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August 1999 Role of Mitogen-activated in Cd40-mediated Activation of Monocyte/ and Vascular Smooth Muscle Cell / Production Denise M. Milhorn East Tennessee State University

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Recommended Citation Milhorn, Denise M., "Role of Mitogen-activated Kinases in Cd40-mediated T Cell Activation of Monocyte/macrophage and Vascular Smooth Muscle Cell Cytokine/chemokine Production" (1999). Electronic Theses and Dissertations. Paper 2950. https://dc.etsu.edu/ etd/2950

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ROLE OF MITOGEN ACTIVATED KINASES IN CD40-MEDIATED T CELL

ACTIVATION OF MONOCYTE/MACROPHAGE AND VASCULAR SMOOTH

MUSCLE CELL CYTOHNE/CHEMOKINE PRODUCTION

A Dissertation

Presented to

the Faculty of the Department of Biochemistry and Molecular Biology

James H. Quillen College of Medicine

East Tennessee State University

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy in Biomedical Sciences

by

Denise M Milhom

August 1999

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPROVAL

This is to certify that the Graduate Committee of

Denise Marie Milhom

met on the

Twenty-fifth of June, 1999

The committee read and examined her dissertation^ supervised her defense of it in an

oral examination, and decided to recommend that her study be submitted to the

Graduate Council in partial fulfillment of the requirements for the degree of Doctor of

Philosophy in Biomedical Sciences.

k / / { Graduate Committee mm

Signed on behalf of the Graduate Council u Dean, School oflGraduate Studies

u

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

ROLE OF MITOGEN ACTIVATED KINASES IN CD40-MEDIATED T CELL

ACTIVATION OF MONOCYTE/MACROPHAGE AND VASCULAR SMOOTH

MUSCLE CELL CYTOKINE/CHEMOKINE PRODUCTION

by

Denise M. Milhom

Atherogenesis is characterized by the infiltration of immune competent cells such as monocytes/ and T cells to the site of lesion formation, resembling a chronic inflammatory response. Histological studies have shown the T cells present at these lesions are activated, CD 154^, CD4^ T cells of the Thl or inflammatory phenotype. The presence of these cells may be critical in regulating vascular due to their capacity to activate proinflammatory activities in CD40^ cells in the vasculature including monocytes/macrophages and vascular smooth muscle cells (SMC), through the CD40:CD1S4 receptor ligand interaction. This dissertation represents efforts to determine the functional consequences acquired by SMC in response to CD40 ligation by activated CD 154^ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC.

To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD 154*, CD4* T cells. The ability of CD40 signaling to initiate cytokine/chemokine mRNA and protein synthesis was assayed by RNAse protection assay, and enzyme linked immuosorbent assay (ELISA), respectively. Western blot analysis was used to determine the activation state of members of the mitogen activated protein (MAPK) family following CD40 stimulation. In addition, the activation of the transcription factor, NFkB, which has been demonstrated to contribute the the induction of cytokine/chemokine expression was analyzed by electromobility shift assay (EMSA). Analysis of SMC cell surface expression of receptors responsive to T cell ligands, and the analysis of CD40-mediated regulation of SMC adhesion and co-stimulatory molecules was analyzed by flow cytometry.

The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were shown to present antigen to, and activate T

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells. In turn T cell stimulus resulted in the activation of proinflammatory function in SMC initiated through the CD154:CD40 interaction. CD40 stimulation of SMC resulted in the production of the 8 (EL-8) and macrophage chemotactic protein-1 (MCP-1), and the upregulation of intercellular adhesion molecule (ICAM). Examination of the intracellular signalingpathways activated through CD40 signaling revealed the involvement of MAPKs in the pathway leading to induction of proinflammatory activity. Evaluation of CD40 signalingin monocytes demonstrated the activation of the MAPK flunily members ERKl/2, but not the MAPK family members p38 or c-jun-N-terminal kinase (INK). In contrast, CD40 signaling in SMC was shown to result in ERKl/2 and p38 activation, and both of these kinases were shown to play a critical role in the induction of chemokine synthesis. An examination of the ability of anti-inflammatory (tytokines to modulate CD40 signalingin monocytes and SMC demonstrated that the anti-inflammatorycytokines IL-4 and IL-10 abrogate CD40-mediated induction of production by monocytes. This inhibition was shown to be a result of a negative influence of IL-4 and IL-10 on CD40 mediated ERKl/2 activation in monocytes. However, IL-4 and IL-10 did not inhibit SMC proinflammatory responses indicating a difference in the intracellular responses to these by the two cell types.

These studies contribute to the understanding of the interaction between SMC and CD4* T cells through the receptor:ligand pair CD40:CD154, and its role in the maintenance of vascular inflammation The identification of the signaling components of the monocyte and SMC CD40 signaling pathways will identify control points that may serve as targets for anti-inflammatory therapies.

IV

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

To my advisor Dr. Jill Suttles, thank you for your guidance and patience. I know I

couldn’t have chosen a better mentor.

To Bob Miller, thank you for all the knowledge you’ve shared with me, teaching me

many immunological techniques, and for the great scientific discussions. Thank yon

for being a friend, for listening, and for your support through many trials.

Finally, I would like to acknowledge the professors that served on my graduate

committee. Dr. Bob Stout, Dr. Mitch Robinson, Dr. Lou Emest-Fonberg, and Dr.

Guha Krishnaswamy. I greatly appreciate your support, and taking the time from your

busy schedules to serve on my committee.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CONTENTS

Page

APPROVAL ...... ü

ABSTRACT...... iü

ACKNOWLEDGEMENTS...... v

LIST OF FIGURES...... vi

ABBREVIATIONS...... xü

CHAPTER

1. INTRODUCTION...... I

Cell Interactions and the Role of Cytokines And Chemokines in ...... 1

Structure and Function of CD40 ...... 5

MAPK Signaling...... 9

2. MATERIALS AND METHODS...... 15

Control of Endotoxin Contamination ...... 15

Inhibitors/Reagents ...... 15

Antibodies ...... 15

Cell L in es ...... 16

CD4* T Cell Purification and Activation ...... 17

Monocyte Isolation and Culture ...... 18

T Cell Plasma Membrane Preparation ...... 19

vi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Flow Cytometric Analysis ...... 19

Electrophoretic Mobility Shift Assay ...... 20 (EMSA)

Western Blot Analysis ...... 21

Analysis of Chemokine Production ...... 22

RNAse Protection A ssay ...... 23

Alloreactive Proliferation Assay ...... 23

3. RESULTS ...... 24

CD40 Signaling in Monocytes ...... 24

CD40 Activation of ERKl/2 in Monocytes ...... 24

IL-4 and IL-10 Inhibit CD40-Mediated Monocyte ERKl/2 Phosporylation ...... 26

Evidence of T cell-SMC Interactions ...... 28

SMC Express CD40 ...... 28

SMC Express CD95 ...... 30

SMC Present Antigen to CD4* T ...... 30

Influence of CD40 Signaling on SMC Chemokine Production ...... 32

CD40 Signaling in SMC Induces Cytokine and Chemokine mRNA Synthesis ...... 35

vu

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD40 Activation of ERKl/2 and p38 in SMC ...... 38

CD40-Mediated SMC IL-8 and MCP-1 Synthesis is Dependent Upon ERKl/2 and p38 MAPK Activity ...... 40

CD40 Signalin in SMC Results in Activation of the Trascription Factor NF-kB ...... 42

4. DISCUSSION...... 46

REFERENCES...... 55

VITA ...... 67

vm

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U ST OF FIGURES

Figure Page

1. Overview of Cell:Cell Interactions Occurring in Vascular Inflammation ...... 2

2. Jak/Stat Signaling Cascade...... 10

3. The ERKl/2, INK, and p38 MAPK Signaling Cascades ...... 11

4. CD40-Mediated Activation of ERKl/2 in Monocytes ...... 25

5. Effects of IL-4 and IL-10 on CD40-Mediated ERKl/2 Activation in Monocytes ...... 27

6. IFNy Up-regulates CD40 Expression on SM C ...... 29

7. SMC Express CD95 ...... 31

8. AUo-Antigen Presentation to CD4+ T Cells by SMC ...... 33

9. Requirement for PTK and PKC Activity in CD40- Mediated SMC IL-8 and MCP-1 Synthesis ...... 36

10. IL-8 Production by SMC is a CD40-Mediated Event ...... 37

11. CD40 Activation of Cytokine/chemokine mRNA ...... 39

12. CD40-Mediated Activation of ERKl/2 and p38 MAPK Family Members in SM C ...... 41

13. Requirement for ERKl/2 and p38 Activity in CD40- Mediated SMC IL-8 and MCP-1 Synthesis ...... 43

14. CD40-Mediated Activation of NF-kB in S M C ...... 45

15. Summary of CD40-Mediated Signaling Events in Monocytes and S M C ...... 54

IX

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABBREVIATIONS

SMC Smooth Muscle Cell

VSMC Vascular Smooth Muscle Cell

IL Interleukin

EMSA Electrophoretic Mobility Shift Assay

ELISA Enzyme-Linked Immunosorbent Assay

PTK Protein

PKC Protein Kinase C

NF-icB Nuclear Factor-kappa B

ERKl, ERK2 Extracellular Signal Regulated Kinase

MCP-1 Monocyte Chemotatic Protein 1

MHC-n Major Histocompatibility Complex Type II

mAb Monoclonal Antibody

Ab Antibody

JNK c-Jim-N-terminal Kinase

M EKl/2 MAPKÆRK Kinase 1,2

LPS

m sr-y -gamma

TmA Plasma Membranes from Activated CD4+ T Cells

TmR Plasma Membranes from Resting CD4-F T Cells

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ICAM Intercellular Adhesion Molecule

RPA RNAse Protection Assay

Grb2 receptor-bound protein 2

Sos Son of sevenless

PAK p2I Activated Kinase

MAPK Mitogen Activated Protein Kinase

MEKK MEK kinase

IkB I kappa B

PMA Phorbol Myristic Acetate

HB-EGF Heparin Binding Epidermal Growth Factor

bFGF Basic Fibroblast Growth Factor

XI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

INTRODUCTION

Cell Interactions and the Role of Cvtoldnes and Chemokines in Atherosclerosis

Atherogenesis is characterized by the infiltration of immune competent cells, such

as monocytes/macrophages and T lymphocytes at the site of a vascular lesion

resembling a chronic inflammatoryresponse. Tissue sections of atherosclerotic plaques

have revealed that the T lymphocytes at the site of the vascular lesion are activated,

CD 154*, CD4*T helper cells of the Thl or inflammatory phenotype (Mach and others

1998). These activated T cells may interact with cells in the vasculature through the

receptor-ligand interaction of CD 154, expressed on activated T cells with its receptor

CD40 expressed on a diverse group of cells including smooth muscle cells and

monocytes/macrophages (Figure 1). This interaction has been shown to result in the up

regulation of costimulatory and adhesion molecule expression, and the induction of

inflammatory cytokine and chemokine synthesis in monocytes, and may result in similar

functional consequences in SMC. We hypothesize that the interaction between

activated T cells and monocytes, and activated T cells and SMC through the receptor-

ligand pair CD40:CD154 results in inflammatorycytokine and chemokine synthesis

contributing to the pathogenesis of atherosclerosis.

