longitudinal incision with

electrocautery and secured by a five-point suture fixation at the penphery. The tissue was

kept moist with warmed bicarbonate buffer pefision (Peristaltic Microperfusion Purnp,

Instecli Laboratories hc., Plymouth Meeting, PA). The testis, epididymus and vas

deferens were gently reduced into the abdominal cavity. The animal was then turned

prone and resecwed ont0 the intravital microsopy specimen board and placed ont0 the

microscope under a heat larnp.

2.4 Measurement of PMN-EC interactions using intravital microscopy

Mice were assigned to three groups: CLP (peritonitis via cecal ligation and puncture),

ORC (orchitis via E. coli injection into the crernaster muscle sheath), O+C (orchitis +

cecd ligation and puncture) or CON (control: neither peritonitis nor orchitis). At time =

0, animals assigned to CLP underwent creation of peritonitis as descnbed in section 2.3.1.

ORC mice underwent anaesthesia and 3 mm transverse skin snip of the scrotum. The

crernaster muscle was extenorïzed and injected with 107 E. coZi cells in 100 pl of 0.9%

saline. The muscle was replaced in the scrotum and the skin closed with a single 3-0

Dermalon suture. Animals in the O+C group underwent both cecal ligation and puncture

as well as E. coli orchtitis at the same time.

Eighteen to 24 hours after creation of peritonitis or orchitis, animals underwent

cremasteric muscle dissection for intravital rnicroscopy as described in section 2.3.4. The

entire cremaster was visually scanned under light microscopy (Nikon Eclipse TE 2000

Inverted Microscope with Epifluorescence, Nikon Canada Inc., PvIontreaI, QC) for

adequate visualization of blood flow in post-capillary veinules. A straight, unbranched

segment approximately 150 pm in Iength and 30-50 prn in diameter was located and

centered. The animal was observed via video rnicroscopy (Cohu 49 15-20 20 CCD

Monochrome Video Camera, Scion Corporation, Fredenck, MD) for 20 minutes to aliow

PMN kinetics to return to baseline values pnor to taking measurement. The animal was

excluded if centerline red blood ce11 velociv, measured continuously by Optical Doppler

Velocimeter (Microcirculation Research Institute, College Station, TX), fell below 3

rnm/second. Vessels were also excluded if mean velocity was greater than 6 mrn/second

at which shear stresses would significantly impact PMN-EC interactions. Vesse1 blood

flow was then recorded for ten minutes by video cassette recorder (RCA Video Cassette

Recorder VR4564, Thomson Consumer Electronics, Indianapolis, IN) with a time-date

generator (Panasonic WJ-8 10, Secaucus, NJ). One to 3 venules per animal were recorded

depending upon adequate vesse1 quality. At the end of the data acquisition, the rnice were

sacnficed in a COz chamber and then underwent percutaneous cardiac punchire and the

blood was stored in a heparinized tube on ice for PMN counting.

2.5 Measurement of vessel kinetics

Off-he video playback analysis of the recorded PMN-EC interactions were

performed using a stage micrometer to calibrate on-screen measurements of vessel

diameter and length. Centerline red bIood ce11 velocity (Vrbc) was deterrnined as

previously mentioned using an Opticai Doppler Velocimeter with the sensors placed in

the center of the vessel. Mean red cell velocity (Vmean) was determined by the formula

Vrbdl.6 and venular blood flow W F ) cdculated by Vmean*cross-sectional area

assuming cylindrical geometry. Venular shear rate (VSR) was calculated by 8*Vmean/Dv

(vessel diameter) and venular wall shear stress by VSRçq where tl is 0.25 poise, the

viscosity of blood (Table 2.2).

PMN rolling fluxes were calculated by averaghg the nurnber of PMNs which

crossed a h e drawn perpendicular to the axis of the vessel per minute over two minutes.

Rolling PMNs were defined by those cells which moved at a constant rate slower than

that of the red blood cells. PMN rolling velocity was calculated by timing twenty PMNs

which moved at a constant rate over a distance of 100 pm and reporting the average

velocity in pn per second. Adherent PMNs were defined as PMNs that become

stationary for at least 30 seconds in a 100 p m segrnent over 5 minutes.

Table 2.2: Formulae for the calculation of vesse1 kinetics

Parameter Formula

VSR

rneasured directly fiom the Optical Doppler Velocimeter

Table 2.2: centerline red ce11 velocity (Vrb,), mean red ce11 velocity Or,,,,), venular blood

flow (VBF), vesse1 diameter (Dv), venular shear rate (VSR), venular wall shear stress

(VWSS) and viscosity (q).

2.6 PMN isolation and counting

Al1 mice undergoing intravital rnicroscopy underwent cardiac puncture and blood

withdrawal after they were sacrificed. The volume of the blood was recorded and the

blood gently layered over 3 ml of Ficoll-Paque and centrifuged (Centra-8R Refi-igerated

Centrifuge, International Equipement Co., Needham, MA) for 25 minutes at 400g (1600

rpm). The supernatant was discarded and the pellet was resusbended in 6 ml of sterile

water for 10 seconds to induce erythrocyte lysis after which the osrnolarity was rapidly

restored with the addition o f 2 ml of 3.6% sodium chloride. M e r centrifugation for 5

minutes at 400g, the supernatant was again discarded and the pellet resuspended in 8 ml

of phosphate buffered saline (FBS). After a final centrifigation at 4003 for 5 minutes, the

pellet was resuspended in 100 pl of PBS and the PMNs were counted as previously

descnied.

