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The role of the p38 mitogen-activated protein kinase in the immune response Salmon, Ruth A. 1998

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The role of the p38 mitogen-activated protein kinase in the immune response. By: Ruth A. Salmon B.Sc: Simon Fraser University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Experimental Medicine Program We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1998 © Ruth A. Salmon, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract The p38 mitogen-activated protein kinases (p38 MAPKs) were originally shown to be activated in response to environmental stress and pro-inflammatory cytokines. This thesis investigates the hypothesis that p38 M A P K would be activated by other stimuli other than stress, such as those involved in the regulation of the immune response. In T cells, crosslinking of the CD3 chains of the T cell antigen receptor (TCR) complex or of Fas resulted in activation of p38 M A P kinase and M A P K A P kinase-2. Crosslinking of CD28 synergized with low doses of anti-CD3 to activate p38 M A P kinase or M A P K A P kinase-2. The in vivo activation of M A P K A P kinase-2 in response to crosslinking of CD3 and CD28 or Fas was shown to be dependent on p38 M A P kinase activity using SB 203580, a specific inhibitor of p38 M A P K a and (3. Crosslinking of the B cell antigen receptor (BCR) or CD40 on B cells also resulted in activation of p38 M A P kinase and M A P K A P kinase-2. Next, a possible role for p38 M A P K in apoptosis triggered by ligation of antigen receptors on lymphocytes was investigated. When used at concentrations which suppressed the in vivo activation of M A P K A P kinase-2, SB 203580 did not inhibit activation-induced cell death in T cells or BCR-induced apoptosis in the immature B lymphoma WEHI 231 cells. We conclude that the activation of p38 M A P kinase and M A P K A P kinase-2 by crosslinking of the TCR, BCR, Fas or CD40 was not correlated with their role in regulating lymphocyte survival. Experiments with SB 203580 also demonstrated a role for p38 M A P K in the synthesis of cytokines. The antigen-initiated production both of IL-12 by splenic antigen-presenting cells (APCs) and of IFNyby CD4 + T cells were regulated by p38 M A P K . In contrast, SB 203580 enhanced the production of IL-12 elicited from macrophages by stimulation with lipopolysaccharide (LPS). The enhancement of IL-12 production correlated with the inhibition Ill of IL-10 by SB 203580, a known negative regulator of IL-12 production in LPS-stimulated macrophages. These results demonstrate that p38 MAP kinase is not only a "stress-activated kinase" but also plays a role in the regulation of both innate and antigen-specific immune responses. Table of Contents iv Abstract i i Table of Contents iv List of Figures viii Preface xi Acknowledgments xii Abbreviations xiii Chapter 1 Introduction 1 1.1 Signaling by antigen receptors 2 1.2 Signaling in T cells: the TCR, CD28 and Fas 3 1.3 Signaling in B cells: the BCR, CD40 and IL-4 6 1.4 The M A P K superfamily 8 1.5 The p38 M A P K family 10 1.6 Stimuli known to activate p38 M A P K 10 1.7 Upstream activators of the p38 M A P K family 11 1.8 Substrates of the p38 M A P K family 12 1.9 The specific inhibitor of p38 M A P K activity, SB 203580 13 1.10 Cytokines involved in the modulation of the immune response: the role of IL-12 16 1.11 Objectives 19 Chapter 2 Materials and Methods 20 2.1 Mice 20 2.2 Cell isolation 20 2.3 Cell culture 22 2.4 Cell stimulations, immunoprecipitations and immunoblotting 23 2.5 Kinase assays 25 2.6 Quantitation of Cell Death 26 2.7 Flow Cytometry 26 2.8 ELISAs 26 Chapter 3 Activation of the p38 M A P kinase pathway in lymphocytes. 28 3.1 Rationale 28 3.2 Results 29 3.2.1 p38 M A P kinase is rapidly activated by crosslinking of the TCR complex on proliferating T cells. 29 3.2.2 p38 M A P kinase is rapidly activated in response to crosslinking of Fas. 30 3.2.3 Crosslinking of the TCR or Fas activates M A P K A P kinase-2, and this activation can be suppressed by a specific inhibitor of p38 M A P kinase activity. 31 3.2.4 Crosslinking of CD28 synergized with crosslinking of CD3 for activation of p38 M A P kinase and M A P K A P kinase-2. 34 3.2.5 Suppression of p38 M A P kinase activity does not inhibit activation-induced cell death. 36 VI 3.2.6 p38 M A P kinase is activated by crosslinking of IgM or CD40 on B cells. 38 3.2.7 M A P K A P kinase-2 activation is induced by crosslinking of the BCR or CD40 and is dependent on p38 M A P kinase activity. 41 3.2.8 Failure of IL-4 to activate p38 M A P kinase. 41 3.2.9 BCR-induced apoptosis of the immature B lymphoma, WEHI 231, is not is not affected by suppression of p38 M A P kinase activity. 43 3.3. Discussion 44 Chapter 4 A role for p38 M A P kinase in the production of cytokines by T cells and APCs. 51 4.1 Rationale 51 4.2 Results 52 4.2.1 Inhibition of p38 M A P K activity suppressed the antigen-initiated, CD40-dependent production of IL-12 by A P C s . 52 4.2.2 The mechanism of inhibition of CD40L-dependent, antigen-initiated IL-12 production by SB 203580. 55 4.2.3 Inhibition of p38 M A P K activity results in decreased production of IFNy by naive CD4 + T cells. 58 4.2.4 Inhibition of p38 M A P K activity affects the production of IFNy to a lesser degree in activated T cells than in naive T cells. 60 vii 4.2.5 The production of IL-12 induced by LPS from PECs is enhanced by SB 203580. 