ABSTRACT

KATHERINE RICCIONE Endosomal maturation in macrophages activated by lacto-N-fucopentaose-III on molecules in schistosome soluble egg antigen (Under the direction of DR. DONALD A. HARN JR.) Lacto-N-fucopentaose (LNFPIII) is a helminth glycan, present on molecules in

schistosome soluble egg antigen (SEA), that activates antigen-presenting cells (APCs) to drive

CD4+ T-cells to the anti-inflammatory T-helper 2 (Th2) type. Knowing the anti-inflammatory

effects of this glycan, its use as a potential Th2 adjuvant and therapeutic compound for transplant

recipients and treatment of autoimmune diseases is currently being studied, and the mechanism of

glycan uptake and activation of APCs is of great interest. The aim of this study was to determine

if SEA containing molecules with LNFPIII takes a comparable endocytic pathway to pure

LNFPIII upon uptake by APCs. To determine the effect on endosome maturation in APCs, a

confocal microscope analysis was done on SEA endocytosis in RAW264.7 macrophages.

Different endosomal inhibitors were used to confirm that SEA proceeds to the endosome, and a

series of time course endocytosis assays were done in conjunction with staining of endosomal

markers and LNFPIII on molecules in SEA. We found that SEA endocytosis does not occur in

APCs treated with endosomal inhibitors, indicating that SEA proceeds to the endosome upon

uptake by RAW cells. Additionally, we found that SEA endocytosis by RAW cells is clathrin-

dependent, and SEA proceeds to the early endosome and lysosome via a pathway that is

comparable to LNFPIII endocytosis.

INDEX WORDS: Lacto-N-fucopentaose (LNFPIII), schistosome soluble egg antigen (SEA), antigen-presenting cell (APC), endosome

ENDOSOMAL MATURATION IN MACROPHAGES ACTIVATED BY LACTO-N-

FUCOPENTAOSE-III ON MOLECULES IN SCHISTOSOME SOLUBLE EGG ANTIGEN

by

KATHERINE RICCIONE

A Thesis Submitted to the Honors Council of the University of Georgia in Partial Fulfillment of the Requirements for the Degree

BACHELOR OF SCIENCE

in BIOLOGICAL ENGINEERING

with HIGH HONORS

and CURO SCHOLAR DISTINCTION

Athens, Georgia

2010

© 2010

Katherine Riccione

All Rights Reserved

ENDOSOMAL MATURATION IN MACROPHAGES ACTIVATED BY LACTO-N-FUCOPENTAOSE-III ON MOLECULES IN SCHISTOSOME SOLUBLE EGG ANTIGEN

by

KATHERINE RICCIONE

 Approved: Donald Harn Jr Dr. Donald Harn Jr, Phd Faculty Research Mentor  Approved:   Leena Srivastava Dr. Leena Srivastava Reader  Approved:    David S. Williams Dr. David S. Williams Director, Honors Program, Foundation Fellows and Center for Undergraduate Research Opportunities 

Approved:   Pamela B. Kleiber Dr. Pamela B. Kleiber Date Associate Director, Honors Program and

   

  5/7/2010 Date

    

5/7/2010 Date 5/7/2010 Date 5/7/2010

Date

Center for Undergraduate Research Opportunities   

 

 

DEDICATION

I would like to dedicate this thesis and the extensive work spent on it to my mother,

Diane Riccione. She has always had the patience and compassionate tolerance for all of my

endeavors, regardless of how overwhelming and far-fetched my aspirations may seem.

iv

ACKNOWLEDGEMENTS

I would like to thank Dr. Donald A. Harn for granting me with the opportunity to work as

an undergraduate in his laboratory for the fall and spring semesters and for his scholarly

mentorship in my research endeavors. I would also like to thank Dr. Leena Srivastava for all of

her excellent guidance throughout my work on this thesis as well as her assistance with data and

image collection and experiment design; without her, this thesis would not have been possible. I

would also like to express my gratitude toward the rest of the researchers in the Harn laboratory

for the assistance they offered in a number of laboratory techniques and for kindly

accommodating the clumsy inexperience of an undergraduate student. Additionally, I would like

to recognize the UGA Honors Program and Center for Undergraduate Research Opportunities

and its wonderful staff for their tireless work in making research so accessible to the

undergraduate community and for presenting me with the means to write my own undergraduate

thesis.