The presence of activated CD4* T cells at atherosclerotic lesions has been well

established (Mach and others 1998). However the role of T cells in the maintenance

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ;;

MONOCYTES C040«(M BLOOD

ENDOTHELIUM

C04(KX}1S« IntandiaiM

-‘'-'I'-A M ED IA ,^: - - - ■

Figure 1. Overview of CeilrCeil Interactions Occurring in Vascular Inflammation. A

cross-sectional view of an artery illustrating cellzcell interactions which may be

occurring through the receptor-ligand pair CD40:CD154 during atherogenesis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and/or exacerbation of atherosclerosis has not been elucidated. Research thus far has

indicated that T cells appear early in the onset of atherosclerosis and localize to the site

of insult or injury to the artery. T cells transmigrate through the endothelium from the

media to the intima of the aorta where they have the ability to interact with many cells

in the vasculature through the receptor-ligand pair CD1S4:CD40. Recently there has

been increasing amounts of evidence that support a critical role of T cells in regulating

vascular inflammation or atherosclerosis (Mach and others 1998). Treatment with

antibodies against murine CD 154 limits atherosclerosis in susceptible mice, and the

atheroma of mice treated with anti-CD 154 antibodies contains significantly fewer

immune competent cells suggesting the involvement of inflammatory pathways and a

role for CD40 signaling in atherosclerosis (Mach and others 1998). Additionally, T

cells have the capacity to produce growth frctors such as basic fibroblast growth factor

(bFGF) and heparin binding epidermal growth factor (HB-EGF) which are potent

mitogens for SMC proliferation (Rolfe and others 1995), and several cytokines

including IFNy, a known co-stimulus for macrophage activation, which enhances

macrophage efrector function and induces or enhances MHC class H expression by

many cells including macrophages, endothelial cells and SMC.

Smooth muscle cells and monocytes/macrophages are thought to play a crucial role

in the process of atherogenesis. Both monocytes/macrophages and SMC have the

propensity to become lipid-laden foam cells which contribute to the formation of the

atherosclerotic plaque. Additionally, monocytes/macrophages and SMC may amplify

the inflammatory process resulting in atherosclerosis due to their ability to express

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. class n histoconqiatibility (MHC class ü) (Warner and Libby 1989). Class H

expression in atherosclerotic lesions in vivo has been well documented (Hansson and

others 1986) suggesting that antigen presentation may be occurring at atherosclerotic

lesions resulting in the activation of T cells. Antigens present in atherosclerotic plaques

may include modified lipoproteins, neoantigens revealed through tissue destruction and

determinants arising from infectious agents (Libby and Hansson 1991). Furthermore,

monocytes/macrophages and SMC have been shown to express CD40 which can

interact with CD 154 present on activated T cells. Recent research has shown that

ligation of CD40 on atheroma-associated cells (i.e. monocytes/macrophages and SMC)

in vitro activates fimctional attributes such as the production of cytokines, chemokines,

and adhesion molecules which are some of the critical components required for the

directional migration of leukocytes during inflammatory processes. The inflammatory

cytokines, (TNF) a and IL-1 p are both know to be involved in

inflammatory diseases and are produced by several cell types including

monocytes/macrophages and SMC. Interleukin (IL)-8 and monocyte chemotactic

protein-1 (MCP-1) are well characterized members of the C-X-C and C-C chemokine

subfamilies, respectively (Beasley and others 1995), are pro-inflammatory in nature,

and are produced by several cell types including vascular smooth muscle cells and

monocytes/macrophages. Histological studies have demonstrated these inflammatory

cytokines and chemokines to be present in atherosclerotic plaques, suggesting that they

may play a crucial role in the development, maintenance, and pathogenesis of

atherogenesis by promoting the directed migration of inflammatorycells (Warner and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Libby 1989; Wang and others 1998; Liu and others 1997; Terkeltaub and others 1998).

MCP-1 mRNA has been detected in several cells including macrophages, endothelial

cells and SMCs in atherosclerotic arteries of patients undergoing a bypass

revascularization (Seino and others 1995). Recent reports have shown that mice

deficient in MCP-1 that were fed a high cholesterol diet deposited less lipids and had

fewer macrophages present within their aortic walls as compared to the wildtype control

(Boring and others 1998). Similarly, mice deficient in the MCP-1 receptor, CCR-2,

had a overall decrease in atherosclerotic lesion size, and had significantly fewer

macrophages and monocytes present in the aortas as compared to the wildtype control

(Gu and others 1998). IL-8 is chemotactic for several cell types including T cells and

neutrophils at picomolar and nanomolar concentrations respectively (Banka and others

1991), and may be involved in the recruitment of T cells to the intima of a blood vessel

contributing to the pathogenesis of atherosclerosis (Larsen and others 1989).

Structure and Function of CD40

CD40 is a transmembrane glycoprotein of approximately 45kDa, a member of the

tumor necrosis receptor superfamily, which also includes the TNF receptor (TNFRl

and TNFR2), nerve growth factor receptor, and Fas (CD95). CD40 is expressed by a

variety of cell types including B cells (Clark 1990), monocytes/macrophages (Alderson

and others 1993), dendritic cells (Caux and others 1994), thymic epithelial cells (Galy

and Spits 1992), endothelial cells (Yellin and others 1995), kératinocytes (Peguet-

Navarro and others 1996), and smooth muscle cells (Mach and others 1998). CD40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. interacts with its ligand, CD 154, a type H membrane protein of approximately 39 kDa,

and a member of the TNF superfamily. CD 154 is preferentially expressed on

activated, but not resting T cells (Lynch and Ceredig 1989; Nbelle and Snow 1992).

Engagement of CD40 by its ligand CD154 has been reported to induce functional

changes on these cell types that may contribute to inflammatory responses, including

the secretion of pro-inflammatory (tytokines and chemokines, the up-regulation of

costimulatory and adhesion molecules (Stout and Suttles 1996), and eflects on cellular

proliferation (Yellin and others 1995). CD40 and its receptor CD 154 has been

identified as a receptor:ligand pair which œntributes to contact dependent signaling

between monocytes and T cells. The importance of CD 154 in vivo was revealed by

studies of X-linked Hyper-IgM Syndrome patients,. Hyper-IgM Syndrome is an

immuncxleficiency characterized by the absence of circulating IgA and IgG and the

absence of germinal centers, which is a result of defects in the gene encxxling CD 154

(Klein and Miedema 1995). CD40 was initially identified on the surface of B cells

Banchereau and others 1994) where CD40 signaling provides critical costimulatory

signals required for proliferation, isotype switching, germinal center formation, and the

generation of memory B cells (Grabstein and others 1993). The signaling pathways

activated through CD40 in B cælls involve activation of thesrc family PTK’s, PKC,

Jak3, serine/theonine kinases, MAPK family members (JNK, p38 and ERKl/2) and

PLCy2 (Aagaard-Tillery and Jelinek 1996; Padmore and others 1997; Hanissian and

Geha 1997).

In monocytes/macrophages, CD40 ligation has been shown to induce monocyte-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. derived inflammatorycytokine synthesis (Wagner and others 1994), nitric oxide

production (Stout and others 1996), and to rescue monocytes from undergoing

(programmed cell ) induced by the deprivation of serum factors (Suttles

and others 1996). Additionally, our laboratory has demonstrated that CD40-mediated

rescue from apoptosis is critically dependent on the activation of PTK but does not

appear to involve the serine/threonine kinase PKC family (Suttles and others 1996).

Our laboratory has also observed activation of the transcription factor Nuclear Factor-

kappa B (NF-kB) in response to CD40 signaling in monocytes. NF-kB is a

transcription frctor involved in the transcription of several genes. It is present in the

cytosol in an inactive state, complexed with the inhibitory I kappa B (IkB) proteins

(Baeuerle and Baltimore 1988; Beg and others 1993; Finco and others 1994).

Activation of NF-kB occurs through phosphorylation of iKB-a at serine 32 and 36,

resulting in the release and nuclear translocation of active NF-kB where it can bind

relevant gene promotors (Brown and others 1995; Brockman and others 1995; Chen

and others 1996). The release of active NF-kB results from the phosphorylated IkB

conjugating with ubiquitin followed by proteaosome-mediated degradation of IkB (Chen

and others 1996).