61 4.2.6 SB 203580 inhibits the production of IL-10 by PECs. 63 4.3. Discussion 66 Chapter 5 Conclusions VI References 76 viii List of Figures Chapter 1 Figure 1.1 Signaling via the TCR and CD28 4 Figure 1.2 Signaling via the BCR 6 Figure 1.3 M A P K cascades 9 Figure 1.4 Chemical Structure of SB 203580 13 Figure 1.5 The ATP-binding pocket of p38 M A P K bound to ATP or SB 2032580 15 Figure 1.6 Regulation of IL-12 and IL-10 18 Chapter 3 Figure 3.1 p38 M A P kinase is activated by crosslinking of the TCR on proliferating T cells. Figure 3.2 p38 M A P kinase is rapidly activated in response to crosslinking of Fas on proliferating T cells. Figure 3.3 In vivo activation of M A P K A P kinase-2 by crosslinking of the TCR on proliferating T cells is inhibited by SB 203580. Figure 3.4 In vivo activation of M A P K A P kinase-2 by crosslinking of Fas on proliferating T cells is inhibited by SB 203580. Figure 3.5 Synergistic activation of p38 M A P kinase by simultaneous crosslinking of CD3 and CD28. 29 30 32 33 35 IX Figure 3.6 Synergistic activation of M A P K A P kinase-2 by simultaneous crosslinking of CD3 and CD28. 36 Figure 3.7 p38 M A P kinase activity is not essential for activation-induced cell death. 37 Figure 3.8 p38 M A P kinase is activated by crosslinking of the BCR or CD40 in freshly-isolated or LPS-activated mature B cells, and in the immature B lymphoma, WEHI 231. 39 Figure 3.9 In vivo activation of M A P K A P kinase-2 by crosslinking of the BCR or CD40 on LPS-activated or WEHI 231 cells is inhibited by SB 203580. 42 Figure 3.10 SB 203580 does not affect anti-IgM-induced apoptosis or anti-CD40-mediated suppression of apoptosis in WEHI 231 cells. 43 Chapter 4 Figure 4.1 Inhibition of p38 M A P kinase activity results in decreased production of IL-12 by APCs. 53 Figure 4.2 SB 203580 does not affect the antigen-initiated up-regulation of CD69 on CD4 + T cells. 55 Figure 4.3 SB 203580 does not affect the up-regulation of CD40L on T cells. 56 Figure 4.4 Inhibition of p38 M A P K activity results in decreased production of IL-12 by A P C s stimulated by crosslinking of CD40. 57 Figure 4.5 SB 203580 inhibits the antigen-initiated production of IFNy by naive CD4+ T cells. 58 Figure 4.6 SB 203580 acts directly on T cells to result in decreased production oflFNy. Figure 4.7 Inhibition of p38 M A P K activity has minimal effects on the production of IFNy by activated T cells. Figure 4.8 Inhibition of p38 M A P K activity enhances the production of LPS-stimulated IL-12. Figure 4.9 SB 203580 inhibits the production of IL-10. Figure 4.10 The effect of SB 203580 on the production of IL-12 in the absence of IL-10. X l l Acknowledgments I have been very fortunate to during my graduate studies to be in an energetic and enthusiastic research environment created by my supervisor, John Schrader. During my time at The Biomedical Research Centre, I have had the opportunity to have excellent scientific interactions with many people. I would especially like to thank Megan Levings and Frances Lee who were always there to give support, friendship and sound scientific advice. Helen Merkens' patient and knowledgeable instruction in tissue culture and flow cytometry techniques was invaluable. James Wieler, Ian Foltz, Rob Gerl and Gbtz Erhardt provided excellent scientific advice and comic relief. I would also like to thank Paul Orban, Sam Abraham, Heather Bone, Leslie Learmonth, John Babcock, Michael Luckach, Yasanna Quin, Melanie Welham, Kevin Leslie and Patricia Orchansky. Frank Jirik and all of the members of his lab have been great friends and enthusiastic colleagues, especially, Susan Andrew, Ken Harder, Ian Melhado, Nicole Jantzen, Jim Peacock, Connie Wong, Janice Penney, Anita Borowski and Chris Ong. There are many people at the BRC who make getting through the work-day a lot easier and more fun, including George G i l l , Tom Yungwirth, Lee Boothby, Sam Kleczkowski, Maureen Mahoney, Maureen Barfoot, Gufmeet Mehroke, Suresh Chand, Mary Bulic and Jane Milner. Thanks to Alan Delany and Ian Jardine for endless help with computers. In the U B C Department of Microbiology and Immunology, I received help, advice and reagents from Mike Gold and Hung-Sia Teh. I was fortunate to receive instruction from Soo-Jeet Teh in the isolation and culture of T cells. Thanks to Claire Sutherland, Oliver Utting and Danielle Krebs for advice, support and friendship. Finally, I would like to thank the people who made reagents available. Peter Young and John Lee at SmithKline Beecham Pharmaceuticals provided anti-p38 M A P K antisera and SB 203580, Maureen Howard from D N A X provided anti-CD40 mAbs and Ian Clark-Lewis of the B R C provided the synthetic peptides, IL-4 and PCC. Abbreviations AICD activation-induced cell death AP-1 activator protein-1 APC antigen presenting cell ATF activating transcription factor BCR B cell antigen receptor C/EBP C C A A T enhancer-binding protein CD cluster of differentiation Cdk cyclin-dependent kinase CREB cAMP response element binding protein CSF colony-stimulating factor D A G diacylglycerol elF elongation initiation factor E R K extracellular-regulated kinases G A D D growth arrest and D N A damage GM-CSF granulocyte-macrophage colony-stimulating factor Hsp heat shock protein IFN interferon lg immunoglobulin IL interleukin IP 3 inositol-1,4,5-tris-phosphate IRS insulin receptor substrate I T A M immunotyrosine activation motifs J A K Janus kinase JNK c-Jun NH2-terminal kinase LPS lipopolysaccharide LT lymphotoxin M A P K mitogen-activated protein kinase M A P K A P M A P K activated kinase M E F myocyte-enhancer-factor M H C major histocompatibility complex M K K M A P kinase kinase M N K M A P kinase interacting kinase NFAT nuclear factor of activated T cells N F K B nuclear factor K B PCC pigeon cytochrome C PEC peritoneal