2.6- I Pniinpuri~,

PMNs were isolated fi-om the blood using a standardized technique described in

the previous section. Purity of the PMNs were assessed in six rnice; three controls and

three with pentonitis by isolation, smear and hematoxylin and eosin staining. The

differential was counted in three hi&-power fields per slide and PMNs were reported as

botti raw number and percentage of total leucocytes. Figure 2.1 dernonstrates a

differential smear of animals with pentonitis and figure 2.2 depicts that of controls. The

number and percent purity of PMNs was 94% for controls and 68% for animals with

pentonitis (CLP).

2.7 Statistical analysis

Al1 analyses of data were performed using Systat 8.0 (SPSS, Chicago, IL). Means

and standard errors of two groups were compared by Student's t-test. Analysis of

variance was used for cornparisons of three or more groups of data with Bonferroni

correction for multiple comparisons. A p value c 0.05 was considered statistically

sigmtlcant.

Figure 2.1. Giemsa-stained smear of PMNs following isolation fiom whole blood fiom a

control animal. PMNs have a characteristic multi-lobed nucleus while lymphocytes have

a prominently stained, large unilobed nucleus.

Figure 2.2. Giernsa-stained smear of PMNs following isolation fiom whole blood fiom a

cecal ligation and puncture (CLP) animal. PMNs have a characteristic multti-lobed

nucleus while lymphocytes have a prominently stained, large unilobed nucleus.

Chapter 3: RESULTS

3.1 A n b a l mortality

Animals which did not survive the penoperative period were excluded fiom the

study and subsequent animals were added to the study until adequate goup size was

achieved. Approximately 170 animals excluding mortalities were used in these studies,

80 in the assessrnent of PMN regionalization and 90 in the intravital microscopy

experïment, ~Mortality rates were on the order of 10%.

3.2 PMN regionalization between peritoneal and skin in jury

The results of the PMNs recovered ffom abdominally placed sponges are

demonstrated in Figure 3. la. In the abdomen, both the mice receiving the cecal Ligation

and puncture and cecal manipulation had significantly greater numbers of PMNs

recovered fiom the sponges wlien compared to the control animals.

CLP CM S P

Figure 3.la. Recruitment of P M . to the peritoneal cavity (x 105) in mice undergoing

cecal ligation and puncture (CLP), mice with cecal manipuIation (CM) and mice

undergoing sponge placement alone (SP). Data are represented as rnedian (horizontal

h e ) , 25 and 75% (box) and 10 and 90% (error bars) confidence intervals with outlying

points. PMN exudation in mice with either CLP or CM were sipnificant-y greater than

those mice undergoing sponge placement alone.

In the sponges placed in the dorsal subcutaneous tissue, there were the greatest

numbers of PMNs recovered in the control group which undenvent no abdominal

intervention (Figure 3.1 b).

CLP CM SP Control

Figure 3.1 b. Recruitment of PMN to the dorsal skin (x 10') in mice undergoing cecal

ligation and punct-are (CLP), rnice with cecal manipulation (CM) and mice undergoing

sponge placement alone (SP). Data are represented as median (horizontal line), 25 and

75% (box) and 10 and 90% (error bars) confidence intervals with outlying points. Mice

with CLP were significantly different fiom those undergoing sharn laparotomy (CM), SP

or contro 1s.

3.3 Circulating PMN counts folIowing orchitis and cecal ligation and puncture

The numbers of circulating PMNs arnong the intravital microscopy study groups

are demonstrated in Figure 3 -2.

Control CLP Orchitis CLP + Orchitis

Figure 3.2. Circulating PMN counts (1 03/ml) in control mice, mice undergoing cecal

ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and

orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 1 0 and

90% (error bars) confidence intervals with outlying points. CLP mice with or without

concomitant orctiitis were significantly different fi-om both orchitis alone and controls.

Mice with orchitis alone were also si,onificantly different kom those with concomitant

CLP.

Within the blood, there was no signincant difference in the numbers of circulating

PMNs between the control group and those mice which undenver~t E. di-induced

cremasteric muscle infection (orchitis). In contrast, when compared to the control mice,

there was a significant reduction in the nurnbers of circulating PMNs in the animds

which undenvent cecal ligation and puncture. In mice receiving both cecal ligation and

puncture and a cremasteric muscle infection, thire was no M e r reduction in the

numbers of circulating PMNs compared to cecal ligation and puncture alone.

3 -4 fntravital microscopy

3.4.1 Circulatory parameters

The circulatory parameters for the groups of mice in the intravital microscopy

study are shown in Table 3.1. Mean vesse1 diarneter and venular blood flow did not differ

signifrcantly between the four groups of anirnals. However mean red blood ce11 velocity

was significantly reduced fiorn 2.9 to 2.3 mm/sec in those mice undergoing cecal ligation

and puncture whether or not this was accompanied by cremasteric muscle id5ection.