CD40 does not contain cytoplasmic sequences with catalytic activity and has been

shown to signal through cytoplasmic adaptor proteins of the TNF receptor-associated

factor family (TRAF), which activate intracellular signaling pathways (Dadgostar and

Cheng 1998). All TRAF family members contain homologous C-terminal TRAF

domains, TRAFs 2 through 6 contain an N-terminal ring finger and zinc finger-like

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8

domains (Dadgostar and Cheng 1998), and TRAF 3 and TRAF 5 contain an isoleucine

zipper domain (Cheng and others 1995; Ishida and others 1996; Nakano and others

1996). TRAFs 2, 5 and 6 interaction with CD40 has been associated with activation of

the transcription frctor NF-kB (Ishida and others 1996), however the disruption of

CD40 cytoplasmic sequences involved in TRAF interaction does not eliminate

NF-kB activation (Using and others 1997). Additionally, signalmg through CD40 can

initiate TRAF mediated activation of MAPK family members (Natoli and others 1997),

however the role of the TRAF family members in CD40-mediated activation of

monocytes/macrophages and SMCs has not been investigated.

Previous research in our laboratory has demonstrated modulation of CD40-mediated

monocyte inflammatory cytokine synthesis by the Th2-derived anti-inflammatory

cytokines IL-4 and IL-10 (Poe and others 1998). Several studies have shown inhibition

of monocyte/macrophage inflammatory function in response to lipopolysaccharride

(LPS) (Moore and others 1993). In vivo, it has been shown that these cytokines reduce

autoimmune inflammatory diseases, and IL-10 is being used in clinical trials to treat

autoimmune diseases (Horsfall and others 1997; Narula and others 1998). The

mechanism by which IL-4 and IL-10 inhibit inflammatorycytokine synthesis has not

been elucidated, however previous research in our laboratory suggested that the

effectiveness of these cytokines may result from the inhibition of the signaling

pathway(s) activated in response to CD40 ligation in monocytes. Our laboratory has

recently shown that IL-4 and IL-10 act early in the CD40 signaling cascade by

modulating the overall PTK induction in monocytes.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Both IL-4 and IL-10 signal through receptors which associate with and activate the

Janus family (Jak) tyrosine kinases upon ligation (O’Shea 1997; Leonard and O’Shea

1998). The Jaks in turn, activate proteins called Signal Transducers and Activators of

Transcription (Stats). The Stats translocate to the nucleus where they regulate the

expression of various genes (Darnell 1997) (Figure 2). The Stats are inducers of a

family of negative regulators of (tytokine signalmg, the suppressor of (tytokine signals

(SOCS) which work as a negative feedback system to inhibit the Jaks either directly or

non-directly (Nicholson and Hilton 1998). CD40-mediated activation of the Jaks in

monocytes has not been investigated, however in B cells studies have shown that CD40

may associate with Jak3 (Hanissian and Geha 1997). The data thus far indicate that IL-

4 and IL-10 inhibit the early PTK activity initiated from CD40 ligation suggesting that

EL-4 and IL-10 may induce protein tyrosine phosphatase (FTP) activity, resulting in the

suppression of the CD40 signaling pathway in monocytes, and thus suppression of

ERKl/2 activity.

MAPK Signaling

The mitogen-activated protein kinases (MAPK) family members are a group of

protein serine/threonine kinases that are activated in response to a variety of

extracellular stimuli and regulate a number of cellular responses. Along with other

signaling pathways, the MAPK hunily members mediate signal transduction by

differentially altering the phosphorylation status of transcription factors (Sivaraman and

others 1997; Das and Vonderhaar 1996; Tong and other 1997) (Figure 3). There are

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10

IL-4 IL-10

IL-4R IL-1 OR

Jaki, Jak3 Jaki, Tyk2

Slat 6

SOCS II Stat homo-hetrodimerization, transloction, and gene transcription

Figure 2. Jak/Stat Signaling Cascade

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11

PD98059

MEK-3 M EK -4 MEK1, MEK2 1 ERKl, ERK2

TRANSCRIPTION FACTOR ACTIVATION

Figure 3. The ERKl/2, JNK, and p38 MAPK Signaling Cascades. The inhibitor

PD980S9 inhibits Raf-1 phosphorylation of MEKl/2, and therefore the phosphorylation

of ERKl/2. The inhibitor SB203S80 inhibits phosphorylation of p38 MAPK.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12

three members of the MAPK family; extracellular signal regulated kinase (ERKl/2), c-

Jun-N-terminal kinase (JNtQ and p38. The ERKl/2 signaling cascade involves the

activation of receptor tyrosine kinases (which have src homology) which provide the

binding site for the adaptor protein, growth receptor-bound protein 2 (GRB2), which

localizes the guanine nucleotide exchange Actor, son of sevenless (SOS), to the plasma

membrane. SOS activates Ras by exchange of GTP for GDP (Winston and Hunter

1996; Sagata 1997; Takeuchi and others 1999; Hildt and Oess 1999). The Ras-GTP

activated Raf phosphorylates a dual specificiQr kinase MAPK/ERK kinasel/2

(MEKl/2), which is phosphorylated on serine 218 and 222 (Avruch and others 1994).

Active MEKl/2 dually phosphorylates ERKl/2 on theonine 183 and Qrrosine 185

(Burack and Sturgill 1997; Cobb and others 1996). Active ERK translocates to the

nucleus to phosphorylated a variety of transcription factors including Elk-1 (Gille and

others 1992; Cheng and others 1996), C/EBPp (NF-IL-6) (Nakajima and others 1993),

ATF-2 (Abdel-Hafiz and others 1992), and NF-kB (Dadgostar and Cheng 1998).

The INK signaling pathway is activated in several ways including exposure to UV

radiation, heat shock, or inflammatory cytokines (Su and Karin 1996; Hibi and others

1993) and is mediated by Rac and cdc42, two small GTP binding proteins (Davis

1994). Activated cdc42 binds to PAK65 which activates MEK kinase (MEKK-1).

MEKK-1 phosphorylates MEKK-4 at serine 219 and serine 223. The active MEKK-4

phosphorylates JNK which translocates to the nucleus and activates a variety of

transcription factors including c-Jun (Kyriakis and others 1994; Derijard and others

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13

1995), ATF-2 (Gupta and others 1995) and Elk-1 (Cavigelli and others 1995).

The p38 MAPK Amily member is activated in response to several stimuli including

inflammatory cytokines, endotoxins and osmotic stress (Tong and others 1997; Han and

others 1994; Rouse and others 1994). P38 is activated by MEK kinase kinase-3

(MKK3) a dual specificity kinase. Following phosphorylation p38 translocates to the

nucleus and phosphorylates and activates transcription Actors including ATF-1, CREB

(Tan and others 1996), Elk-1 (Price and others 1996), CHOP (Wang and Ron 1996),

and ATF-2 (Raingeaud and others 1996).

The sites of phophorylation are conserved between the MAPK family

members, however, these sites are located within distinct dual specificity

phosphorylation motifs; TEY for ERK, TPY for JNK, and TGY for p38 (Davis 1994;

Derijard and others 1994; Raingeand and others 1995). Additionally, the MAPK

family members share sequence homology -45% sequence homology between JNK and

ERK (Moriguchi and others 1996), and -50% homology between p38 and ERK (Tong

and others 1997) .

This dissertation represents the results of studies designed to evaluate the role of

CD40 signaling in two key cells found at atherosclerotic lesions, SMC and

monocytes/macrophages, which contribute to the maintenance and/or exacerbation of

vascular inflammation. We investigated the functional activities expressed by SMC in

response to CD40 ligation, and critical components involved in CD40-mediated SMC

chemokine synthesis. Additionally we examined the role of MAPK family members in

CD40-mediated synthesis of inflammatory cytokines and chemokines in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14

monocytes/macrophages and SMC. and evaluate the role of anti-inflammatory qrtokines

on CD40-mediated activation of ERKl/2 MAPK family member(s), a critical

component in CD40-mediated induction of mono

monocytes and IL-8 and MCP-1 synthesis in SMC. Gaining a better understanding of

CD40-mediated activation of specific kinases, transcription Actors and inflammatory

cytokines and chemokines, and how these events can be modulated by natural medAtors

will help identify potential targets for drug intervention.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER!

MATERIALS AND METHODS

Control of endotoxin contamination

All cell culture reagents used were either certified as low endotoxin when

purchased, or were ensured low endotoxin as determined by chromogenic limulus assay

(BioWhittaker, Walkersville, MD). Stock solutions containing > 1 ng/ml (10

endotoxin units/ml) were considered unacceptable. Stock solutions were diluted in

assays such that endotoxin levels did not exceed 1 pg/ml.

Inhibitors/ Reagents

The MEK1/MEK2 inhibitor PD98050 was obtained firom New England BioLabs,

Inc. (Beverly, MA). The p38 MAPK inhibitor SB203580, the PTK inhibitor

Herbimycin A and the PKC inhibitor RO-31-75499 were purchased from Calbiochem

(La Jolla, CA). H7 was purchased from Sigma Chemical Co. (St, Louis, MO). IL-4

and IL-10 were purchased from R&D Systems (Minneapolis, MN). Sodium

orthovanadate (Na^VOJ was acquired from Fisher Scientific, (Fair Lawn, NJ).

Antibodies

The following mAB's were prepared form culture supernatants of hybridomas

purchased form American Type Culture Collection (ATCC, Rockville, MD): IgG

15

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16

mouse anti-human CD3 (OKT-3), IgG mouse anti-human CD8 (OKT-8), IgG mouse

anti-human monocyte (3C10), IgG mouse anti-human B cell (LYM-1), IgM mouse anti­

human NK cell (hNK-1), and IgG mouse anti-human CD40 (G28-5). BioMag* goat

anti-human IgG and IgM was obtained from PerSeptive Diagnostics, Inc. (Cambridge,

MA). FITC-conjugated donkey anti-mouse IgG (H + L) was purchased from Jackson

Immunoresearch Laboratories, Inc. (West Grove, PA). IgG mouse anti-human CD154

mAh was obtained from Genzyme (Cambridge, MA). Rabbit antibodies recognizing

the active, phosphorylated (Thr‘“ , Tyr‘“) form of ERKl/2 were acquired from

Promega (Madison, WI) and New England BioLabs. Rabbit antibodies recognizing

phosphorylated p38 (Thr‘“ , Tyr‘“ ) and phosphorylated JNK (Thr^“ , Tjrr*“) were

purchased from New England BioLabs. Horseradish peroxidase-conjugated F(ab ')2

donkey anti-rabbit AB was purchased form Jackson ImmimoResearch Laboratories

(West Grove, PA). Monoclonal mouse anti-human CD 154 was prepared from culture

supernatant of the hybridoma 5C8 (American Type Culture Collection, Rockville,

MD). The monoclonal mouse IgG anti-human CD40 used for flow cytometry was

prepared from culture supernatant of the hybridoma G28-5 (American Type Culture

Collection, Rockville, MD). The FITC-conjugated donkey anti-mouse IgG (H 4- L)

was purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA).