exudate cells PI3K phosphatidylinositol-3' kinase PLC phospholipase C P R A K p38 related/activated kinase SH2 SRC homolgy domain 2 SLF steel locus factor STAT signal transducers and activators of transcription TCR T cell antigen receptor Th T helper TNF tumour necrosis factor TRAF TNF-receptor associated factors WEHI Walter and Eliza Hall Institute Chapter 1 Introduction 1 The cells of the immune system have evolved complex signaling pathways to regulate responses to infection. These signals, transmitted from the receptors exposed to the extracellular environment through the cell membrane to the nucleus control an orchestrated response to pathogens that is mediated by many different types of cells. Each type of cell carries out distinct functions while also regulating and being regulated by other immune cells, thereby mounting a concerted attack on the pathogen. Within minutes, an infection will initiate an innate immune response. This is a rapid and non-antigen specific reaction triggered by invariant structural components of the pathogen that are recognized by non-clonal receptors on myeloid cells such as macrophages, dendritic cells and neutrophils. The innate immune response constitutes the first line of defense against infection and can be thought of as an ancient "danger response", having evolved prior to "the adaptive immune response (Fearon and Locksley, 1996; Medzhitov and Janeway, 1998). The innate immune response triggers the adaptive or antigen-specific immune response which develops over the course of days and is mediated by a vast clonal repertoire of antigen receptors expressed on lymphocytes. The antigen receptors expressed by T and B cells have an enormous diversity conferred by the somatic recombination of variable, joining and diversity region genes encoded by the closely-related T cell antigen receptor (TCR) genes and Immunoglobulin (Ig) genes. This process can create up to 10" distinct clones of T and B cells (McBlane et al., 1995). Despite the fact that TCRs and B cell antigen receptors (BCR) are structurally and genetically related to one another, these cells recognize antigens in different ways. Immunoglobulins made by B cells can 2 be expressed both as membrane-bound antigen receptors, or as secreted antibodies both of which can bind directly to an antigen. In contrast, the TCR recognizes antigen only after it has been processed into short peptides presented in the context of major histocompatibility molecules (MHC) expressed on the surface other cells. The great diversity of antigen receptors allows the body to respond to a shifting universe of pathogens, however, it is inevitable that such diversity will create some receptors that will also recognize components of the body's own tissue. These dangerous self-reactive lymphocytes must be culled from the repertoire while maintaining the lymphocytes that do not react with self. The activation and clonal expansion of lymphocytes in response to stimulation by antigen must be precisely controlled to maintain self-tolerance, and mechanisms such as clonal deletion and anergy of self-reactive lymphocytes prevent autoimmune responses. Signals delivered through the antigen receptors on T or B cells can result in completely different outcomes, inducing proliferation, anergy or apoptosis (Goodnow et al., 1995; Matzinger, 1994; Nossal, 1994; von Boehmer, 1994). The choice between these fates is influenced by the developmental stage of the cell, its past encounters with antigen, and the presence or absence of co-stimulatory signals during encounter with antigen. 1.1 Signaling by antigen receptors Upon encounter with antigen, signals are transduced from the extracellular environment, through the plasma membrane and to the nucleus, eventually resulting in the transcription of new genes. In lymphocytes, kinase cascades as well as calcium and lipid-mediated signals all act as important cytosolic intermediates between the plasma membrane and the nucleus (Fig. 1.1 and 1.2). When antigen receptors on lymphocytes encounter their cognate antigen, tyrosine residues within immunotyrosine activation motifs (ITAMs) in accessory chains of the antigen receptor 3 complexes become phosphorylated. Kinases such as the Zap 70 or Syk are recruited to the ITAMs via Src homology 2 (SH2) domains that bind to phosphotyrosine residues in the context of a conserved sequence. The initiation of this kinase cascade will result in the recruitment of adapter molecules, the activation of GTPases and the activation of other cytoplasmic kinases, such as the mitogen-activated protein kinases (MAPKs). The MAPK pathway will be described in more detail later in this chapter. The binding of antigen also activates phospholipase C (PLC) y which cleaves a lipid component of the plasma membrane into two second messengers, diacylglycerol (DAG) and inositol-1,4,5-tris-phosphate (IP3). The former is needed for the activation of protein kinase C, which can also act upstream of MAPKs and the latter triggers the release of calcium from intracellular stores. Calcium signals are important for the activation of a calcium-dependent phosphatase, calcineurin, that activates nuclear factors of activated T cells (NFATs), transcription factors involved in the transcriptional control of many genes in lymphocytes, including those encoding cytokines (Gold and Matsuuchi, 1995; Robey and Allison, 1995). 