Fwthermore, venular wall shear stress was si,onificantly reduced in those animais which

undenvent orchtitis compared to controls and this was observed both in the presence and

absence of cecal ligation and puncture.

Table 3.1. Baseline circulatory parameters (expressed as mean i- SEM) in mice

undergoing cremaster muscle infection (orchitis, ORC), cecal ligation and puncture

(CLP) or both (ORC + CLP). * p < 0.05 compared to control mice (CON).

Group n Mean rbc Vesse1 Venular Venutar wall velocity diameter blood flow s hear stress ( d s e c ) ( ~ m ) (1 0' ml/sec) ( 1 o5 dyn/cm2)

Control 24 2.9 k 0.1 35.9 t 1.2 29.6 + 2-1 16-8 t 1.1

ORC 18 2.6 t 0.1 38.7 t 1.3 31.1 -t- 2-0 13.9 t 0.7*

CLP 21 2,S-tO-l* 35.1-i-1.1 24.3t1.9 14.4 t 0.8

ORC+CLP 10 2,3+0.1* 38.7I1.7 16.6t2.0 12.0 +_ O.L*

3 - 4 2 Kinetics of PMN-endothelid ceZZ interactions

The results for PMN rolling flux, which was defïned as the number of PMNs

rolling past a stationary point per minute, are represented in Figure 3.3a Alth~ugh the

greatest PMN rolling flux was observed in the control group of mice, these values did not

sipificantly differ from those animals undergoins cremasteric E. coli infection. In

contrast, the mice which undenvent cecal ligation and puncture showed a significant

reduction in rate of PMN rollin,o compared to the control mice. Moreover, this rolling rate

was M e r reduced in those animals which had a concomitant muscle infection.

20

O

Control C LP Orchitis CLP + Orchitis

Figure 3.3a. Rolling PMN flux (ceilshin) in control mice, mice undergoing cecd

ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and

orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 10 and

90% (error bars) confidence intervals with outlying points. CLP mice with or without

concomitant orchitis were significantly different fiom both orchitis alone and controls.

Mice with orchitis alone were also significantly different from those with concomitant

CLP.

Since circulating PMN concentrations was demonstrated to be different among

the groups, this rnight serve as a confounding variable in the determination of rolling

flux. In order to account for any effect of PMN concentration, an adjustment factor for

each data point was created by the ratio of circulahng PMN concentration to group mean

PMN circulating concentration. The adjusted rolling count was determined by the product

ofthe rolling nurnber and the adjustment factor (Figure 3 -3b).

Con trol CLP Orchitis CLP + Orchitis

Figure 3.3 b. Adjusted roliing flux in which values are adjusted for circulahng PMN

concentration in control mice, mice undergoing cecai Ligation and puncture (CLP), mice

with orchitis and mice undergoing both CLP and orchitis. Data are represented as rnedian

(horizontal line), 25 and 75% (box) and 10 and 90% (error bars) confidence intervals with

outlying points. CLP rnice with or without concomitant orchitis were significantly

diifferent fi-orn both orchitis alone and controls. Mice with orchitis alone were also

sigdicantly different from those with concomitant CLP.

Adjushg the rolling numbers of PMNs for the effect of circulating PMN

concentration did not signihcantly alter the results.

Mean PMN rolling velocities for the four groups of rnice are represented in Figure

L I

Controi CLP Orchitis CLP + Orchitis

Figure 3.4. Rolling PMN velocity (prnkec) in control mice, mice undergoing cecal

Ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and

orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 10 and

90% (error bars) confidence intervals with outlying points. Orchitis mice with or without

concomitant CLP were significantly different f?om controls. Mice with CLP alone were

also significantly different fkom those with concomitant orchitis.

The highest rolling velocity was observed in the control group of mice, although

this did not significantly differ f o m the animals undergoins cecal ligation and puncture.

There was however a significant reduction in rolling velocity in those mice with orchitis.

Animals which underwent cremasteric muscle infection with E, coZi and had a

concomitant cecal ligation and puncture were not sigdicantly different fiom those with

cremasteric muscle infection alone, however they did have a reduced rolling velocity

compared to the cecal ligation and puncture group.

The results of PMN adherence in the four groups of mice, defined as the number

of cells which becarne adherent to a 100 Fm section of the venule endothelium over a 5

minute period, are demonstrated in Figure 3.5. in the group of r i c e with cremasteric

muscle infection (orchitis), there was a significant increase in the numbers of adherent

PMNs compared to the control animals.