v

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.............................................................................................................v

LIST OF FIGURES......................................................................................................................viii

CHAPTER

1 INTRODUCTION.....................................................................................................1

2 MATERIALS AND METHODS...............................................................................8

RAW Cell Culture...........................................................................................8

Antibodies........................................................................................................8

Time Course Endocytosis with Staining of Endosomal Markers....................8

Dynasore Inhibitor Assay...............................................................................10

Methyl-β-cyclodextrin Inhibitor Assay..........................................................10

3 RESULTS................................................................................................................11

SEA Endocytosis with Rab5 Staining...........................................................11

SEA Endocytosis with MPR Staining...........................................................13

SEA Endocytosis with LAMP-1 Staining.....................................................14

SEA Endocytosis with Clathrin Staining......................................................15

Inhibition with Dynasore...............................................................................16

Inhibition with Methyl-β-cyclodextrin..........................................................18

4 DISCUSSION..........................................................................................................20

vi

WORKS CITED............................................................................................................................24

vii

LIST OF FIGURES

Page

Figure 1: LNFPII/LewisX signaling via C-type lectin receptors and Toll-like receptors................4

Figure 2: General endosomal pathway of clathrin dependent endocytosis of signaling receptors..6

Figure 3: SEA endocytosis with Rab5 staining.............................................................................13

Figure 4: Co-localizations of Rab5/E5 in RAW cells....................................................................13

Figure 5: SEA endocytosis with MPR staining.............................................................................15

Figure 6: SEA endocytosis with LAMP-1 staining.......................................................................16

Figure 7: Co-localizations of LAMP-1/E5 in RAW cells..............................................................16

Figure 8: SEA endocytosis with clathrin staining..........................................................................17

Figure 9: Co-localizations of clathrin/E5 in RAW cells................................................................17

Figure 10: SEA endocytosis with differing concentrations of dynasore.......................................18

Figure 11: Close-up of RAW cells treated with 80 μM and 120 μM dynasore.............................19

Figure 12: SEA endocytosis with differing concentrations of methyl-β-cyclodextrin..................20

viii

1

CHAPTER 1 INTRODUCTION

The cellular mechanisms by which parasitic infections regulate host immune responses

have been of increasing interest. Initial studies were guided by an interest in how the host can

control and/or eliminate parasitemia, but more recent fascination concerns immune modulation

by parasite molecules and specifically, the mechanisms by which these molecules have strong

immune modulatory effects (1). For instance, the protozoan parasite, Leishmania, expresses

lipophosphoglycans and proteophosphoglycans, which inhibit maturation of the macrophage

endosome (2), and glycans from schistosomes, a helminth parasite of man and animals, have

been observed to drive strong CD4+ T-cell biasing towards T helper 2 (Th2)-type (3).

In the context of schistosome glycans, of particular interest is Lacto-N-fucopentaose III

(LNFPIII) which is found in schistosome eggs. This pentasaccharide contains the Lewisx

trisaccharide, the immune modulatory glycan expressed by schistosomes that activates antigen-

presenting cells (APCs) to drive CD4+ T-cells toward the anti-inflammatory Th2-type (4,5).

APCs stimulated by LNFPIII co-cultured with naive CD4+ T-cells induced a CD4+ Th2-type

response (6). In experimental murine schistosomiasis, a polarized shift from Th1 to Th2 takes

place in correlation to egg deposition, and schistosome eggs alone were capable of inducing a

Th2 bias (7,8). Additionally, schistosome soluble egg antigen (SEA) has been shown to contain

the molecules that drive this Th2 bias (9).