1.2 Signaling in T cells: the TCR, CD28 and Fas Engagement of antigen receptors on naive T cells by peptide-MHC complexes on APCs induces the activation of the T cell and results in the initiation of cellular proliferation and the production of cytokines. These processes are greatly enhanced by the presence of a co-stimulatory signal delivered through CD28 on the T cells that ligates B7 / CD80/86 expressed on APCs (Bluestone, 1995; Jenkins, 1994). As seen in Figure 1.1, the TCR is composed of the a and (3 chains, involved in binding of peptide-MHC antigen, and the chains of the CD3 complex which contain ITAMs. In addition to the TCR complex, the CD4 or CD8 co-receptors expressed by T cells also play a role in signal transduction by binding Src family kinases such as Lck (Robey and Allison, 1995). 4 IL-2 gene Figure 1.1: Signaling via the TCR and CD28. Adapted from Robey and Al l i son , 1995. 5 One of the major biological roles of CD28 is enhancing the transcription and stabilization of cytokine messenger RNAs (mRNAs), such as the IL-2 mRNA. The intracellular portion of CD28 contains a consensus binding motif for the lipid kinase, phosphatidylinositol-3-kinase (PI3K) and for adapter proteins. In addition, co-ligation of CD28 and the TCR have been shown to activate c-Jun NH2-terminal kinases (JNKs) (Rudd, 1996; Su et al., 1994), as will be discussed later. The combined signals transmitted through the TCR and CD28 will result in the clonal expansion of T cells in response to a specific antigen. In contrast to the response of naive T cells to antigen, crosslinking of the antigen receptor on T cells that have previously encountered antigen results in activation-induced cell death (AICD) (Green and Scott, 1994; Russell, 1995) in a Fas-dependent manner. Fas is a member of the TNF receptor family and was originally identified as the target of cytotoxic monoclonal antibodies. Engagement of Fas by crosslinking with antibodies or by its natural ligand, the Fas ligand, results in the rapid induction of apoptosis (Nagata, 1997; Nagata and Golstein, 1995). Peripheral T cells from Ipr mice, which carry a mutation in the gene encoding the Fas/CD95 molecule, or from gld mice, which do not express functional Fas ligand, are resistant to AICD (Bossu et al., 1993; Russell et al., 1993; Russell and Wang, 1993). Consistent with these observations, AICD has been shown to occur in a cell-autonomous manner due to the up-regulation of Fas-ligand expression on Fas-positive T cells (Brunner et al., 1995; Dhein et al., 1995; Ju et a l , 1995). 6 1.3 Signaling in B cells: the BCR, CD40 and IL-4 Upon engagement of the BCR, mature, peripheral B cells enter the cell cycle and proliferate. Within the bone marrow, however, immature B cells become anergic or are deleted in response to antigen encounter, processes which silence auto-reactive B cells (Goodnow et al., 1995; Hartley et al., 1993; Nemazee and Buerki, 1989). The WEHI 231 B-lymphoma cell line is the transformed counterpart of an immature B cell, and has provided a model for BCR-mediated deletion, as crosslinking of surface IgM (slgM) induces growth arrest (Boyd et al., 1981) and apoptosis (Benhamou et al., 1990; Hasbold and Klaus, 1990). The B C R is composed of an lg molecule which is involved in recognition and binding of antigen and two associated chains, Iga and Ig(3, each containing ITAMs (Fig. 1.2). As described above, these become tyrosine phosphorylated upon antigen binding and recruit downstream signaling molecules, such as Syk, to the receptor complex (Weiss and Littman, 1994). The Ras/MAPK pathway, PI3K and PLCy are also activated after ligation of the BCR (Gold and Matsuuchi, 1995). Figure 1.2: Signaling via the BCR The molecules indicated in boxes become phosphorylated on tyrosine following crosslinking of the BCR. From DeFranco, 1997. 7 One of the most important co-stimulatory signals on B cells is delivered by CD40. CD40 is a member of the TNF receptor family and is engaged by CD40 ligand, a homologue of TNF expressed on activated T cells. Ligation of CD40 promotes long-term proliferation of B-cells, germinal center formation, Ig class switching, IL-6 production and up regulation of molecules such as LFA-1 and CD23 (Foy et al., 1996). CD40 co-stimulation also suppresses the induction of apoptosis induced by crosslinking of the BCR (Tsubata et al., 1993) on immature B cells, such as WEHI 231 cells. Crosslinking of CD40 has been shown to induce the kinase activity of Lyn and PI3K (Ren et al., 1994) and of the JNKs (Berberich et al., 1996; Sakata et al., 1995; Sutherland et al., 1996). The intracellular domain of CD40 associates with TNF-receptor associated factors (TRAF)-2, TRAF-3 , TRAF-5 and TRAF-6 which are involved in the activation of JNKs and the transcription factor nuclear factor K B ( N F - K B ) (Cheng and Baltimore, 1996; Cheng et al., 1995; Hu et a l , 1994; Ishida et al., 1996; Ishida et al., 1996; Lee et al., 1997; Rothe et al., 1995; Sato et al., 1995; Yeh et al., 1997). Janus kinase 3 (JAK3) also associates with the cytoplasmic tail of CD40 following ligation (Hanissian and Geha, 1997), however, B cells which are deficient in JAK3 do not appear to have defects in many biological functions regulated by CD40 (Jabara et al., 1998). The role of the Ras-MAP kinase pathway in CD40 signaling appears complex, as extracellular-regulated kinases (ERKs) have been shown to be activated by CD40 in resting primary mouse B-lymphocytes and B cells lines (Kashiwada et al., 1996; Kashiwada et al., 1998; L i et al., 1996; Purkerson and Parker, 1998) however, in other studies both in human lymphoma-derived B-cell lines and mouse B cell lines, no activation of ERKs was detectable after crosslinking of CD40 (Berberich et al., 1996; Sakata et al., 1995; Sutherland et al., 1996). The cytokine IL-4 is another regulator of B-cell development. Mice with homozygous disruptions of the IL-4 gene are defective in Ig class switching, with a complete lack of IgE and greatly diminished levels of IgGi (Kuhn et al., 1991). IL-4 synergises with CD40 in promoting long-term growth and class switching of primary B-cells in vitro (Clark and Ledbetter, 1994; Foy et al., 1996). IL-4 stimulation activates PI3K, JAKs, signal transducers and activators of 8 transcription (STAT) 6 and induces phosphorylation of insulin receptor substrate (IRS-2) (Keegan et al., 1994). In contrast to most other cytokines which regulate the growth of hemopoietic cells, IL-4 fails to activate the Ras/ERK pathway (Duronio et al., 1992; Satoh et al., 1991; Welham et a l , 1994). 