Control CLP Orchitis CLP + Orchitis

Figure 3.5a. PMN adherence (cells) in control rnice, mice undergoing cecal ligation and

puncture (CLP), mice with orchitis and mice undergoing both CLP and orchitis. Data are

represented as median @orizontal line), 25 and 75% (box) and 10 and 90% (error bars)

confidence intervals with outlying points. Orchitis rnice were significantly different fiom

those with concomitant CLP or controls,

There was no difference in the nurnbers of PMNs adhering to the venular

endothelium in the anirnals which had cecal ligation and puncture when compared to

control @ > 0.05). Those mice which underwent orchitis with concomitant cecal ligation

and puncture had significantly fewer adherent PMNs than the orchitis alone group @ c

0.001). However the concomitant cecal Ligation and puncture with orchitits rnice were not

significantly different in their numbers of adherent PMNs to the cecal ligation and

puncture alone group (p > 0.05).

In order to account for any effect of PMN concentration on adherence, the same

adjustment factor was used to calculate the adjusted adherence count. This value was

detennined by the product of the adherent PMNs and the adjustment factor (Figure 3.5b).

Control CLP Orchitis CLP + Orchitis

Figure 3.5b. Adjusted PMN adherence (cells) in which values were adjusted for

circulating PMN concentration in control mice, mice undergoing cecal ligation and

puncture (CLP), mice with orchitis and mice undergoing both CLP and orchitis. Data are

represented as median (horizontal line), 25 and 75% (box) and 10 and 90% (error bars)

confidence intervals with outlying points. Orchitis mice were significantly different fiom

those with concomitant CLP or controls.

Adjusting the adherence data for the effect of circulating PMN concentration did

not significantly alter the results.

Chapter 4: DISCUSSION

These studies were designed to examine the local recruitment of neutrophils in a

murine mode1 of pentonitis and secondary injury. PMNs delivered to polyvinyl sponge

discs in the extravascular space were studied in a manner encompassing the sequential

interactions with the endothelium of capture, rolling adhesion, £hm adhesion and

transendothelial migration. The resdts demonstrated a regionalization of neutroptiils to

the prïmary site of inflammation with a concomitant reduction in neutrophil recruitrnent

to site of secondary inflammation. With the aid of the intravital microscope, the

component parts of PMN-EC interactions prior to vascular emigration were examined.

These included capture (ro lling adhesion), ro lling velocity and adherence of PMNs.

Using the post-capillary venules of the cremaster muscle as a reference point, the

interactions between PMNs and ECs in each of the following four perspectives were

investigated: at the site of injury, at a point remote fiom the site of injury, in the absence

of any injury and at the site of a secondary injury in the face of a severe primary injury.

We demonstrated that at the site of injury, both PMN rolling and firm and adherence

were greatest and P M ? velocity was reduced corresponding to enhanced recruitment.

Conversely, at a site remote fiom significant injury, fewer numbers of rolling and

adherent PMNs were present but they rolled at a higher velocity likely corresponding to

an enhanced delivery of PMNs to a distant site of injury. At a site of an injury of Iesser

severity when a significant primary injury is present distantly, PMN rolling and fim

adherence rates were the lowest and the PMNs that were present rolled at the slowest

rates. This irnplies a triage of PMNs away fiom the secondary injury for enhanced

delivery to a distant site. Together these hdings suggest important implications

regarding PMN recruitment to injury and raise M e r questions about m e c h ~ s m s of

recruitment.

4.1 PMN delivery to polyvinyl sponge discs

In d l animais regardless of the site or degree of injury, PMNs were recruited

primarily to the peritoneum, the site of major injury. The degree of recmitment at the

prïmary site of injury corresponded to the extent of injury. A simultaneous secondary

injury to the skin of lesser severity yielded fewer PMNs to an extent inversely

proportional to the degree of injury to the primary site. These results were corroborated

by circulating PMN counts in mice with cecal ligation and punctwe-induced primary

injury and E.coli-induced cremaster muscle uifection as a secondary injury. Significantly

fewer PMNs were circulating in anirnals with peritonitis compared with cremaster muscle

infection or controls, and even fewer were counted in the presence of both injuries,

however the latter value was not statistically significant,

A consistent neutropenia was observed in mice in response to infectious injury

while similar infections in human patients dcmonstrate a consistent neutrophilia.

Furthemore, other investigators have reported a hypothermie response in rnice

undergoing cecal ligation and puncture while patients with peritonitis generally manifest

a fever. These differences either relate to either the rodent mode1 itself which may

manifest a different sephc response than that found in hurnans or that the cecal ligation

and puncture provides an extrernely severe injury which ovenvhelms the immune system

in the acute period. Our data supports the latter hypothesis since al1 measurements were

taken within 24 hours of this injury after which the animals were sacrificed since

preliminary data revealed that virtually al1 animals succurnbed in the following 12-24

hours. The presence of a neutrophilia implies that an adaptive compensatory myeloid

response to injury has occurred- Further studies are warranted to determine if a

compensatory neutrophilia occurs following a less severe injury.

Recruitment of PMNs to the sponge discs occurred in mice whose only injury was

the subcutaneous irnp lantation of the po lyvinyl sponge discs alone. The mechanisms

responsible for the induction of PMN recruitment following sponge placement have been

previouçly described elsewhere 151. It is clear that the polyvinyl sponge discç are more

than passive vehicles for the collection of PMNs since the cells rnust undergo al1 the steps

of recruitment including vascular exudation before they contact the sponges. Upon

sponge removal, there was never any evidence of bleeding or sanpinous fluid suggesting

vascular disruption tlius PMN exudation is a prerequisite to collection by the sponge. The

polyvuiyl mesh serves as an irritant leading to a consistent, reproducible recruitment of

PMNs sirnilarly documented 52.