Cell Lines

Cell lines used in these studies included 293 (human embryonic kidney, ATTC),

stable transfectants of 293 which express high levels of CD154 (Yellin and others

1994), the human T cell leukemia line Jurkat (ATCC) and the CD 154^ subclone of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17

Jurkat, D l.l (Yellin and others 1991). Both D l.l and 293-CD154 transfectants were

gifts of Dr. Seth Lederman, ColumbA Universify. Cell lines were maintained in RPMI

1640 (Hyclone, Logan, UT), containing l(X)mM HEPES, SO pg/ml gentamicin, and

5 % fetal bovine serum (FBS) (R-5). The 293-CD154 transfectants (created by co­

transfection with pcDNA 1-CD154 and pRSVneo) were periodically passaged in 200

pM G418 (Gibco/BRL). The hiunan aortic smooth muscle cell line (SMC) was

obtained from Clonetics Corporation (San Diego, CA). The SMC line designated

AOSMC 2634-3 by Clonetics, was characterized phenotypically as SMC and found to

be virus-free. The line was maintained in SmGM*-3 BuUetKit* (Clonetics) containing

0.5pg/ml human recombinant Epidermal Growth Factor, 5mg/ml Insulin, 1 pg/ml

human recombinant Fibroblast Growth Factor, 50mg/ml Gentamicin, and 2% FBS

(Clonetics Corporation). The SMC cell line was rested in Human Endothelial-SFM*

Gibco/Life Technologies, Grand Island, NY). SMC were cultured in 25cm tissue

culture flasks (Coming, Incorporated, Coming NY).

CD4^ T cell Purification and Activation

CD4^ T cells were purified by negative magnetic panning from elutriation-enriched

T cell populations. Cells were incubated in R-5 with mAB's against cell surface

molecules generated from the hybridomas OKT-8 (anti-CD8^ T cell), 3C10 (anti­

monocyte), LYM-1 (anti-B cell), and hNK-1 (anti-NK cell), used as culture

supematants at dilutions of 1:10, for 30 min at RT. Cells were then treated with

BioMag® iron-conjugated antibodies to murine IgG and IgM (PerSeptives Diagnostics,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18

Cambridge, MA) for 30 min with gentle shaking at 4°C. Cells were diluted with DPBS

in 75 cm^ flasks (Fisher Scientific) and the CD4 populations were removed via 27

megagauss Oerstead magnets (PerSeptives DAgnostics). A sample of the purified

population was stained with an FITC conjugated anti-CD4 mAh and analyzed by flow

cytometry on a FACSTAR* flow cytometer (Becton Dickinson, San Jose, CA).

Resulting populations were typically found to be greater than 95% CD4 . CD4^ T

cells were then rested in R-5 alone, or activated for 6 h in R-5 by mcubation with

lOng/ml phorbol myristic acetate (PMA) (Sigma) and 0.5pM ionomycm (Calbiochem,

San Diego, CA). Expression of CD 154 on activated, but not restmg, CD4^ T cells was

then confirmed by flow cytometric analysis on a FACSTAR* plus flow cytometer

(Becton Dickinson).

Monocvte Isolation and Culture

Blood was collected from normal, healthy human volunteers and PBMC's were

isolated over a Ficoll densify gradient (Fico-Lite-LymphoH, Atlanta Biologicals,

Norcross, GA). PBMC's were plated at a density of 5 x 10® cells/well in 24-well tissue

culture plates (Falcon Primaria, Lmcoln Park, NJ) in RPMI 1640 (Hyclone,

Logan, UT), containing 100 mM HEPES, 50 pg/ml gentamicin, and 5% FBS (R-5).

Monocytes were isolated by plastic adherence for 1 h at 37 °C after which nonadherent

cells were removed by Pasteur pipetting during 2 washes with Dulbecco's PBS (DPBS).

Cells were maintained in R-5 overnight prior to treatment.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19

T-Cell Plasma Membrane Preparation

For the preparation of purified plasma membranes, resting and activated purified

CD4^ T cells were resuspended in a hypotonic buffer containing SO mM Tris-HCL, pH

7.4, 25 mM KCl, 5 mM MgClz, and 50 pg/ml phenolmethylsulfonyl fluoride (PMSF)

for 30 min on ice. The cells were then homogenized using a PowerGen 35

homogenizer (Fisher Scientific) until completely disrupted as determined

microscopically. Disrupted cells were centrifuged at 500 x g for 5 min to remove

nuclei, then centrifuged at 95,(X)0 x g for 30 min using a Ti-50 rotor in a Beckman L5-

65 Ultracentrifuge. Cell debris was resuspended in 35% (wt/vol) sucrose/hypotonic

buffer then layered on 73% (wt/vol) sucrose/hypotonic buffer. Hypotonic buffer was

layered on the 35% sucrose and the samples were centrifuged using a SW50.1 rotor at

130,000 X g for 1 h to separate plasma membranes. The plasma membrane layer (at the

73%-35% interface) was collected and diluted 1:5 with hypotonic buffer, then

centrifuged again for Ih at 130,000 xg to pellet purified plasma membranes. The

membrane pellets were resuspended in PBS and total protein was determined by

microtiter plate protocol of the bicinchoninic acid (BCA) protein assay (Pierce,

Rockford, IL). The BCA protein assay was read on a Biotek Instruments microtiter

plate reader at 561nm.

Flow Cvtometric Analvsis

Smooth muscle cells were harvested from tissue culture flasks with .025%

Trypsin/EDTA (Clonetics Corporation). SMC were labeled with the appropriate

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20

dilution of the primary antibody 30 min at room tenq>erature, washed in DPBS, and

stained with a FITC-conjugated donkey anti-mouse IgG (H + L) secondary antibody

for 30 min at RT. Cells were washed in DPBS and analyzed for CD40, CD95 or

ICAM surface expression on a FACSTAR* plus flow cytometer (Becton Dickinson, San

Jose, CA).

Electrophoretic Mobility Shift Assay (EMSA)

After treatment or stimulation as required by the particular assay, cells were

collected and lysed to isolate nuclei based on the method of Bturas and others (1994).

Briefly, cells were lysed in hypotonic buffer containing 10 mM KCl, 0.3 M sucrose, 10

mM P glycerol phosphate, 0.2 mM EDTA, 0.4% nonidet P-40, 1 mM PMSF and 1

;fg/ml each of leupeptin and pepstatin, on ice for 30min with gentle agitation.

Following lysis, nuclei were collected by centrifugation at 11,500 x g for 5min.

Nuclear proteins were extracted by incubating nuclei in a buffer containing 25%

glycerol, 0.3 M KCl, 1.5 mM MgCl;, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM

PMSF, and 1 /tg/ml each of leupeptin and pepstatin for 30 min on ice. Extracts were

centrifuged at 11,500 x g and supematants were collected as nuclear extracts. Protein

determinations were performed by the BCA micro protein assay. Extracts were

aliquoted and stored at -80° C. Binding assays were performed using a gelshift assay

consensus oligonucleotide containing the binding element for NF-kB (Geneka,

Quebec, Canada) that was end-labeled using ^P-gamma ATP (Amersham Pharmacia

Biotech, Inc., Piscataway, NJ). Nuclear extracts were incubated with gel shift binding

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21

buffer and end-labeled oligonucleotide at approximately 5x10® cpm. Binding

reactions were done per Geneka protocol. Following binding reactions, samples were

analyzed on 5% polyacrylamide gels. Following electrophoresis, gels were dried onto

Whatman filter paper and exposed to x-ray film fi-om several hours to overnight at

room temperature. X-ray films were analyzed by scanning densitometry using the UN-

SCAN-IT-gel automated digitizing system. Silk Scientific Corp., Orem, UT.

Western Blot Analvsis

Prior to stimulation, monocytes were pretreated with 50#tM Na^VO,* for 20 min to

negate FTP effects on tyrosine-phosphorylated cellular proteins during stimulation.

After monocyte or smooth muscle cell treatment/stimulation in 24-well plates, cells

were lysed in 50 /xl boiling treatment buffer (125 mM Tris, pH 6.8, 2% SDS, 20%

glycerol, 1% beta-mercaptoethanol, and 0.003% bromophenol blue) containing 1 mM

each PMSF and sodium orthovanadate. Samples were boiled an additional 5 min prior

to protein separation by SDS-PAGE on 15% minigels. Gels were equilibrated in

transfer buffer (48 mM Tris, pH 9.2, 39 mM glycine, 1.3 mM SDS, and 20%

methanol) for 15 min prior to transfer. Protein transfer to nitrocellulose membranes

(Coming Costar Corp., Kennebunk, ME) was performed at 15V for 30 min using a

Trans-Blot* SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Richmond, CA).