Knowing its ability to drive CD4+ T-cells towards the anti-inflammatory Th2 type, the

potential for using LNFPIII in immunotherapy has come into question. Atochina et al. (10)

2

explored the potential therapeutic use of LNFPIII with the ‘flaky-skin’ Fsn/Fsn mouse model of

psoriasis. LFNPIII glycoconjugates or a carrier control were injected into Fsn/Fsn mice prior to

disease development, and mice aged without further intervention. By 8-9 weeks of age, Fsn/Fsn

mice injected with the carrier control developed severe psoriatic lesions, while Fsn/Fsn mice

injected with LNFPIII glyconjugates did not develop lesions and appeared as normal, control

littermates. Fsn/Fsn mice injected with LNFPIII glyconjugates also had normal skin thickness,

CD4+/CD8+ ratios, and IL-10 levels, when compared with the control littermates, while Fsn/Fsn

mice injected with the carrier control were observed to have these same parameters altered in a

way that is correlated to the disease. In a separate set of experiments, LNFPIII was used to

successfully treat psoriatic lesions. These studies suggest that LNFPIII could potentially be used

for the treatment of diseases caused by inflammation, including autoimmune diseases,

cardiovascular disease, and obesity (6).

Macrophages activated with LNFPIII conjugates have been shown to produce lower

levels of pro-inflammatory cytokines, including IL-12 and IFN-γ, in concert with higher levels of

anti-inflammatory cytokines including IL-10 and TGF-β (11). In a study done with heart

allograft transplants, mice injected subcutaneously with LNFPIII experienced prolonged survival

compared to the control group, further suggesting that LNFPIII has immunomodulatory effects

in transplant recipients (D. Harn, personal communication).

Other studies are currently underway to test the therapeutic potential of LNFPIII

glycoconjugates, including one on the prevention of Type 1 diabetes and one on a murine model

of multiple sclerosis, and in both of these studies, LNFPIII glyconjugates have been protective

(D. Harn, personal communication). Additionally, it may be possible to use LNFPIII, as well as

3

other helminth-derived sugars, as adjuvants driving the Th2 type bias. Noting the potential

therapeutic uses of LNFPIII, and to further understand the immune modulatory mechanisms of

helminth parasites, it is of great interest to understand the immune modulatory signaling cascade

that LNFPIII drives, starting specifically with APC uptake of and activation by this glycan.

Helminthes and/or helminth derived molecules have been shown to activate APCs via

Toll-like receptors (TLRs) and/or C-type lectin receptors (CLRs). Helminthes and helminth

molecules specifically activate B-1 B cells, macrophages, and dendritic cells (DCs) to result in

production of anti-inflammatory mediators (6). Activation and maturation of DCs by LNFPIII

glycoconjugates was shown to be dependent on interaction with a number of C-type lectins as

well as TLR4 and resultant TLR4 antagonism downstream in the signaling pathway (6).

Lepper et al. (12) demonstrated that Helicobacter pylori lipopolysaccharide (LPS)

expressing the Lewisx trisaccharide activated APCs via TLR-2, leading to an antagonism of

TLR4 pro-inflammatory signaling, while H. pylori LPS that did not express Lewisx activated

APCs by the accepted pro-inflammatory signaling pathway. This study thus aids in confirming

that LNFPIII glycoconjugates require the expression of TLR4 on DCs to drive them to an anti-

inflammatory phenotype (6).

Liempt et al. (13) demonstrated that saline soluble egg antigens (SEA) from schistosomes

drive anti-inflammatory maturation of DCs by binding to three distinct C-type lectins: DC-

specific (intercellular adhesion molecule-3)-grabbing non-integrin (DC-SIGN), the mannose

receptor (MR), and macrophage galactose lectin-1 (MGL-1). In this study, antibody blocking of

any one of these three C-type lectins was not enough to inhibit SEA activation of human DCs,

while blocking of any two of these three lectins did significantly inhibit SEA activation of

human DCs. This study concluded that there is a redundancy in the CLRs that recognize

LNFPIII, and that ligation of any one of these three C-type lectins was enough to induce a

signaling cascade that drives DCs to mature into anti-inflammatory APCs. The study is also one

of many to suggest that activation of human DCs via glycan interaction with these C-type lectins

eventually results in TLR4 antagonism. In comparing peritoneal macrophages from wildtype

mice with macrophages deficient in DC-SIGN and MGL-1, Harn et. al. (6) found that

macrophages deficient in these two C-type lectins responded similarly, if not identically, to

macrophages from wildtype mice, further corroborating that there is a redundancy in C-type

lectins that are necessary for LNFPIII activation of APCs.