With respect to the intraperitoneally-placed sponges there was no significant

difference between nurnbers of PMNs recruited by sponges placed in cecal ligation and

puncture anirnals or those with cecal manipulation. This suggests that either the creation

of peritonitis adds little to the degree of injury caused by sharn lapamtomy with

subsequent placement of sponges or that a maximum PMN response was achieved by the

lesser injury which has depleted a finite reserve of PMNs. Our data supports the latter

(Le. hypothesis #1) since exudated PMNs collected Gom the sponges placed in both the

abdomen and dorsal subcutaneurn demonstrate a trend of sequential increase of PMNs

delivered to the primary site of injury (the abdomen) depending on the degree of injury

with a concomitant reduction of PMNs dehered to the sponges placed at a secondary site

of injury. Because the cecal ligation and puncture arimals dernonstrated signincantly

fewer PMNs in the sponges in the back than the sharn laparatomy group (cecal

manipulation), it appears that peritonitis does add a significant "hit" above that of a sham

laparatomy. These data support hypothesis #1 in that they sugsest the likelihood for a

relatively finite reserve of PMNs available within 24-30 hours d e r inciting event for

delivery to one or more sites of injury. The mechanism by which this pool of PMNs is

triaged to one or more injuries deserves fùrther investigation.

The unifjhg concept fkom the data represented in Figures 3.1 a and 3.1 b support a

regionalization phenornenon by which a reserve pool of PMNs are triaged to sites of

injury depending on theie relative severities. The clinical analogy to this situation is the

ICU patient with peritonitis who is not only at increased risk of developing a secondary

infection, such as a pneumonia, but the morbidity and mortality associated with a

secondary pneurnonia are increased over a pneurnonia developing in othenivise healthy

patients. A recent study in murine models of bacterial-induced peritonitis demonstrated

that neutrophil recruitment is associated with intrapentoneal granulocyte and

granulocyte-rnonocyte colony stimulating factors (G-CSF and GM-CSF, respectively).

Since intrapentoneal injection of these hematopoietic factors failed to yield enhanced

PMN recruitrnent into the peritoneum, they likely senre as systemic markers of an up-

regulated response in the bone marrow of granulocyte production within three hours 153.

Such hematopoietic factors in response to injury serve to increase PMN production to up

to 10" cells per day 6. It is unclear how long before the increased production impacts on

the immediate pool of PMNs, but in the mode1 of murine peritonitis this time hime is at

least 24-30 hours.

4.2 Intravital microsocpic analysis of PMN fluxes, rolling adhesion and firm adhesion

In the intravital microscopy study we exarnined the numbers of PMNs undergoing

rolling adhesion (PM? flues), the mean velocity at which PMNs roll as well as the

numbers undergoing firm adherence in the selected groups in order to determine whether

differences were due to changes in circulatory parameters or another mechanism such as

adhesion molecule expression. With respect to baseline hemodynamic circulatory

parameters measwed for each group, we demonstrated that cecal ligation and puncture in

mice was associated with a reduction in mean red blood ce11 velocity. This effect

occurred whether or not a secondary injury, E. coli-induced cremasteric muscle iniCection

(referred to herein as "orchitis"), was present. Furthermore, orchitis, with or without cecal

Iigation and puncture, was associated with a reduction in the venular shear stress. These

changes occurred despite no differences in vessel diarneters or venular blood flow among

the four groups of animals.

To determine the extent of any relationship between mean RBC velocity and

venular wall shear stress, and the kinetics of PMN-endothelial ceIl interactions, we

performed correlational analyses. These analyses demonstrated that there were significant

relationships between both the mean RBC velocity and vessel shear stress, and PMN

rolling (2 = 0.39 and r2 = 0.56, respectively; p c 0.001). No other signincant correlations

between baseline hemodynamic parameters and kinetics of PMN-endothelid ce11

interactions were observed. This implies that only differences in numbers of rolling

PMNs between the groups, and not PMN rolling velocity or adherence, could be

attributed to differences in RBC velocity or vesse1 wall shear stress.

Our data supports that changes in mean red ce11 velocity act systernically while

changes in venular wall shear stress acts only at the site of local injury without affecthg

PMN delivery elsewhere. A reduction in mean red ceIl velocity was seen only in the

groups of animals with the most severe insults: peritonitis with or without orchitis. The

reduced velocity seen in animals with a major injury in a site rernote fiom the point of

reference implies a systemic effect has occurred that may account for differences in

numbers of rolling PMNs. The reduced venular wall shear stress seen in only in the

groups where a local injury occurred: orchitis with or without peritonitis sugsests that the

change in wall shear stress was simply a local phenornenon since the most severely

injured group (peritonitis and orchitis) did not manifest these changes. Thus while it is

unlikely that changes in venular wall shear stress occurred systemically in the face of

injury, we cannot exclude a reduction in mean red ce11 velocity as a cause of differences

found in numbers of rolling PMNs. Factors responsible for the reduction in mean RBC

velocity in our experirnentd groups rernain to be established. However, a reduction in

biood pressure which comrnonly occurs in association with SIRS, may have been

conûibuting factors. Further experiments to assess blood pressure in these animals and to

institute masures to prevent differences in baseline hemodynamic variables are

warranted.