1.4 The MAPK superfamily The M A P kinase superfamily includes the ERKs, the JNKs and the p38 MAPKs . Each of these families of M A P kinases are part of functionally distinct, but structurally conserved kinase cascades which result in a diversity of downstream events including the regulation of transcription and translation, the activation of transcription factors resulting in the expression of new genes (Fig. 1.3), and the modulation of the cytoskeleton. The exact nature of the pathways resulting in the activation of M A P K s downstream of different receptors on the surface of cells remains to be elucidated. However, the canonical cascade described in Fig. 1.3 has been conserved from yeast to mammals and can be considered as a conceptual framework. The GTPases, near the top of the signaling hierarchy are thought to act as molecular switches, changing into an "active conformation" by binding GTP. The conformational change triggered by binding GTP allows the activated GTPases to bind to effector molecules that contain GTPase-binding domains. The Saccharomyces cerevisiae ste 11 homologues, the Raf kinases, contain GTPase binding domains and are thought to initiate kinase cascades upstream of ERKs, while ste 20 homologues such as the p21-activated kinases (PAKs) may be involved in the activation of JNKs and p38 M A P K s (Fanger et al., 1997; Robinson and Cobb, 1997). A l l M A P K s are characterized by a conserved T X Y motif which is phosphorylated on the threonine and tyrosine residues by dual-specificity M A P kinase kinases (MKKs), the mammalian homologues of S. cerevisiae ste 7 genes seen in Figure 1.3. Once activated, M A P K s phosphorylate many 9 substrates, including other kinases present in the cytosol. Importantly, activated MAPKs can also translocate to the nucleus where they can phosphorylate and activate transcription factors. All MAPKs activate the ternary complex factors Elk and the SAPs, which associate with serum response factor (SRF) to regulate the transcription of the c-fos gene. The JNKs phosphorylate Jun proteins which homodimerize with other Jun molecules or heterodimerize with members of the Fos family to form activator protein-1 (AP-1) (Karin et al., 1997). Many genes contain AP-1 sites, including genes encoding cytokines, as seen in Fig. 1.1. The substrates activated by p38 MAPK will be discussed in greater detail later in this chapter. GTPase: Ras Rac/Cdc42 Ste20: Stell: Mos Raf Tpl2 PAK65 PAK 2,3 G C K MEKK1-4 MLK1-3 ASK1 TAK1 Ste7: MEK1 MEK2 MKK4 MKK7 MKK3 MKK6 i l K Kssl/Fus3: ERK1/2 ERK3 JNK1/2/3 p38a p38P p38y/5 Mnk2 p90 r s k Mvc Mnkl Elkl Sapl iEF4 ATF-2 MAPKAP PRAK1 ATF-1 Kinase 2/3 CREB c-Jun MEF-2C ATF-2 CHOP Hsp25 Figure 1.3: MAPK cascades In black, on the far left are the S. cerevisiae homologues of the mammalian MAPKs, in gray on the near left are the ERK and JNK pathways, and on the right, in black, are molecules thought to be involved in the p38 MAPK pathway, as described in the text. This figure was designed with I. Foltz. 10 1.5 The p38 MAPK family The p38 mitogen-activated protein (MAP) kinases are the mammalian homologues of the HOG-1 kinase of S. cerevisiae (Brewster et al., 1993) and the Spc-1 kinase of Schizosaccharomyces pombe (Shiozaki and Russell, 1995), both of which are activated in response to stressful environmental conditions. The p38 M A P K s belong to a multigenic family and thus far four members have been identified: p38a (Han et al., 1994; Lee et al., 1994; Rouse et a l , 1994); p38p (Enslen et al., 1998; Jiang et al., 1996; Stein et al., 1997); p38y/ERK6/SAPK3 (Lechner et al., 1996; L i et al., 1996; Mertens et al., 1996) and p385/SAPK4 (Goedert et al., 1997; Kumar et al., 1997; Wang et al., 1997). 1.6 Stimuli known to activate p38 MAPK The role of p38 M A P K in responding to environmental dangers in yeast (Brewster et al., 1993; Shiozaki and Russell, 1995) has been conserved in mammals where p38 M A P K has been shown to be activated in response to "stressful" stimuli including heat, high osmolarity, U V irradiation, protein synthesis inhibitors, endotoxin and pro-inflammatory cytokines (Kyriakis and Avruch, 1996). When I began the work described in this thesis, these were the only known stimuli that had been shown to activate p38 M A P K . The JNKs are also strongly activated by stress, while the ERKs are activated by growth factors, but not in response to stress (Kyriakis and Avruch, 1996). The JNKs and p38 M A P K s therefore collectively came to be known as "stress-activated kinases". More recently however, it has become clear both from the work described in this thesis and from that of many others in the literature that p38 M A P K s are activated not only by stress, but also by many molecules involved in the regulation of growth and antigen-specific immune responses. In T cells, p38 M A P K is activated synergistically by ligation of the TCR and CD28 11 (Fig. 3.5) (DeSilva et al., 1997; Salmon et al., 1997), and by ligation of Fas (Fig. 3.1 and 3.2) (Huang et al., 1997; Juo et al., 1997; Salmon et al., 1997). p38 M A P K is activated in B cells by crosslinking of the BCR (Fig. 3.8) (Graves et al., 1996; Salmon et al., 1997) or CD40 (Fig. 3.8) (Salmon et al., 1997; Sutherland et al., 1996). The activation of p38 M A P K by the BCR is enhanced by co-ligation of CD19 and inhibited by co-ligation of CD22 (Tooze et al., 1997). Crosslinking of the Fc y receptor (FcyR) on myeloid cells also results in activation of p38 M A P K (Rose et al., 1997). Furthermore, many hemopoietic growth factors including Interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin and Steel locus factor (SLF) acting on myeloid cells (Foltz et al., 1997; Nagata et al., 1997), and IL-2 and IL-7 acting on lymphoid cells (Crawley et al., 1997) have been shown to activate p38 M A P K . 