Figure 1: Combined LNFPIII/LewisX signaling via C-type lectin receptors (CLRs) and Toll-like receptors (TLRs). Studies have identified TLR-4 as a receptor target for LNFPIII induced signaling along with its ability to activate APCs through various CLRs. LNFPIII downstream activation involves primarily MAPK/ERK activation and transient NF-κB translocation independent of NF-κB degradation. Dashed arrows indicate putative pathways. (6)

4

5

Overall, the aforementioned studies, taken together with other reports, suggest that there

are at least two classes of receptors to which SEA glycans and/or LNFPIII bind to activate APCs,

each of which causes a down-regulation of TLR4 pro-inflammatory responses. Figure 1 shows a

summary of the combined CLR and TLR signaling pathways (6).

Subsequent to interaction with any of the three C-type lectins, Liempt et al. (13) showed

that endosome formation is initiated, and subsequent to endosome formation, a Ras-dependent

signaling cascade is induced, and TLR4 antagonism occurs. Our study specifically explores the

endosomal pathway by which endocytosis of SEA occurs with immature APCs by utilizing a

number of endosomal markers in conjunction with SEA endocytosis by macrophages.

Endocytosis can occur through either a clathrin-independent or a clathrin-dependent

pathway (14). In clathrin-dependent endocytosis (CDE), the cytoplasmic domains of plasma

membrane proteins are recognized by adaptor proteins and packaged into clathrin-coated vesicles

that are subsequently brought into the cytoplasm of the cell (15). By contrast, clathrin-

independent endocytosis (CIE) comes in many forms, including actin-driven pathways such as

macropinocytosis and phagocytosis (15). There is now evidence to suggest that virtually every

family of signaling receptor, including CLRs and TLRs, undergo CDE, excluding perhaps G-

protein coupled receptors, receptor tyrosine kinases, transforming growth factor-β, and Wnt and

Notch receptors (16).

Regardless of whether cargo is endocytosed via CDE or CIE, it is typically delivered to

the early endosome for sorting, where it can thereby be routed to the late endosome and

lysosomes for degradation, to the trans-Golgi netowrk (TGN), or to recycling endosomal carriers

that bring the cargo back to the plasma membrane (15). Various markers are associated with each

stage of the endosome, and these markers will be utilized in this study to further understand the

path that endocytosed SEA takes.

Figure 2: The best-studied pathway of receptor internalization is mediated by clathrin-coated pits. Receptors are recruited to clathrin coated pits by directly interacting with the clathrin coat adaptor complex, AP2, or by binding to other adaptor proteins. Clathrin-coated pits invaginate inwards with the help of several accessory proteins and pinch off to form a clathrin-coated vesicle in a process that requires GTPase dynamin. Endocytic vesicles then fuse with early endosomes, and endosomal trafficking is controlled by several Rab proteins. (18)

Generally, the endocytosis of signaling receptors is stimulated by ligand-induced

activation, and signaling receptors can use the same basic endocytic machinery as other cargo

(16, 17). Figure 2 shows a detailed depiction of the endosomal pathways potentially taken upon

receptor endocytosis, along with various endosomal markers that are associated with each of the

endosomal stages. The best-studied pathway of receptor internalization, and the one most often

observed with signaling endocytosis, is mediated by clathrin-coated pits. Receptors are recruited

to clathrin-coated pits by directly interacting with adaptor proteins. These pits invaginate

inwards and pinch off to form a clathrin-coated vesicle in a process that requires the GTPase

6

7

dynamin. These endocytic vesicles may then fuse with early endosomes, and endosomal

trafficking is controlled by a number of Rab proteins wherein each Rab protein resides in a

particular endosomal stage and functions by recruiting specific effector proteins. After fusing

with Rab5-containing early endosomes, receptors can recycle back to the plasma membrane by a

Rab4-dependent mechanism, traffic to the recycling compartment that contains Rab11A, or

remain in endosomes, which mature into multivescular bodies (MVBs) and late endosomes,

involving Rab7. Late endosomes and MVBs ultimately fuse with lysosomes carrying proteolytic

enzymes, thereby resulting in cargo degradation (16).