Since mean red ce11 velociv differences may contribute to changes in numbers of

PMNs undergoing rolling adhesion, we wondered if differences that occurred were due to

the actual adherence properties themselves (Le. adhesion molecule expression and

hypotheses #2 and 3) or simply due to a change in overall numbers of PMNs in the

circulation. Simply put, did we rneasure dserences in defivery of PMNs to the segment

of venule being studied? In order to resolve this question, we adjusted each group for the

effect of circulating PMN concentration by calculathg an adjustment factor fiom the

quotient of the group mean circulating PMN count into the individual animal's number of

circulating PMNs. The product of the adjustment factor and either the rolling or the £ïrm

adherence nurnber deterrnined the respective rolling or fïrm adherent value adjusted for

the effect of circulating concentration of PMNs. When adjusted values were p lotted

against each group, no alteration in statistical significance for each group were noted

when compared to unadjusted data. Therefore any results which occurred could not be

attributed to differences in PMN concentrations in the circulation. By virhie of this fact,

the differences in hemodynamic parameters seen did not significantly alter the rolling or

firm adherence of the PMNs.

Anunals with pentonitis with or without orchitis (CLP and CLP + Orchitis

groups) demonstrated a marked reduction in numbers of rolling PMNs compared to

controls. These differences correspond to similar results reported elsewhere 154. The

greatest nurnbers were associated with the orchitis and control anirnals. Surprisingly the

creation of a local injury, orchitis, did not result in increased nurnbers of rolling PMNs

over controls. These results when taken together imply that either the addition of a local

infection has no fiirtlzer stimulus above baseline PMN recruitment or that changes in

recrutirnent are not manifested in the numbers of rolling PMNs. It is unlikely that orchitis

does not add to PMN recruitment over controls since PMNs fcom animals with orchitis

roll at a slower velocity and adhere in greater numbers than controls. It appears that the

minor injury of orchitis does not lead to increased numbers of rolling PMNs over control

animals, however increased local recmitment of PMNs to injury is demonstrated in the

reduced velocity and increased firm adhesion seen in orchtitis animals. Specific selectin

rnolecule expression has been associated with changes in either rolling adherence or

velocity or both and this rnay account for increased PMN recmitment to infected

cremasteric tissue. Other investigators have demonstrated elevated systemic CDllb

expression in CLP animals compared to controls in a sirnilarly-designed study 154.

The possibility that a minor local infectious injury does not result in increased

PMN fluxes in a finite pool of PMNs must also be considered. In other words the

quiescent control state is associated with maximum numbers of PMN rolling penpherally

and the presence of an injury serves to reduce numbers of rollhg cells elsewhere without

increasing them locally. The PMNs recruited &om the peripheral maiginated pool may

appear as adherent or sIowly rolling PMNs at the site of injury. Since adherent PMNs are

not included in the rolling numbers and that the reduced velocity may result in fewer

fluxes as well, it is possible that the changes in PMN-EC interactions seen in sepsis are

manifested in PMN velocity and adherence alone.

PMN rolling velocity was reduced in the mlce with orchitis with or without

concomitant cecal ligation and puncture (Orchitis and CLP + Orchitis groups). Rolling

velocity of PMNs reflects the presence of adhesive interactions between selectins and

their ligands on the PMN and the underlying endothelial cells. In a mode1 of acute

inflammation such as orchitis, these adhesion molecules would be upregulated both on

the PMNs and vascular endothelium. The time course of Our experiments, 18 - 24 hours

following intra-cremasteric injection of the E. Coli organisms, suggested that E-selectin

wodd have been the predominant selectin expressed 353 507 51. The predominance of

this adhesion molecule may account for the reduced rolling velocity of the PMNs within

the cremasteric vasculature since in vitro studies of E-selectin have demonstrated slower

rolling than P or L selectin s3. Further studies to address this hypothesis using specific

anti-E-selectin antibodies in this mode1 are necessary.

Those animals with cecal Iigation and puncture alone did not demonstrate any

significant difference in PMN rolling velocity fiom controls. This was to be expected

since there was no locaiized inflammation occwrïng within the cremasteric muscle in

these animals and therefore no stimuli for the recruitment of PMNs in this region.

Although the specific adhesion molecule mediating PMN rollirg in the crernaster muscle

in the absence of local injury (CLP or controls) was not determined in these experiments,

evidence fiorn previous investigations suggests that rollinj in these venules is most likely

mediated by L-selectin. Although L-selectin is the only selectin to be constitutively

expressed, induction of P-selectin can occur within minutes following cytokine-induced

EC stimulation. It is possible that the trauma of general anesthesia and crernastenc

muscle dissection alone c m be a sufficient stimulus for P-selectin upregulation. Baseline

recording of PMN-EC interactions took place 20 minutes after dissection was completed

and rnounted ont0 the microscope stage in order to eliminate srnall peiturbations in

cytokines and other mediators of adhesion rnolecule expression and circulatory dynamics.