Dried membranes were blocked by gentle agitation in PBS containing 0.1% Tween 20

and 1 % BSA (blocking buffer) for 30min at 37° C. Membranes were incubated with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22

the relevant primary Ab in blocking buffer for 30 min at 37° C, and then washed for 10

min x2 washes in PBS containing0.1% Tween 20 (wash buffer), followed by

incubation with the relevant (HRP-conjugated) secondary Ab for 30 min at 37 °C (if

primary antibody not HRP-conjugated), and then washed for 10 min x3 in wash

buffer. Ab-bound proteins were detected using an ECL™ Western blotting analysis

system (Amersham Corp.. Arlington Heights, IL) and the membranes were exposed to

Kodak X-Omat LS X-ray film (Eastman Kodak, Rochester, NY). X-ray films were

analyzed by scanning densitometry using the UN-SCAN-IT-gel automated digitizing

system.

Analvsis of Chemokine Production

Analysis of IL-8 and MCP-1 production by SMC stimulated through CD40, was

examined by ELISA. SMC were plated in 96 well microtiter plates and rested for 10

days in serum free medium. The cells were then treated with IFNy for 5 days to up-

regulate CD40 expression. SMC were co-incubated with activated T cell membranes

(TmA), resting T cell membranes (TmR), 293 parent cell line, 293-CD154

transfectants, Jurkat cell line, or D l.l cells. Supematants were harvested after an 18-h

incubation and assayed by enzyme-linked immunosorbant assay (ELISA) using R&D

system’s Quantikine™ human enzyme linked immunosorbant assay system or OptEIA™

sets for human IL-8 and MCP-1, PharMingen (San Diego, CA).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23

RNAse Protection Assay

Following stimulation of SMC, cells were lysed and RNA was isolated using TRIzol*

Reagent (Life Technologies/Gibco) per TRIzol* protocol. The Riboquant* Multi-Probe

(PharMingen) RNAse protection assay system was used to detect mRNA species. The

multi-probe was labeled with a-®^P-UTP (Amersham), and hybridized to the RNA.

RNAse treatment and purification of protected probes was performed per Riboquant*

protocol. Samples were analyzed on ()uickPoint™ precast gels (NOVEX, San Diego,

CA), and exposed to Kodak X-Omat LS X-ray film.

Alloreactive Proliferation Assay

A total of 4x10® SMC per well were plated in 96-well tissue culture plated in SMGM*-

3, and lOOU/ml recombinant human IFNy was added after overnight culture. On day

3, cultured cells were washed with PBS, and treated with SO ^g/ml mitomycin C

(Sigma Chemical Co.) to ensure that they were unable to contribute to the

measurement of [®H]-thymidine incorporation used to assay T cell proliferative

responses. In some experiments, the SMC were fixed, with freshly prepared 0.8%

paraformaldehyde at room temperature (RT) for 10 minutes, rather than mitomycin C-

treated. The cells were washed with PBS prior to addition of 2x10* human CD4^ T

cells per well with or without IL-1 (1% P388D1 supernatant) as a source of costimulus.

Three days after addition of the T cells, the cells were pulsed with 0.5 /iCi/well

[®H]thymidine for 3 h and harvested on glass fiber filters for scintillation counting.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTERS

RESULTS

CD40 Signaling in Monocvtes

CD40 Activation of ERKl/2 in Monocvtes

Previous research done in our laboratory demonstrated that CD40-mediated

monocyte inflammatory cytokine synthesis was dependent on activation of PTKs (Poe

and others 1998). To further investigate the activation of PTKs, the role of MAPK

family members in T cell activation of monocytes through CD40 signaling was

evaluated. Stimulus was provided by purified plasma membranes from CD4^ T cells

which had been activated for 6 hours (a time point at which CD 154 expression was

optimal) designated TmA. Purified plasma membranes from resting (CD 154 ) T cells,

designated TmR, were used as a control. Plasma membrane preparations were titrated

for activity based on membrane protein concentrations. In the experiments presented in

this dissertation, membranes were used at lOpg of protein/ml. The ability of TmA to

induce MAPK phosphorylation was evaluated over a 2 hour time period postactivation.

ERKl/2 phosphorylation was evident at 10 minutes, and declined at 2 hours (Figure

4A, top panel). The blot was stripped and reprobed with antibody recognizing both

phosphorylated and nonphosphorylated forms of ERK present demonstrating that the

differences observed were not due to differences in the level of ERKl/2 protein, or

artifacts of gel loading (Figure 4A, bottom panel).

24

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Phospho*ERK1/2

Total ERKl/2

B r Phospho-p38

Phospho-JNK

Figure 4. CD40-Mediated Activation of ERKl/2 m Monocytes. A, analysA of ERKl/2

phosphorylation . Monocytes were left unstimulated or stimulated with LPS for 30

minutes, or TmA for 10, 30, 60 or 120 minutes. After stimulation lysates were

harvested and analyzed by western blot using an antibody specific for the

phosphorylated form of ERKl/2 (top panel) or antibodies recognizing total ERKl/2

(bottom panel). B, analysis of p38 and JNK phosphorylation. Monocytes were

stimulated as in A, lysates were harvested and analyzed by western blot using

antibodies specific for the phosphorylated forms of p38 and JNK.

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Although LPS stimulation resulted in the activation of the MAPK family members p38

and JNK, treatment of the monocytes over the same time period examined in 3A did not

induce or enhance p38 or JNK phosphorylation (Figure 4B). These data suggested that

CD40 signaling in monocytes resulted in the phosphorylation of the MAPK Amily

member ERKl/2, but not in the phosphorylation of p38 or JNK over the time period

examined (Suttles and others 1999).

IL-4 and IL-10 Inhibit CD40-Mediated Monocvte ERKl/2 Phosphorvlation

In previous work, IL-4 and IL-10 were found to inhibit CD40-induced activation of

PTK activity and the subsequent synthesis of inflammatory cytokines (Poe and others

1997). Additionally our previous work indicated a role of MEK and ERK activation in

this pathway, therefore we hypothesized that IL-4 and IL-10 acted by interference of

the signaling cascade leading to ERKl/2 activation. To test this hypothesis, monocytes

were pretreated for an 18-h period with IL-4 or IL-10 prior to TmA stimulation and the

cell lysates were assayed for phosphorylation of ERKl/2. ERKl/2 activation was

decreased in a dose-dependent manner with both IL-4 and IL-10 (Figure S).

Quantification by scanning densitometry indicated that a dose of 50 ng/ml IL-4 resulted

in 79% decrease in the level of phosphorylation observed over background in response

to TmA. A 90% decrease was observed with treatment of IL-10 at 50 ng/ml. Neither

of these cytokines affected the level of expression of ERK’s during this time period as

shown by Western blots of the same samples, reprobed with an antibody reactive with

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TmA •!> IL-4 TmA + IL-10 I______////// Phospho-ERKI Pho*pho-ERK2

800

600

ë 400 -

"5 200 -

Total ERK1 Total ERK2

Figure 5. Effects of IL-4 and IL-10 on CD40-Mediated ERKl/2 Activation in

Monocytes. Monocytes were pretreated with IL-4 or IL-10 at 5, 10, and 50 ng/ml, as

indicated, then left unstimulated (control) or stimulated with TmA for 30 minutes. Cell

lysates were harvested and analyzed by western blot for level of ERKl/2

phosphorylation using anti-phospho-ERKl/2 antibodies (top panel). The film was

scaimed and the digitized image analyzed for band density. The histogram represents

density (total pixels minus background x 10 ^) of the ERK2 bands (middle panel). The

membrane was stripped and reprobed using antibodies recognizing total ERK (bottom

panel).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28

total ERKl/2, which show equivalent levels of ERKl/2 regardless of treatment (Fig

5,bottom panel).

Evidence of T Cell-SMC Interactions

SMC Express CD40

Monocytes can interact with activated CD4^ T cell through several receptorrligand

pairs including the interaction of CD40 on monocytes with its natural ligand CD 154

present on activated CD4^ T-cells. This interaction results in the up-regulation of pro-

inflammatory cytokine synthesis. Both monocytes and SMC appear to play a role in

vascular inflammation by interacting with T cells. We next examined SMC for CD40

and other receptors which have the ability to interact with T cells similar to monocytezT

cell interactions. Using the monocytezT cell model we hypothesized that vascular

smooth muscle cells may interact in a similar way contributing to the development of

vascular inflammation. To address the hypothesis we phenotyped the SMC to

determine the expression of the cell surface molecule CD40. SMC were treated over a

period of 0 to 5 days with Thl cytokine IFN-y, which is a costimulus for macrophage

activation and has been shown to up-regulate MHC-II expression in other cell lines.

Cells were harvested and stained with anti-CD40 antibodies and analyzed by flow

cytometry for CD40 expression (Figure 6). Untreated cells were negative for CD40

expression. However, IFNy treatment induced CD40 expression which increased over

the five day period. The experiment was carried out through day 7

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ISO

lu

FLUORESCENCE (CO40 EXPRESSION) B

ri d iÿ l

î / v j S itf li* iÿ il/ itf t ‘ lcr 10 IF it FLUORESCENCE (CO40 EXPRESSION) FLUORESCENCE (CO40 EXPRESSION) FLUORESCENCE (CO40 EXPRESSION)

•0 * ’ 10 10* 10* l £ f 1 0 W " i o * i t 'If i5 i6* w* FLUORESCENCE (CO40 EXPRESSION) FLUORESCENCE (0040 EXPRESSION) FLUORESCENCE (CO40 EXPRESSION) Figure 6. IFNy Up-regulates CD40 Expression on SMC. A, CD40 surface expression

on SMC. SMCs were left untreated or treated with lOOU/ml of IFNy for 3 days. Cells

were harvested and stained for CD40 surface expression. Cells were analyzed by flow

cytometric analysis. B, Kinetics of IFNy treated on CD40 surface expression on SMC.

SMC were left untreated or treated with IFNy over time period of 1 to 5 days. Cells

were harvested and analyzed by flow cytometric analysis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30

(data not shown), however, expression declined consistently after day 5.