1.7 Upstream activators of the p38 MAPK family Ectopic expression of dominant-inhibitory forms of the GTPases, Cdc42 and Rac, but not Ras, block the activation of p38 M A P K (Fanger et al., 1997), suggesting that these molecules could be required as upstream activators of p38 MAPKs . p38 M A P kinases are directly activated by phosphorylation on the threonine and tyrosine residues of a conserved T G Y activation motif by the dual specificity M K K s (Kyriakis and Avruch, 1996). Phosphorylation of p38 M A P kinase on the threonine and tyrosine of the T G Y activation motif has been shown to be an absolute requirement for activation of its kinase activity (Doza et al., 1995). Two members of the family of dual-specificity kinases have been shown to be activators of the p38 M A P kinase family, M K K 3 and M K K 6 (Derijard et al., 1995; Han et al., 1996; Moriguchi et al., 1996; Raingeaud et al., 1996). M K K 3 activates p38a, p38y and p385 (Wang et al., 1997) but may not be a strong activator of p38(3 (Enslen et al., 1998; Jiang et al., 1996). M K K 6 activates all four forms of p38 M A P K s (Enslen et al., 1998). 12 1.8 Substrates of the p38 MAPK family Members of the p38 M A P K family are known to act on two classes of substrates: kinases and transcription factors (Fig. 1.3). The kinases include M A P K activated kinase-2 and 3 (MAPKAP) , as well as the related molecule p38 related/activated kinase (PRAK) all of which phosphorylate and activate heat shock proteins (Hsp) 25/27, (Ludwig et al., 1996; McLaughlin et al., 1996; New et al., 1998; Rouse et al., 1994). Hsp 25 is an actin-capping protein and phosphorylation of Hsp 25 lowers the affinity of its interaction with actin, thus allowing actin polymerization (Lavoie et al., 1995), suggesting a role for Hsp 25 in control of cell morphology. p38 M A P K also activates M A P kinase interacting kinase-1 (MNK-1) which in turn phosphorylates elongation initiation factor-4E (eIF-4E), a protein involved in the initiation of translation (Fukunaga and Hunter, 1997; Minich et al., 1994; Waskiewicz et al., 1997). The transcription factors phosphorylated by p38 M A P K include activating transcription factor-1 (ATF-1), ATF-2, and the cAMP response element binding protein (CREB). CREB is phosphorylated in a p38 MAPK-dependent manner by M A P K A P kinase-2, enhancing its ability to activate transcription in response to cAMP (Tan et al., 1996). A T F and CREB can enhance the transcription of the c-fos gene. p38 M A P K also phosphorylates the ternary complex factors Elk-1 and SAP-1 (Janknecht and Hunter, 1997; Price et al., 1996; Whitmarsh et al., 1997). The transcription factor growth arrest and D N A damage (GADD) also acts downstream of p38 M A P K . It is expressed in response to cellular stresses and binds to members of the C A A T T enhancer-binding protein (C/EBP) family of transcription factors and acts as a dominant negative regulator of C/EBP signaling, or to direct C/EBP to bind other sequences (Wang and Ron, 1996). Finally, myocyte-enhancer-factor (MEF-2C) is activated by p38 M A P K in monocytes following stimulation with lipoploysaccharide (LPS) and results in enhanced transcription of c-jun (Han et al., 1997). 13 1.9 The specific inhibitor ofp38 MAPK activity, SB 203580 A key insight into the function of p38 M A P K in immune cells came from the identification of a group of pyridinyl imidazole compounds that bind to and specifically inhibit p38 M A P K activity. These molecules, typified by compounds such as SB 203580 (Fig. 1.4), were identified in an empirical drug screen for compounds that blocked the release of pro-inflammatory cytokines from human monocytes stimulated with LPS and have thus implicated p38 M A P K in the control of inflammatory responses. Figure 1.4: Chemical Structure of SB 203580 4-(4-Fluorophenyl) - 2 ( 4 -methylsulfinylphenyl)-5-(4-pyridyl) imidazole 14 SB 203580 blocks the release of TNFaand IL-lp from monocytes stimulated with LPS (Lee et al. 1994) and inhibits processes initiated by IL-lp and TNFa signaling (Beyaert et al., 1996; Foey et al., 1998; Guan et al., 1997; Pouliot et al., 1997; Ridley et al., 1997). For instance, SB 203580 blocks the IL-lp- or TNFa-induced production of prostaglandin E 2 and IL-6 (Beyaert et al., 1996; Pouliot et al., 1997; Ridley et al., 1997), the TNFa-induced production of GM-CSF (Beyaert et al., 1996) and the production of nitric oxide and inducible nitric oxide synthase stimulated by IL-lp from chrondrocytes (Badger et al., 1998). Inhibitors of p38 M A P K have also proven effective in vivo in reducing inflammation and tissue damage in models of collagen-induced arthritis (Badger et al., 1996; Badger et al., 1989; Griswold et al., 1988; Olivera et al., 1992). The mechanism, targets and specificity of SB 203580 have been widely studied. Analysis