This is a standard technique employed in similar intravital microscopie investigations 52,

155-157- Using a laminar flow assay to measure rolling velocity of PMNs on g l a s slides

with P-, E-selectin or a L-selectin ligand, L-selectin-mediated rolling velocity was double

that of the other two selectins 53. Our data similarly demonstrated significantly increased

velocity of control animals (where L-selectin is likely to predomioate) as compared to

animals with orcilitis both with and without cecal Ligation and puncture (Orchitis and

CLP + Orchitis groups) where E-selectin is likely to predominate-

PMN £km adherence to the endothelium requires the sequential involvement of

selectins, chemoattractants and integins under physiologie conditions of blood flow.

Animals undergoing local injury (orc hitis) dernonstrated significantly greater numbers of

adherent PMNs in the cremasteric vascuIature than animals undergoing cecal ligation and

puncture or controls. This was to be expected since adhesion molecule up-regulation is

present locally in the orchitis group and not in the others. Tliat there was no dinerence in

CLP and contrai groups with respect to £km adherence (thus rejecting hypothesis #2) has

been demonsîrated both here as well as elsewhere 154- Althouph we expected that

pentonitis would have been associated with reduced firm adherence at remote sites

Oîypothesis #2), it is possible that the circulatins and marginating pool of PMNs buffer

such a change. Furthemore, despite the likelihood of increased adherence at the

mesenteric venules, we saw no change frorn baseiine peripherally- This stands in contrat

to rolling adhesion which is significantly reduced peripherally in the face of peritonitis.

When a primary injury is present remotely combined with a relatively minor

injury locally (CLP + Orchitis group), we observed a drarnatic reduction in PMN

adherence within the cremaster muscle. This suggests a picture of competing sites of

injury for a relatively finite pool of PMNs available within the f i s t 24 hours of acute

injury. AIthough the precise mechanism this effect was not investigated, it is likeIy due to

differences in levels of chemoattractants and clustering of P2 integrhs. Regardess of the

actual mechanisrn, this hding m e r supports hypothesis #1 that a regionalization

p henomenon exists among stimuli of cornpeting injuries.

Chemoattractants function to alter adhesiveness of integins and are produced and

released in concenûations which correlate with the severity of the inciting injury 158.

The degree of P M . delivery has also been demonstrated to correlate zh vivo with

chernokine concentration in a dose-dependent rnanner 159. Thus it is possible that the

major injury (peritonitis) caused a greater magnitude of chemokine syntiesis and release

than the minor injury (orchitis) and resulted in an increased PMN recruitment to the

peritoneum with fewer cells available for recniitment by the cremasteric vasculature.

Sirnilarly increased clustering of P2 integrins on the PMN ce11 surface has been

demonstrated in response to chemoattractants carising increased numbers of adherene

PMNs as well as increased sîrength of adherence to endothelial in vivo Io. This correlated

with our data which demonstrated a drarnatic reduction in adherence of PMNs to the

cremasteric vendes in the presence of orchitis and peritonitis.

The concentration of PMNs in the circulation of these anirnals M e r supports

this hypothesis. Circulating PMN counts in control mice or mice with orchitis were

significantly greater than cecal Ligation and punchlre mice with or without orchitis (CLP

and CLP + Orchitis groups). The concentration of circulating PMNs for each group of

mice appears inversely proportional to the nurnbers of adherent PMNs. This finding

supports the presence of a relatively finite pool of circulating PMNs available for

recruitment and fûrther suggests a possible role for adhesion molecule and

chemoattractant as regulators of these alterations in the kinetics of PMN-EC interactions.

These results were further corroborated by the PMN smears fiom CLP and control

animals used for pmity analysis. In the photographs (which were consistent findings in al1

six animals who undenvent purity analysis of PMN preparations), the raw number of

lymphocytes per high power field was maintained while a 90% reduction in raw PMN

counts were obtained between control and CLP anirnals.

4.3 Future directions: a potential role for shed L-selectin

The results fiom these investigations suppoas the existence of a relatively finite

pool of PMNs available for the immediate period following injury- In a murine mode1 of

cecal ligation and puncture-induced peritonitis, this pool of PMNs is present for at least

24-30 hours following injury. The duration and extent of compensatory hematopoietic

responses by the bone marrow warrants further investigation. The myeloid response is

likeiy mediated by endogenous colony stimulating factors. The potential benefit of

administering exogenous hemopoietic colony stimulating factors in the early period

followirg injury also merits investigation.