SMC Express CD95

Monocytes can interact with activated T ceils through several receptorrligand pairs

in addition to the CD40:CD1S4 interaction. Monocytes express CD95 (Fas), a

member of the TNF receptor superAmily. CD95 binds to its ligand CD95L (Fas

ligand) expressed on T cells. This interaction can lead to programed cell death or

apoptosis. Apoptosis of SMC has been demonstrated in vivo and in vitro (Bennett and

others 1995). Apoptosis of SMC observed in atheroma suggest that this process

contributes to vascular remodeling that results in intimai thickening and fibrous cap

formation. We hypothesized that SMC express CD95, which could interact with T cells

through the CD95:CD95L receptorrligand interaction similar to the monocyte-T cell

model, resulting in apoptosis, and vascular remodeling. To address this hypothesis,

SMC were analyzed for CD95 expression by flow cytometry (Figure 7).

SMC Present Antigen to CD4^ T cells

Monocytes have the abiliQr to display peptide-MHC complexes in a form that can be

recognized by T cells, and are known as classical antigen presenting cells (APC). The

interaction of T cells with monocytes through antigen presentation results in reciprocal

activation involving both contact-dependent and cytokine-mediated signals.

Since the possible SMC-T cell interactions seem to resemble the monocyte-T cell

model, we were interested in exploring the possibiliQr of antigen presentation by SMC

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31

64 (£. UJ OQ 2 3 Z _l _l UJ Ü Ui >

5 UJ 0:

10" 10' 10" 10' 10* FLUORESCENCE (FAS EXPRESSION)

Figure 7. SMC Express CD95. Analysis of cell surface expression of CD95(Fas) on

SMC. SMC were left untreated or treated with IFNy for 5 days. After 5 days of

treatment the cells were were harvested, stained for sur&ce CD95, and analyzed by

flow cytometric analysis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32

to T cells. Previous work done in the murine system has shown that pure populations

of vascular smooth muscle cells (VSMC) clones are capable of antigen processing and

presentation via the exogenous pathway to syngeneic or histocompatibili^ matched

CD4^ T cells. We have hypothesized that human SMC are also capable of antigen

presentation to allogeneic CD4^ T cells. To determine this human SMC were treated

with lOOU/ml IFN-y for 3 days, which had been shown in previous experiments to

optimally induce expression of class H MHC in monocytes. SMC were IFN-y primed

for three days, washed with DPBS and treated with mitomycin C to ensure that the cells

were unable to contribute to the measurement of [^H]-thymidine incorporation or fixed

with 0.8% paraformaldehyde. The SMC were co-cultured with human allogeneic

CD4^T cells in the presence or absence of IL-ip as a source of co-stimulus. On day 3

of culture, wells were pulsed with [^H]-thymidine, and SMC were evaluated for their

ability to activate the T cells by allo-antigen presentation. CD4^T cells incubated in the

presence of fixed allogeneic SMC proliferated excessively as compared to the

mitomycin C treated group (Figure 8). These data indicate that the SMC are capable of

allo-antigen presentation to CD4^ T cells, and therefore reciprocal activation.

Influence of CD40 Signaling on SMC Chemokine Production

Our laboratory has demonstrated that CD40 signaling in monocytes results in the

activation of inflammatory cytokine synthesis and rescue from apoptosis. We have also

demonstrated that both of these events are critically dependent on the activation of PTK

and but does not exhibit dependence on the activi^ of the PKC family (Poe and others

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Fixed AOSMC + T cells Mito-C treated AOSMC + T cells CD4* T cells, alone

+ IL-1

1000 2000 3000 4000 5000 CPM ^H-Thymidine Incorporation (T cell proliferation)

Figure 8. Alio-Antigen Presentation to CD4^ T Cells by SMC. SMC were plated in

96-well culture plates, IFNy treated, and either paraformaldehyde fixed or treated with

mitomycin C. 10* allogeneic CD4^ T were added to the SMC cultures in the presence

or absence of IL-ip (1%P388D1 supernatant). On the third day T cell proliferation

was assayed by [*H]thymidine incorporation. The average cpm [*H]thymidine

incorporation ± standard deviation of triplicate samples.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34

1997) However, the activi^ of PTK and the activity of the serine/threonine PKC

family has been demonstrated in CD40 signaling in B cells. We were interested in

determining the nature of kinase involvement in CD40 signalingin SMC. CD40-

mediated IL-8 and MCP-1 production were evaluated in SMC pretreated with inhibitors

of PTK and PKC activiQr. SMC were treated either with herbimycin A, a potent, broad

PTK inhibitor, or H-7, an inhibitor of PKC, prior to CD40 stimulation. The inhibitors

were used at concentrations based on previous experiments (Suttles and others 1996),

and caution was exercised to ensure that the concentrations used did not affect cell

viabiliQr and total protein synthesis. Stimulus was provided by plasma membranes

purified from CD4^ T cells which had been activated for 6 hours (a time point at which

CD 154 expression was optimal) as well as by co-culture of SMC with CD 154

transfectants, or the CD 154^ Jurkat T cell variant, D l.l (Yellin and others 1991;

Yellin and others 1994). SMC were pretreated with or without 0.5 pg/ml HerbA and

2 pg/ml H-7 for -18 hours and then stimulated through CD40 with plasma membranes

from either activated (CD 154^ ) CD4^ T cells designated TmA or CD 154-transfected

293 cells. Supematants were collected for analysis at 18 hours. Controls consisted of

supematants from SMC stimulated with resting (CD 154 ) CD4^ T cells designated

TmR or the 293 parent cell line. SMC EL-8 and MCP-1 synthesis was

induced by co-culture with TmA and the CD 154 transfectants, but not by co-culture

with TmR or the control 293 population. Furthermore both IL-8 and MCP-1

production was suppressed by both HerbA and H-7 which suggests that both PTK and

PKC activity is required for CD40-mediated activation of SMC IL-8 and MCP-1

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35

production (Figure 9). Additionally, we demonstrated that activation of SMC induced

by co-incubation with CD154 transfectants is inhibited by the addition of anti-CD 154

antibodies confirming that the primary activating component of this interaction is the

CD40 ligand, CD154 (Figure 10). These data suggest that CD40-mediated IL-8 and

MCP-1 synthesis in SMC is a CD40-mediated event and dependent on both PTK and

PKC activity.

CD40 Signaling in SMC results in Cvtokineand Chemokine mRNA Svnthesis

SMC have the capacity to play an active role in vascular inflammation by their

ability to produce a variety of cytokines including IL-1, TNF-a, IL-6, GM-CSF, IL-8,

MCP-1, and monocyte chemotatic and activation factor (MCAF) (Ross 1993). The

production of these cytokines by SMC may contribute to the recruitment and activation

of leukocyte populations, resulting in vascular inflammation and plaque formation.

Since we observed that CD40 signaling in SMC resulted in the induction of the

inflammatory chemokine synthesis, we next addressed the question how specific is

CD40-mediated chemokine and cytokine synthesis in SMC. To address this question

SMC were stimulated with TmA for various time points, and the mRNA was extracted

and evaluated for a panel of inflammatory cytokines and chemokines by an RNAse

protection assay as described in materials and methods. We previously observed that

PTK, PKC, MEK/ERK and p38 were involved in CD40-mediated induction of IL-8 and

MCP-1 in SMC, therefore we hypothesized that activation of these kinases had an effect

on CD40-mediated cytokine/chemokine mRNA synthesis. To determine the

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Control TmA TmR H-7 HerbA

0 15003000 4500

Picograms/ml IL-8

Control

HerbA

Picograms/ml MCP-1

Figure 9. Requirement for PTK and PKC Activity in CD40-Mediated SMC IL-8

and MCP-1 Synthesis. SMC were pretreated with the PTK inhibitor, HerbA

(0.5^g/ml) or the PKC inhibitor H-7 (2^g/ml) for 18 hours. SMC were either

stimulated with 293-CD154 cells or the control parent 293 cell line. After 18 hours

stimulation, supematants were removed and analyzed by ELISA.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37

CD40-MEDIATED SMC IL-8 PRODUCTION

Control 293 2 9 3 -0 0 1 5 4 lOpg/ml anti-CD154 isotype control

5 0 0 1000 1 5 0 0

pg/ml IL-8

Figure 10. IL-8 Production by SMC is a CD40-Mediated Event. SMC were left

unstimulated (control) or stimulated for 18 hours with 293 parent cell, CD 154

transfectants, or CD 154 transfectants + antibody, as indicated. Supernatants were

assayed for lL-8 production by ELISA.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38

involvement of PTK, PKC, ERKl/2 and p38 activiQr in CEMO-mediated induction of

cytokine/chemokine mRNA synthesis, SMC were pretreated with or without the PTK

inhibitor. Herb A, the PKC inhibitor H-7, the MEK/ERK inhibitor PD98059, and the

p38 inhibitor SB203S80, and stimulated at several time points with TmA (figure 11).

Synthesis of TGPp-l, lL-8 and IL-IRA was observed with SMC stimulated with TmA.

Inhibition of the cytokines/chemokines was seen with PD98059, SB203580 and HerbA

treatment, but not with H-7. These data indicate that CD40 signaling in SMC results in

the induction of cytokine/chemokine mRNA synthesis, and that activation of the

MEK/ERK, p38 and possibly other pathways are involved.