The aim of this study was to better understand the endocytic pathway that SEA takes

upon uptake by and activation of antigen-presenting cells. Specifically, we hypothesized that

SEA would proceed to the endosome in a comparable pathway to LNFPIII.

8

CHAPTER 2 MATERIALS AND METHODS

RAW Cell Culture

RAW264.7 cells (mouse leukaemic macrophage cell line) were obtained from ATCC.

Cells were cultured in DMEM/high glucose media (HyClone) containing penicillin and 10%

fecal bovine serum and grown for two days at 37°C. RAW cell lines were split every 48-60

hours. For all endocytosis experiments, cells were seeded on glass coverslips at a density of

1x106 cells/well in 12-well plates and left overnight at 37°C.

Antibodies

Primary antibody against LNFPIII on molecules in purified schistosome soluble egg

antigen (SEA) was a mouse IgM (E5) generated in the laboratory of Dr. Donald Harn.

Specificity was tested by ELISA. Antibodies against Rab5, mannose 6-phosphate receptor

(MPR), and other endosomal markers were purchased from Abcam. Alexa488 conjugated goat

anti-mouse IgM and Alexa594 conjugated goat anti-rabbit secondary antibodies were purchased

from Invitrogen. Nuclei were stained with Hoechst.

Time Course Endocytosis with Staining of Endosomal Markers

Several separate time course endocytosis assays were done in conjunction with Rab5

staining, MPR staining, lysosomal-associated membrane protein 1 (LAMP-1) staining, and

clathrin staining. Before endocytosis, seeded cells were starved for 2 hours in serum-free

9

DMEM. Wells were brought to a volume of 500 μL and left on ice for 10 minutes. Cold SEA

was added to cells at a volume of 30 μL and remained on ice for another 10 minutes.

A 0 minute control was collected in a fresh well and placed in 500 μL of 3% PFA/PBS

for 10 minutes at room temperature for fixation. The remaining samples were incubated at 37°C

for varying time intervals (15-45 minutes). After endocytosis, each coverslip was placed in a

fresh well and fixed in 500 μL of 3% PFA/PBS for 10 minutes. After fixation, cells were

washed 3 times with 1 mL of PBS. Cells were then permeabilized with 0.5% triton for 4 minutes

and again washed with 1 mL PBS 3 times.

Cells were incubated over two sets of primary/secondary antibodies, and coverslips were

washed three times in PBS on low speed between stainings with each of the four antibodies.

Coverslips were incubated for 20 minutes over 40 μL of 5% BSA/PBS on parafilm at room

temperature to block non-specific binding. Coverslips were then placed over 40 μL of E5

primary antibody for 1 hour at room temperature on parafilm, and subsequently placed over 40

μL of 1:300 Alexa488 goat-antimouse-IgM secondary antibody for 1 hour at room temperature.

Coverslips were then incubated for 1 hour over 40 μL of a second primary antibody (rabbit

antibody against Rab5, MPR, clathrin, or LAMP-1) on parafilm at room temperature and were

subsequently incubated over Alexa594 secondary antibody for 1 hour at room temperature. Cells

were then stained in 30 μL Hoescht over parafilm for 5 minutes and mounted on blank slides

with Prolong Gold.

Confocal LSM 510 images were taken under 100X objective. Sequential scanning was

performed for each antibody, and images were analyzed by LSM software for co-localization.

10

Dynasore Inhibitor Assay

The effect of dynasore on SEA endocytosis was analyzed in a concentration dependent

manner. Dynasore interferes in vitro with the GTPase activity of dynamin1 and dynamin2 as

well as mitochondrial dynamin and acts as a potent inhibitor of endocytic pathways known to

depend on dynamin by blocking coated vesicle formation (18).

Dynasore was obtained from Sigma Life Sciences. Prior to endocytosis, seeded RAW

cells were starved for 1 hour in serum-free DMEM and treated with DMSO or dynasore (80 μM

and 120 μM) for one hour. After starvation, 30 μL SEA was added to each well and incubated

for 30 minutes at 37°C. An E5 control (no SEA) and SEA control (no dynasore or DMSO) were

also collected. Fixation and staining protocol was followed as mentioned previously with 5%

BSA used to block non-specific binding and Alexa488 goat anti-mouse IgM used to stain the

E5/LNFPIII antibody complex.