We have also demonstrated the presence of a regïonalization phenornenon by

which PMNs fiom the relatively f i t e pool are triaged to the site of major injury. No

attempt was made here to determine the mechanism of this effect but it is likely that

alterations in adhesion rnolecule expression play conîributory roles. Chemoattractants

such as IL-8, f M U , and LTB4 have been previously shown to increase the adherence and

exudation as well as influence chernotactic migration of PMNs to and across HLNECs in

viho 63 and rabbit demis 60 and hamster cheek pouch in vivo 64. When PMNs were

pretreated with IL-8, CSa, or MLP, adherence to HLJVECs under flow conditions was

inhibited in a dose-dependent rnanner 160. The explanation for why chemoattractants at

times can either facilitate or inhi'bit PMN adherence to ECs is that these chernoattractants

induce shedding of L-seleciin 72. Shedding of L-selectin is a concomitant step in

transition fiom r o l h g to firm adhesion and is associated with up-regulation of the P2

integrin molecules on the PMN 67. When PMNs are pretreated with IL-8 and shed their

L-selectin prior to interaction with the endothelium, adhesion does not occur 160.

Little is known of the fate or the subsequent role played by these soluble L-

selectin molecules on PMN-EC interactions downstream. It is postulated that once shed,

the soluble L-selectin may bind to its receptors peripherally and competitively block

quiescent PMN rolluig on the endothelium, This would lead to a reduced PMN flux and

fïrm adhesion at sites remote from injury (hypothesis #3) and increased numbers at sites

of injury where the soluble L-selectin would less effectively compete with up-regulated

L-selectin receptors. Using a murine thioglycollate-induced peritonitis model, Watson

and CO-authors demonstrated that the intravenous administration of a soluble

immunoglobulin chimera containing the extrallular L-selectin domains significantly

reduced PMN ernigration to the peritoneum 161. It is possible that shed L-selectin may

contribute to the regionalization effect of PMlV delivery to sites of injury. The sites of

major injury such as pentonitis would presurnably recruit a greater proportion of the pool

of available PMNs since shed L-selectin would compete less effectively with the

associated up-regulated endothelial expression of selectin receptors. Conversely,

uninjured tissue or less severe sites of injury such as an E-coli-induced crernaster muscle

infection would accumulate fewer PMNs since the relatively fewer selectin receptors

would be more efficiently blocked by soluble L-selectin.

Further studies are indicated to determine the role of soluble L-selectin on PMN

delivery to sites of injury. Tech icd and ethical bamers need to be overcome in order to

produce the necessary quantities of murine shed L-selectin as few investigators have been

able to achieve this end. Without the data to support or contradict the shedduig of L-

selectin as well as the role of soluble L-selectin on rolling and fum adherence, we can not

at this time test hypothesis #3.

4.4 Conclusion

Using polyvinyl sponge discs implanted in the peritoneum and skin of the back in

mice with or without peritonitis, a relatively f i t e pool of PMNs was demonstrated to be

present with PMNs delivered to each site in proportion with the severity of injury to the

major site. In this mode1 this pool exists for at least 24-30 hours following injury before a

compensatory response by the myeoloid organ occurs. These results support hypothesis

#l however the precise mechanism of PMN delivery, or regionalkation, remains uncIear.

With intravital microscopy we demonstrated a that numbers of PMNs involved in

rolling adhesion with the endotheliurn are significantly reduced at a site remote f?om

peritonitis and this effect was unchanged if a local injury was present as well-

Correlational analyses revealed that differences in mean red blood ceIl velocity, a rnarker

of intravascular hemodynarnic change, could account for differences in rolling PMNs by

altering number of PMNs arriving at the post-capillary venule. However, adjusting for

circulating P M . concentration and replotting the data, we found that there were no

differences significant outcomes. Since liemodynamic changes do not account for these

differences, the mechanism of altered PMN-EC interactions in the presence of a two-kont

injury mode1 is likely due to alterations in both ceU surface-expressed and soluble

adhesion molecuIes. In addition, PMNs rolled at sIower velocities in the presence of local

injury whether or not peritonitis was present. The presence of peritonitis did not result in

reduced numbers of finrily adherent PMNs to the cremasteric vasculature, thus rejecting

hypothesis #2, and this reIationship was not altered by the addition of a Iocal injury.

Further studies investigating the role that shed L-selectin plays on PMN-EC interactions

downstream fkom a primary site of injury are warranted to determine whether or not this

results in enhanced delivery of the marghating PMN popu1ation to the prirnary injury

(hypothesis #3).

Chapter 5: CONTRIBUTION TO ORIGINAL KNOWLEDGE

1, PMNs are regionalized in the acute period fdlowing a severe injury. In a murine

peritonitis model, PMN delivery to a second site when peritonitis is present is

inverseIy proportional to the degree of injury to the primary site,

2. PMN adherence is greatest in the presence of a localized injury (orchitis) than when

the injury is distant (peritonitis) or in the presence of a cornbined localized and

massive distant injury (pentonitis-orchitis).

3. Numbers of rolling PMNs were greatest in the absence of injury and reduced in the

presence of distant, massive injury (peritonitis) and virtually abolished in the

cornbined presence of a localized and a massive, distant injury (peritonitis-orchitis).

4, The PMNs rolled most rapidly in the absence of injury and at a slower velocity in the

presence localized injury and even slower in the combined presence of a localized and

massive, distant injury.

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