of co-crystals of p38 M A P K and SB 203580 (Tong et al., 1997) as well as mutagenesis studies of p38 M A P K (Young et al., 1997) have shown that SB 203580 acts as a competitive inhibitor of ATP binding. SB 203580 has a narrow specificity within the p38 M A P K family and has been shown to inhibit p38a (Cuenda et al., 1995; Kumar et al., 1996; Lee et al., 1994; Young et al., 1997) and p38p (Goedert et al., 1997; Kumar et al., 1997; Stein et al., 1997), but not p38y (Cuenda et al., 1997; Kumar et al., 1997) or p385 (Kumar et al., 1997; Wang et al., 1997). The specificity of SB 203580 is conferred by three residues (Thr-106, His 107 and Leu-108) in the ATP binding pocket which are conserved in p38a and p38p, but not in p38y or p385 (Fig. 1.5) (Gum et al., 1998). Northern blot analysis of human lymphoid tissues including spleen, thymus, peripheral leukocytes, lymph nodes and bone marrow indicate that p38a and p388 are the most abundantly expressed, while there is low or undetectable expression of p38p and p38y (Wang et al., 1997). 15 p38 MAPK bound to ATP p38 MAPK bound to SB 203580 Figure 1.5: The ATP-bindingpocket ofp38 MAPK bound to ATP or SB 2032580. From Gum etal, 1998 When used to treat cells at concentrations of 10 nM or less, SB 203580 appears to be a specific inhibitor of intracellular p38 M A P K activity (Chen et al., 1998; Jacinto et al., 1998; Whitmarsh et al., 1997). At higher concentrations (20-40 uM) however, SB 203580 has been shown to inhibit the activity of JNKs (Chen et al., 1998; Jacinto et al., 1998). High concentrations of SB 203580 (100 yM), when added directly to in vitro kinase assays has been shown to have no effect on the activity of a number of other closely related kinases, including ERK-2, M A P K A P kinase-2, Cyclin A-cyclin-dependent kinase (cdk) 2, Cyclin E-cdk2, c-Raf, M A P kinase kinase, phosphorylase kinase, and casein kinase-2 (Cuenda et al., 1995). 16 1.10 Cytokines involved in the modulation of the immune response: the role of IL-12 Given that p38 M A P K had been identified as playing an important role in the production of cytokines by LPS (Lee et al., 1994), an important trigger of the innate immune response, it was of interest to investigate whether p38 M A P K was also involved in the production of cytokines during the antigen-specific immune response. The cytokines produced by both T cells and the APCs during the interaction of CD4 + T cell with APCs presenting cognate peptide antigens play a critical role in determining the nature of the immune response. CD4 + T helper (Th) cells can differentiate into distinct Th subsets which secrete characteristic cytokines. Th l cells produce interferon y (IFNy) and lymphotoxin (LT), "inflammatory" cytokines that help to regulate cell-mediated immune responses such as the clearing of intracellular pathogens. However, Th l cells are also implicated in the pathology of many autoimmune diseases. Th2 cells are characterized by the production of IL-4, IL-5 and IL-10 which contribute to the development of humoral immune responses by acting on B cells. Many factors influence the production of cytokines during the priming of T cells, including the nature of the pathogen, the genetic make-up of the infected host and the type of APC (Coffman and von der Weid, 1997; O'Garra, 1998). The regulation of cytokine production at the molecular level is the subject of intense study. Signaling via kinases such as JAKs and transcription factors including STATs, GATA-3 and c-Maf has been shown to be critical in determining whether a naive, CD4 + T cell differentiates into a Thl or Th2 effector cell (O'Garra, 1998). One factor that strongly influences the development of effector T cells towards a Th l phenotype is the cytokine IL-12. IL-12 is produced by APCs such as dendritic cells and activated macrophages in response to bacteria or viruses, or in a T cell-dependent manner by CD40 ligand (CD40L) (Trinchieri, 1997). Biologically active IL-12 is a heterodimer consisting 17 of a p35 and a p40 subunit. Many cells, including those that are not known to produce IL-12, constitutively express p35 mRNA, (D'Andrea et al., 1992), but secretion of the p35 protein alone is difficult to detect, even from cells that have been transfected with a p35 gene (Trinchieri, 1995). The expression of the p40 gene, however, is restricted to hemopoietic cells and is inducible and highly regulated (D'Andrea et al., 1993; Murphy et al., 1994). The p40 subunit can be produced in a 10-1000 fold excess of the p70 heterodimer (Aste-Amezaga et al., 1998; Stern et a l , 1990). The production of IL-12 triggered by bacterial products during the innate immune response is tightly controlled. When macrophages and dendritic cells encounter infectious agents, the production of many cytokines is triggered (Fig. 1.6) (Unanue, 1997). Among these is IL-10, which has potent anti-inflammatory properties and is the most physiologically significant inhibitor of IL-12 production (D'Andrea et al., 1993; Koch et al., 1996). In mice deficient in the production of IL-10, infection with Toxoplasma gondii triggers an uncontrolled production of IL-12 that results in death by toxic shock and these animals are also much more sensitive to LPS (Berg et al., 1995; Gazzinelli et al., 1996). IL-12 is also regulated by a potent positive feedback loop. As shown in Figure 1.6, the production of IL-12 by macrophages induces the production of IFNy by natural killer cells, which acts to further enhance the production of IL-12 by macrophages. In addition, IL-12 also stimulates T cells to produce IFNy (Trinchieri, 1997). 