Methyl-β-cyclodextrin Inhibitor Assay

The effect of methyl-β-cyclodextrin (MβCD) on SEA endocytosis was also analyzed.

MβCD selectively extracts cholesterol from the plasma membrane, resulting in inhibition of

calveoli lipid raft and clathrin-coated pit formation.

MβCD was obtained from Sigma Life Science. Seeded cells were starved for 1 hour in

serum-free DMEM and treated with varying concentrations of MβCD (5 μM, 10 μM, and

20 μM) for 40 min. After starvation, 30 μL SEA was added to each well and incubated for 30

minutes at 37°C. An E5 control (no SEA) and SEA control (no inhibitor) were also collected.

Fixation and staining protocol was followed as mentioned previously with 5% BSA used to

11

block non-specific binding and Alexa488 goat anti-mouse IgM used to stain LNFPIII containing-

molecules in SEA.

12

CHAPTER 3 RESULTS

SEA Endocytosis with Rab5 Staining

To determine if SEA is endocytosed by RAW cells and proceeds through the early

endosome, cells were stained with antibodies against Rab5, a known early endosomal marker. To

determine optimal times of RAW cell endocytosis of SEA, a time course assay was done,

wherein RAW cells were incubated with SEA for 0 minutes, 15 minutes, 30 minutes, and 45

minutes (data not shown). As incubation time increased, SEA endocytosis and co-localization

with Rab5 (yellow) increased. Co-localization with Rab5 was observed at the 30 minute time-

point, indicating that early endosome formation occurs at 30 minutes, so images from this time

point are shown (Figure 3).

Figure 3: After 30 minutes of SEA endocytosis (30 uL), RAW cells were stained with Alexa488 goat-antimouse IgM specific for E5 bound to LNFPIII-containing molecules (green). Cells were then stained with rabbit anti-Rab5 primary antibody and Alexa594 secondary antibody (red). Hoescht was used for nucleus visualization (blue).

Figure 4: After 30 minutes (A) and 45 minutes (B) of SEA endocytosis, co-localization of E5 with Rab5 is seen in RAW cells (yellow), indicated by white arrows.

B A

13

14

In addition to the 30 minute time point, co-localization with Rab5 continues to be seen on

RAW cells cultured with SEA for 45 minutes (Figure 4), while no co-localization was seen with

RAW cells cultured with SEA for 15 minutes. These data suggest that LNFPIII on molecules in

SEA proceeds to the early endosome 30 minutes after the start of phagocytosis, and the early

endosome may continue forming 45 minutes after the start of phagocytosis.

SEA Endocytosis with MPR Staining

To determine if SEA molecules proceed to the late endosome, staining of MPR, a known

late endosomal marker, in conjunction with a time course of SEA endocytosis was done (Figure

5). RAW cells were incubated with SEA for 0 minutes, 15 minutes, 30 minutes, and 45 minutes

(data not shown). Incubation for 45 minutes resulted in the most SEA uptake when compared

with the other three time intervals. After 30 minutes and 45 minutes of endocytosis, no

discernable MPR/LNFPIII co-localization was observed. However, we were able to see clear

staining of MPR or LNFPIII after 45 minutes of endocytosis. This suggests that LNFPIII on

molecules in SEA does not proceed to the late endosome after 45 minutes of endocytosis.

Figure 5: After 45 minutes of SEA endocytosis (30 uL), RAW cells were stained with Alexa488 goat-antimouse IgM specific for E5 bound to LNFPIII-containing molecules (green). Cells were then stained with rabbit anti-MPR antibody and Alexa594 goat-antirabbit secondary antibody (red). Hoescht was used for nucleus visualization (blue).

SEA Endocytosis with LAMP-1 Staining

To determine if SEA molecules proceed to the lysosome, RAW cells were stained with

rabbit anti-LAMP-1 antibody subsequent to 30 minutes of SEA endocytosis. SEA endocytosis

was compared with an E5 control (no SEA) to check for background staining. A significant

amount of co-localization with LAMP-1 was observed within the cytoplasm of RAW cells

(Figure 7), suggesting that SEA proceeds to the lysosome.