CD40 Activation of ERKl/2 and p38 in SMC

Previous studies firom our laboratory demonstrated that CD40 signaling in

monocytes is dependent on the activation of the ERKl/2 MAPK family members

(Suttles and others 1999). In contrast, in B cells previous research has shown that

CD40 signalinginvolves activation of the MAPK family members INK, p38 and

ERKl/2 (Swantek and others 1997; Sanghera and others 1996). We next addressed the

question are the MAPK signalingpathway(s) activated in response to CD40 ligation in

SMC. All three MAPK family members; ERKl/2, p38 and INK were considered as

candidates. To evaluate the ability of CD40 stimulation to activate the MAPK family

members, SMC were treated with TmA or TmR over a 2 hour time period post­

activation. After a 10 to 120 minute incubation period, cell lysates were harvested and

analyzed for MAPK activation by Western blot using antibodies specific for the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39

1 2 3456789

IL1-P IL1-RA

IL-8

TGF-p

L32

Figure 11. CD40 Activation of Cytokine/Chemokine mRNA. SMC were left

unstimulated or stimulated with TmA for 3, 6, or 19 hours in the absence or presence

of 30mM PD98059, 30mM SB203S80, 0.5mg/ml HerbA, or 2mg/ml H-7. Lanel,

unstimulated; lane2, TmA 3hour; lane 3, TmA 6 hour; lane 4, TmA 19 hours; lane 5, 3

hour TmA + HerbA; lane 6, 6 hour TmA + HerbA; lane 7, 6 hour TmA + H-7; lane

8, 6 hour TmA + PD98059; lane 9, 6 hour TmA 4- SB203S80.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40

phosphorylated (active) fonns of ERKl/2, p38, and JNK. ERKl/2 and p38

phosphorylation was evident at 10 minutes and declined at 2 hours (Figure 12A and B).

Treatment of SMC with TmA over the same 2 hour time period examined did not

induce JNK phosphorylation (Figure 12C). Probing the blot with antibody recognizing

the nonphosphorylated forms of JNK present revealed that the absence of

phosphorylated JNK present was not due to artiActs of gel loading (data not shown).

Lysates from UV-treated 293 cells (New England Biolabs) were used as a positive

control for anti-phospho-JNK reactive proteins.

CD40-Mediated lL-8 and MCP-1 Svnthesis in SMC is Dependent Upon ERKl/2 and

p38 MAPK Activitv

Since both ERKl/2 and p38 MAPK family members appear to be involved in CD40

signaling in SMC we next wanted to determine if the activation of ERKl/2 and p38

MAPKs have a functional significance in CD40-mediated induction of lL-8 and MCP-1

synthesis. The role of CD40-mediated activation of ERKl/2 and p38 in the induction

of inflammatory chemokine synthesis was explored via upstream blockade of the

ERKl/2 and p38 pathways. Activation of ERKl/2 is catalyzed by MEKl/2, which,

itself, is activated through serine phosphorylation catalyzed by Raf family kinases

(Davis 1993). Use of a specific MEKl/2 inhibitor, PD98059, which prevents Raf-

mediated activation of MEKl/2 (Allessi and others 1995), (Figure 3), allowed us to

evaluate the role of the MEK/ERK pathway in CD40-mediated induction of SMC lL-8

and MCP-1 synthesis. Additionally, use of a specific p38 inhibitor, SB203580, (Figure

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41

Phospho-ERKI Phospho-ERK2

B

^

Phospho-p38

Phospho-JNK

Figure 12. CD40-Mediated Activation of ERKl/2 and p38 MAPK Family Members in

SMC. SMC were either left unstimulated (control) or stimulated with LPS for 30

minutes, or TmA for 10, 30, 60 or 120 minutes as indicated. Cell lysates were then

harvested and analyzed by Western Blot with antibodies recognizing the phosphorylated

form of A, ERKl/2; B, p38; and C, JNK.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42

3), allowed us to evaluate the role of the p38 signaling pathway in CD40-mediated

induction of SMC IL-8 and MCP-1. The influence of PD98059 and SB203S80 on

CD40-mediated SMC lL-8 and MCP-1 synthesis was analyzed by enzyme-linked

immunosorbant assay of SMC supemantants. SMC were stimulated for 18 hours by

CD 154-transfected 293 cells (293-CD154) in the presence or absence of PD98059 and

SB203580. Supernatants were then collected for analysis. Controls consisted of

supernatants from SMC stimulated with the 293 parent cell line. SMC lL-8 and MCP-1

synthesis was induced in response to stimulation with the 293-CD154 cells, but not with

the control 293 parent line. Additionally IL-8 and MCP-1 synthesis was suppressed by

the addition of PD98059 and SB203580 (Figure 13A and B), and had no adverse efrects

on cell viabiliQr in a concentration range of l-lOOpM tested. These data suggest that

MEK activation of ERKl/2 and the activation of p38 are critical elements

in the signalingpathways of CD40-mediated induction of SMC lL-8 and MCP-1

synthesis.

CD40 Signaling in SMC Results in Activation of the Transcription Factor NF-kB

The transcription factor NF-kB is in part critical for gene expression of many

cellular products involved in inflammatory responses, such as TNF-a, IL-ip, lL-6, IL-

8 and MCP-1 (Sharma and others 1998). Previous observations have shown that

CD40-mediated lL-8 and MCP-1 require activation of ERKl/2 and p38, we

hypothesized that CD40 signaling in SMC results in activation of NF-kB and that

activation of both ERKl/2 and p38 is required. To determine the role of CD40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43

Control

+10nM PD98059 +10pM SB203580

1500 3000 4500

Pfcograms/mi IL-8 B

Control

TmA

+10nM PD98059 +10^M SB203580

0 500 1000 1500 Picograms/ml MCP-1

Figure 13. Requirement for ERKl/2 and p38 Activity in CD40-Mediated SMC IL-8

and MCP-1 Synthesis. SMC were left untreated (control) or stimulated with TmA in

the presence or absence of the MEK/ERK inhibitor PD980S9 or the p38 inhibitor

SB203S80. After 18 hours supernatants were harvested and analyzed by ELISA for: A,

IL-8 production or B, MCP-1 production.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44

signaling in the activation of transcription factors contributing to inflammatorycytokine

and chemokine synthesis in SMC, nuclear localization of NF-kB was evaluated by

electrophoretic mobilier shift assay (EMSA) as described in materials and methods.

Additionally, the involvement of the MAPK family members ERKl/2 and p38 in

CD40-mediated activation of NF-kB was examined. SMC were pretreated with or

without the MEK/ERK inhibitor PD980S9 and the p38 inhibitor, SB203S80, and

stimulated with TmA for 30 minutes. NF-kB was activated in SMC cells stimulated

with TmA, and the amount of activation was decreased in cells pretreated with

PD98059 and SB203580 (figure 14). These data suggest that CD40 signaling in SMC

results in the activation and/or nuclear localization of the transcription factor NF-kB,

and that the activation of the MEK/ERK and p38 pathways are critical.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 6

NF-kB

6000

5000 -

5 4000 -

g 3000 -

: 2000

1000 -

Figurel. CD40-Mediated Activation of NF-kB in SMC. SMC were left unstimulated

(control) or stimulated with Img/ml TmA for 30 minutes in the absence or presence of

30mM SB203580 or PD98059. Cells were then lysed and nuclear proteins extracted

and an EMSA was performed for NF-kB activation. Band densities were determined

by scanning densitometry. Lanel, unstimulated,; lane 2, Tm; lane 3, TmA +

SB203580; lane 4, TmA + PD98059; lane 5, cold competition assay of 30 minute

TmA stimulation; lane 6, positive control; lane 7, mutant oligo.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4

DISCUSSION

The interaction of CD40 with its natural ligand CD 154 contributes to normal cell-

mediated responses as well as to chronic inflammatorydiseases (Stout and Suttles

1996).Atherosclerosis, or vascular inflammation is characterized by the migration of

immune cells (i.e. monocytes, T cells) to the site of injury, which transmigrate through

the endothelium from the media to the intima of an artery. There are many cells which

contribute to the development of the disease, however the presence of activated,

CD 154^ T cells at these atherosclerotic lesions may play a crucial role in the

maintenance and/or exacerbation of inflammation. There are several known functional

attributes acquired in response to CD40 ligation in monocytes/macrophages including

the up-regulation of inflammatorymediators, and the increase of monocyte/macrophage

longevity, both events contributing to the maintenance and/or exacerbation of

inflammatorydisease. Studies of tissue sections of atherosclerotic plaques have found

the accumulation of activated (CD154^), CD4^ T lymphocytes (Mach and others 1997)

which have the capacity to interact with CD40 expressing cells in the vasculature

including endothelial, monocytes/macrophages, and smooth muscle cells. Previous

research has shown that both monocytes/macrophages and SMC have the ability to

contribute to vascular inflammation by the production of inflammatory

cytokines/chemokines, tissue factor, the up-regulation of adhesion molecules, and both

46

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47

have the propensi^ to become lipid-laden foam cells (Poe and others 1997; Mach and

others 1997), In vascular inflammation the role of CD40, and the interactions that occur

between major cell Qrpes contributing the formation of atherosclerotic plaques is

currently under investigation. Experiments described in this dissertation were

performed to determine the role of CD40 signaling in monocytes and SMC in the

development of atherosclerosis. We examined the functional attributes acquired by

SMC in response to CD40 ligation, and the role of MAPK signaling pathways activated

in response to CD40 signalingin both monocytes and SMC.

Previous research done in our laboratory investigated the signaling pathways

activated in response to CD40 ligation in monocytes resulting in the production of

inflammatory cytokine synthesis. These studies revealed that the activation of PTK is a

critical component in CD40-mediated activation of monocyte inflammatory cytokine

synthesis, as well as rescue from apoptosis (Poe and others 1998). We evaluated the

role of the MAPK family members as potential downstream mediators of CD40

signalingin monocytes and SMC. MAPK ftunily members ERKl/2, p38 and JNK were

all considered as likely candidates since previous reports have demonstrated that all

three members are activated in response to CD40 signaling in B cells, and in LPS

activation of monocytes (Li and others 1996; Sakata and others 1995; Sanghera and

others 1996). The experiments presented in this dissertation employed primary human

monocytes and primary aortic smooth muscle cells (of less than passage 6). Stimulus

used in these studies included plasma membranes purified from activated (CD 154^)

CD4^ T cells and CD 154 transfectants.

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We observed CD40 signaling in monocytes resulted in the rapid phosphorylation

and activation of the MAPK ftunily member, ERKl/2, however over the same time

period of stimulation, phosphorylation of p38 and JNK was not increased above

background (Figure 4). Additionally, CD40-mediated activation of ERKl/2 in

monocytes was accompanied by enhanced kinase activi^ and is a critical event in

CD40-mediated monocyte IL-ip and TNFd synthesis (Suttles and others 1999). How

ERKl/2 contributes to the transcriptional control of CD40-mediated induction of

inflammatory cytokine synthesis is currently being investigated. Both IL-ip and TNFa

genes are, in part, regulated by several transcription factors including NF-tcB and NF-

IL-6. NF-IL-6 is a direct substrate of ERKl/2 catalytic activity.

The intent of our research is not only to determine how CD40 signalingin

monocytes results in the activation of inflammatorycytokine synthesis, thus leading to

inflammatory diseases such as vascular inflammation, rheumatoid arthritis, multiple

sclerosis, and inflammatory bowel disease, but also in how to suppress the

inflammatory process. Previous studies suggested that the anti-inflammatory cytokines

IL-4 and IL-10 inhibited CD40-induced monocyte PTK activity, and CD40-mediated

monocyte IL-ip production and together worked in a synergistic manner. Additionally,

inhibition of CD40-mediated IL-ip synthesis, and activation of ERKl/2 in monocytes

was observed in response to pretreatment with an PTK inhibitor suggesting that PTKs

were a critical element in the monocyte CD40 signaling pathway (Poe and others

1997). IL-4 and IL-10 were evaluated for their ability to suppress CD40-mediated

activation of ERKl/2 in monocytes. Both IL-4 and IL-10 modulated CD40-mediated

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ERKl/2 phosphorylation in monocytes, suggesting that the MAPK, ERKl/2 may be

their target of inhibition (Figure S). Both IL-4 and IL-10 signal through receptors

which associate with and activate Jaks upon ligation (O’Shea 1997; Leonard and

O’Shea 1998). The Jaks in turn, activate the Stats which induce negative regulators of

cytokine signaling SOCS, which work as a negative feedback system to inhibit the Jaks

either directly or non-directly (Darnell 1997; Nicholson and Hilton 1998). IL-4 and IL-

10 appear to act early in the signaling cascade since they inhibit PTK activity in CD40-

mediated activation of monocyte inflammatory cytokine synthesis (Poe and others

1998). These data suggest that IL-4 and IL-10 may induce tyrosine phosphatase

activity, resulting in the suppression of the CD40 signaling pathway in monocytes, and

thus suppression of ERKl/2 activity.

Recent reports have shown that vascular inflammation involves the participation of

immune competent cells and the activation of inflammatorypathways and mediators.

Both monocytes and SMC are present at atherosclerotic plaques and may be the major

progenitors of vascular inflammation (Mach and others 1997). We observed that SMC

express CD40 (Figure 6) and other receptors that interact with T cells, similar to the

monocytes including cell surftice expression of CD95 (Figure 7), intercellular adhesion

molecule (ICAM), the costimulatory molecules B7-1(CD80) and B7-2 (CD86) (data not

shown), and both have the ability to present antigen to CD4^ T cells (Suttles and others

1994). These data suggest that CD40 signaling in SMC results in the activation of

signaling pathways and induction of inflammatory mediators in a manner similar to

what occurs in monocytes.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50

The induction of chemokine synthesis may play an crucial role in the pathogenesis of

atherosclerosis by promoting directed migration of immune competent cells to the site

of injury. The observation that CD40 ligation in SMC induced the inflammatory

chemokines MCP-1 and IL-8 suggested that SMC may play a critical role in the

pathogenesis of atherosclerosis by enhancing the migration of immune competent cells

to the atherosclerotic plaque. We further confirmed that the production MCP-1 and IL-

8 was a CD40-mediated event by employing anti-CD 154 antibodies and blocking the

synthesis of the chemokines as compared to treatment with an isotype control antibody

(Figure 10). Furthermore, we observed that CD40-mediated induction of MCP-1 and

IL-8 synthesis was partially dependent on both PTK and PKC activity (Figure 9),

suggesting that the CD40 signaling pathway in SMC may also have similarities to the

signaling pathway(s) activated in B cells in response to CD40 ligation. MCP-1 induces

tissue factor (TF), a transmembrane glycoprotein that initiates coagulation, mRNA

synthesis in SMC (Schecter and others 1997). The exposure of TF during plaque

rupture may induce acute thrombosis resulting in myocardial infarction. Further

investigation of MCP-1 production and its effects on SMC may lead to a greater

understanding of the pathways involved and provide important targest for inhibition of

vascular inflanunation and plaque thrombosis. The observation that CD40-

mediated IL-8 and MCP-1 synthesis was, in part, dependent on PKC activity suggests

that the PKC signaling pathway may play an important role in contributing to vascular

inflammation and may be a potential target for modulation.

We observed that CD40 signaling in SMC resulted in the phosphorylation of the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51

MAPK family members ERKl/2 and p38, however we did not see activation of JNK

over the time course examined (Figure 12). The critical dependence of both ERKl/2

and p38 activation on CD40-mediated EL-8 and MCP-1 were also important

observations (Figure 13), suggesting that both pathways may contribute to the

regulation of transcription factor activation resulting in the synthesis of these

chemokines. These data suggMt that CD40-mediated activation of SMC chemokine

synthesis implements a distinctly different signaling pathway from that in monocytes

and B cells. The ability of the MEKl/2 inhibitor PD98059 and the p38 inhibitor

SB203580 to inhibit CD40-mediated SMC chemokine synthesis suggests that both

MAPK signalingpathways are involved in the downstream activation of transcription

factors resulting in chemokine production. Both IL-8 and MCP-1 are in regulated by

several transcription factors including NF-kB (Roebuck and others 1999). SMC

stimulated with TmA resulted in the activation of the transcription factor NF-kB

(Figure 14). Additionally we observed that inhibitors of the MEK/ERK and the p38

signaling pathways suppressed activation of NF-kB suggesting that both of the MAPKs

are involved in CD40-mediated activation of this transcription factor. Activation of

NF-kB in collaboration with the transcription Actors NF-IL-6 (C/EBP) and AP I

regulate both IL-8 and MCP-1 transcription in most cells (Sharma and others 1998).

These data suggest that the CD40 signaling in SMC results in the activation of the

MEK/ERK and p38 MAPK signaling pathways which in turn activate transcription

factors leading to the synthesis of the chemotactic factors IL-8 and MCP-1 at

atherosclerotic lesions. We have demonstrated that CD40-mediated induction of VSMC

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52

chemokine synthesis was in part regulated by PTK and PKC (Figure 9), this suggests

that both of these pathways may be involved in activation of the MAPK pathways.

Activation of PTK results in activation of ERKl/2, and activation of PKC may result in

the activation of the MAPK p38.

Finally, these data suggest that monocytes/macrophages and SMC present at

atherosclerotic lesions may be critical regulators of vascular inflammation. Both of

these cells are capable of interacting with activated T cells through CD40:CD154

receptor-ligand pair resulting in the synthesis of inflammatory mediators.

Accumulation of activated T cells and monocytes/macrophages at plaque formation sites

and in the neointima, and the activation and proliferation of SMC contribute to the

inflammatory reaction that takes place inside the plaques. The production of

inflammatory cytokines including IL-ip and TNFa, and the secretion of chemotactic

factors including IL-8 and MCP-1 facilitate aberrant migration of immune competent

cells to that site. The delineation of the signaling pathways activated in response to

CD40 signaling in monocytes and SMC will help us to gain a better understanding of

the pathways activated resulting in the onset of inflammation, and will help us define

targets of therapeutic intervention.

The ability of the Th2 cytokines IL-4 and IL-10 to abrogate CD40-mediated

monocyte cytokine synthesis, and ERKl/2 phosphorylation suggests that there may be

direct interaction between the Jak/Stat and ERKl/2 signaling pathways. However,

significantly different to the observations in the monocyte system, neither IL-4 nor IL-

10 suppress CD40-mediated SMC chemokine production or MAPK phosphorylation

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53

(data not shown).

Monocytes and SMC exhibit many similar characteristics, but are distinctly

unique in signaling pathways implemented in response to CD40 ligation (Figure 15).

CD40 signaling in monocytes results in the activation of the inflammatory cytokines IL-

ip and TNFa, which are both critically dependent on the activation of the MEK/ERK

pathway, and can be suppressed by the anti-inflammatory cytokines IL-4 and IL-10.

CD40 signaling in SMC results in the synthesis of the chemotactic Actors IL-8 and

MCP-1, which are both dependent on the activation of the MEK/ERK and p38

pathways suggesting divergence in the signaling pathway. However, CD40 signaling in

both monocytes and SMC may result in the activation of divergent signaling pathways

but with the downstream outcome being the activation of transcription Actors which

contribute to the production of inflammatory cytokines and chemokines.

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Figure 15. Summary of CD40-Mediated Signaling Events in Monocytes and SMC

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VTTA Denise M. Milhom

Personal Data: Date of Birth: April 14, 1961 Place of Birth: Norristown, Pennsylvania Marital Status: Married

Education: East Tennessee State UniversiQr, Johnson City, Tennessee; B.S., Chemistry, ACS approved, 1994 East Tennessee State University, Johnson City, Tennessee; James H. Quillen College of Medicine Ph.D. in Biomedical Science, 1999

Professional East Teimessee State UniversiQr Experience: Department of Biochemistry and Molecular Predoctoral Fellow Biology, 1994-1999

Department of Health Sciences Graduate Immunology Laboratory, 1996

East Tennessee State University Department of Chemistry Undergraduate Research 1993-1994

Publications: Suttles, J., Milhom, D M., Miller, R.W., Poe, J.C., Wahl, L.M., Stout, R.D., "CD40 stimulation of monocyte inflammatory cytokine synthesis through an ERKl/2-dependent pathway: a target of IL-4 and IL-10 anti-inflammatoryaction”. J.Biol.Chem.274:5835-5842

Research East Tennessee State University Quillen College of Medicine Awards: 13"' Annual Student Medical Research Forum 2"" Place Graduate Division H, 1997

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