15

A B A

Figure 6: After 30 minutes of SEA endocytosis (30 μL), RAW cells were stained with Alexa488 goat-antimouse IgM specific for E5 bound to LNFPIII-containing molecules (green). Cells were then stained with polyclonal rabbit anti-LAMP-1 and Alexa594 goat-antirabbit secondary antibody (red). Hoescht was used for nucleus visualization (blue). SEA endocytosis (B) is compared to an E5 control with no SEA (A).

Figure 7: After 30 minutes of SEA endocytosis, E5/LAMP-1 co-localization in RAW cells (yellow) is seen, indicated by white arrows.

SEA Endocytosis with Clathrin Staining

To determine if SEA undergoes endocytosis via a clathrin-dependent pathway, RAW

cells were stained with rabbit anti-clathrin antibody following incubation of RAW cells for 30

minutes. SEA endocytosis was compared with E5 antibody only as a control to check for

16

background staining (Figure 8). A significant amount of co-localization with clathrin was seen,

suggesting that SEA undergoes a clathrin-dependent pathway (Figure 9).

B A A

17

Figure 8: After 30 minutes of SEA endocytosis (30 μL), RAW cells were stained with Alexa488 goat-antimouse IgM specific for E5 bound to LNFPIII-containing molecules (green). Cells were then stained with rabbit anti-clathrin antibody, and Alexa594 goat-antirabbit secondary antibody (red). Hoescht was used for nucleus visualization (blue). SEA endocytosis (B) is compared to an E5 control with no SEA (A).

Figure 9: After 30 minutes of SEA endocytosis, E5 co-localization with clathrin is seen at the surface of RAW cells (yellow), indicated by white arrows.

Inhibition with Dynasore

To explore the role of dynamin in SEA endocytosis, RAW cells were treated with

varying concentrations of dynasore (dissolved in DMSO), a known inhibitor of dynamin (18), in

an SEA uptake assay. Treated cells were compared to an E5 antibody only control, SEA control,

and a DMSO vehicle only control. When compared with these controls, endocytosis of SEA is

markedly decreased in RAW cells treated with 80 μM or 120 μM dynasore (Figure 10).

Figure 10: RAW cells were treated with differing concentrations of dynasore: 80 μM (D) and 120 μM (E). SEA endocytosis was compared with an SEA control with no inhibitors (A), an E5 control wherein no SEA or inhibitors were introduced to RAW cells (B), and a DMSO control (C). Alexa 488 goat anti-mouse -IgM was used to stain E5 bound to LNFPIII-containing molecules.

B A

C

D E C

18

Figure 11: SEA endocytosis by RAW cells treated with 80 μM dynasore (A) compared with RAW cells treated with 120 μM dynasore (B).

A B

Comparing cells treated with 80 μM and 120 μM dynasore, cells treated with 120 μM had

the greatest decrease in SEA uptake (Figure 11). RAW cells treated with the lower dynasore

concentration have notably more E5 bound to LNFPIII-containing molecules (green) scattered

throughout the cytoplasm, evidenced by the more visible green coloration in images from this

sample. These data show that dynasore directly inhibits SEA endocytosis, suggesting that

dynamin plays a role in endocytosis.

Inhibition with Methyl-β-cyclodextrin

To determine how plasma membrane cholesterol affects SEA endocytosis, the effect of

MβCD, a compound known to selectively extract cholesterol from the plasma membrane (19),

was observed in SEA endocytosis. RAW cells were treated with varying concentrations of

MβCD (5 μM, 10 μM, and 25 μM), and SEA endocytosis was compared to an E5 control to

check for background staining and an SEA control (Figure 12).

19

SEA endocytosis is markedly decreased as the concentration of MβCD is increased.

Endocytosis continues to occur when only 5 μM of MβCD is added to RAW cells, but SEA

uptake is substantially decreased upon the addition of 10 and 25 μM MβCD to RAW cells.

C D E

Figure 12: RAW cells were treated with MβCD inhibitor at differing concentrations: 5 μM (C), 10 μM (D), and 25 μM (E). SEA endocytosis and cell viability were compared with an E5 control wherein no SEA or inhibitors were introduced to RAW cells (A) and to an SEA control with no inhibitors (B).

A B

As MβCD concentration is increased to 25 μM, RAW cell viability begins to decrease

significantly. This is evidenced by loss of shape in the nucleus (blue) and decreased cytoplasm

area. These data suggest that MβCD may have an inhibitory effect on SEA uptake by RAW

cells, by either directly inhibiting endocytosis or through its cytotoxic effects on RAW cells.

20

21

CHAPTER 4 DISCUSSION

The ultimate goal of this study was to explore the endocytic pathway that SEA takes in

RAW264.7 macrophage cells. We hypothesized that SEA would proceed to the endosome and

follow a pathway similar to that of LNFPIII and that thus SEA is responsible for APC activation.

The study is part of a broader goal in determining how APCs are alternatively activated by

LNFPIII, and specifically the role that CLRs and endosome formation play in this activation. The

findings suggest several details concerning this endocytic pathway: SEA endocytosis is

comparable to LNFPIII endocytosis in that it is clathrin-dependent and proceeds to the early

endosome as well as the lysosome.

The experiments done with each of the two inhibitors, dynasore and MβCD, both aid in

suggesting that LNFPIII endocytosis is clathrin-dependent. Dynasore is a known inhibitor of

dynamin, and dynamin is essential for the pinching off of a variety of vesicles from a parent

membrane, including clathrin-coated pits. Dynasore interferes with the GTPase activity of

dynamin, thus blocking complete vesicle formation (18). Therefore, any clathrin-dependent

endocytosis would be inhibited by the addition of dynasore, thus the data suggest that SEA

endocytosis is clathrin dependent.

MβCD inhibits the formation of calveoli lipid rafts as well as clathrin-coated pits. This

compound works by extracting cholesterol from the plasma membrane, resulting in an

accumulation of shallow pits that are not fully-formed (19). The results of inhibition by MβCD,

22

in conjunction with the results of inhibition by dynasore and co-localization with clathrin, further

indicate that SEA endocytosis occurs via a clathrin-dependent mechanism. These results are not

particularly surprising, as it is accepted that the uptake of virtually every signaling receptor,

particularly CLRs, occurs by clathrin-dependent endocytosis (16).

Upon entrance into the cell via a clathrin-coated vesicle, SEA proceeds to the early

endosome and lysosome. The extensive co-localization seen with Rab5 and LAMP-1 staining

corroborate this. However, the time points at which each of these events occurs is not completely

understood by the data. Rab5 and LAMP-1 co-localizations both occur substantially at the 30

minute time point, but early endosome formation should occur before material proceeds to the

lysosome. Furthermore, late endosome formation should occur subsequent to early endosome

formation but prior to fusion with the lysosome, but the data indicates that no late endosome

formation occurs at any time before 45 minutes. Further studies could be done with another late

endosomal marker, such as Rab7, to better understand the time points associated with this

pathway. Experiments could also be done with a number of other endosomal markers, including

Rab4 for recycling back to the plasma membrane and Rab11A for entrance into recycling

compartments, using essentially the same protocol as that done with Rab5, LAMP-1, clathrin,

and MPR staining.

To confirm that the SEA endocytic pathway is representative of the LNFPIII endocytic

pathway, analogous experiments were done using pure LNFPIII, rather than using SEA as a

source of LNFPIII containing molecules. Results of these experiments align well with the results

of experiments done with SEA, indicating that SEA endocytosis is a viable method of studying

the LNFPIII endocytic pathway (data not shown).

23

To further understand signaling of DCs by LNFPIII, subsequent studies should be done

that specifically explore the role of the various CLRs through which activation occurs. It has

been shown that LNFPIII binds to three CLRs: MR, MGL-1, and DC-SIGN, in addition to

MSIGNR1 (13). In order to discern the role of these receptors, Hek cells and mice deficient in

these lectins, or singly transfected with each of them, could be utilized.

Additionally, the downstream signaling cascade that occurs upon APC activation by

LNFPIII should be further studied. Harn et al. has shown that activation by LNFPIII leads to

extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)

activation (20) and that LNFPIII activates DCs via TLR-4, resulting in transiently activated

NFκB (21). To further elucidate the downstream signaling pathways, candidate genes and the

role of these candidate genes in APC activation are currently being studied.

24

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