18 O Figure 1.6: Regulation of IL-12 and IL-10. From Unanue, 1997 IL-12 is also produced during the antigen-specific phase of the immune response. When T cells encounter their cognate antigen presented by APCs, such as dendritic cells or activated macrophages, signaling via the TCR results in the up-regulation of CD40L. The interaction between CD40L on the T cell and CD40 on the macrophage or dendritic cell triggers the production of IL-12 by the APC (Cella et al., 1996; Kennedy et al., 1996; Koch et al., 1996). IL-12 acts on CD4 + T cells both to enhance the production of IFNy and to directly promote their differentiation into Th l effector cells (Hsieh et al., 1993; Manetti et al., 1993). Mice that are deficient in the expression of CD40L (Campbell et al., 1996; Kamanaka et al., 1996; Soong et al., 1996), IL-12 p40 (Magram et al., 1996) or IFNy (Dalton et al., 1993) have impaired Thl responses and difficulty clearing intracellular bacterial parasites (Campbell et al., 1996; Dalton et al., 1993; Kamanaka et al., 1996; Soong et al., 1996; Wang et al., 1994). 19 1.11 Objectives When we began this work, we had evidence that the simple notion that p38 M A P kinase was activated only in response to "stressful" stimuli, as described above was incomplete. This was based on our observation that one of the molecules that became phosphorylated on tyrosine following stimulation of myeloid cells with the hemopoietic growth factors IL-3, SLF and G M -CSF was p38 M A P K (Welham and Schrader, 1992; Foltz et al., 1997). This thesis set out to examine the hypothesis that p38 M A P K would be activated by other stimuli involved in the regulation of the immune response. We were encouraged by the fact that the ERKs had already been identified as important molecules involved in signaling by antigen receptors (Gold and Matsuuchi, 1995; Robey and Allison, 1995) and JNKs had been found to be activated in T cells by co-stimulation of the TCR and CD28 (Su et al., 1994). One objective of this thesis was to determine whether p38 M A P K was activated in lymphocytes in response to ligation of antigen receptors or co-stimulatory molecules. Furthermore, given that p38 M A P K activity had originally been shown to be involved in the production of cytokines by macrophages following stimulation with LPS (Lee et al., 1994), a second objective of this thesis was to determine whether p38 M A P K played any role in the production of cytokines triggered by antigen. Chapter 2 Materials and Methods 20 2.1 Mice: CD4 + T cell populations were derived from A N D TCR transgenic mice that recognize pigeon cytochrome C fragment 88-104 (PCC) presented by I-E K (Kaye et al., 1989). These mice have been backcrossed onto the BIO.BR (H-2K) background for more than 6 generations. C D l l b 7 C D l l c + APC populations were derived from the spleens of either A N D mice or B10.BR mice. (CBA x C57BL/6) F, mice were used as a source of splenic CD4 + T cells and APCs in some experiments involving polyclonal activation of T cells. For experiments involving T cells in Chapter 2, splenic T cells were obtained from C57BL/6 mice and B cells were from the spleens of Balb/C nu/nu mice. 2.2 Cell isolation: To obtain CD4 + T cells from A N D mice, popliteal, inguinal and mesenteric lymph nodes were harvested, pooled and single cells suspensions were made by passing the cells through a wire screen. CD4 + T cells were obtained either by positive or negative selection, with similar results using either method. Positive selection was carried out by incubating lymph nodes cells with biotinylated anti-CD4 mAbs (H129.19) (Pharmingen, San Diego, CA) followed by magnetic separation using streptavidin-coated ferro-magnetic particles (Miltenyi, Sunnyvale, CA). The resulting T cells were 96-98% CD4 + , as determined by flow cytometry. CD4 + T cells were also obtained by negative selection by depleting lymph nodes of CD8 + T cells and B cells using anti-CD8 mAbs (53-6.7) (Pharmingen, San Diego, CA) followed by removal of CD8 + T cells and B cells using para-magnetic beads coated with sheep anti-mouse IgG (Dynal, Lake Success, N Y ) . Using this technique, T cells were 90-95 % CD4 + , as determined by flow cytometry. 21 In experiments using freshly isolated T cells, lymph nodes were dispersed into a single cell suspension, adherent cells were removed by culture on tissue-culture treated plastic for 1 hour, and B cells were removed using magnetic beads coupled to sheep anti-mouse Ig (Dynal, Lake Success, NY) . To obtain proliferating T cells, spleen or lymph node tissue was dispersed and T cells were activated polyclonally as described below. Positive selection of splenic C D l l b 7 C D l l c + APCs from A N D mice was performed as follows. Splenic fragments were digested in 5 mg/mL collagenase D (Boehringer Mannheim) for 45 minutes at 37° C and passed through a wire mesh screen to obtain a single-cell suspension. Cells were washed once and FcR-mediated binding was blocked by incubation with anti-Fey RII/III (2.4G2) (American Type Culture Collection, Rockville, MD) for 15 minutes at 4° C. Cells were stained with biotinylated anti-CDllb (Ml/70) and ant i -CDllc (HL3) (both from Pharmingen, San Diego CA), followed by magnetic separation using streptavidin-coated ferro-magnetic particles (Miltenyi, Sunnyvale, CA). APCs isolated using this procedure expressed I-E K , had the morphological characteristics of dendritic cells or macrophages and formed clusters with T cells. Splenic APCs from BIO.BR mice were negatively selected by passing spleen fragments through a wire screen, followed by removal of red blood cells with ammonium chloride. T cells and B cells were depleted by staining with anti-CD4 and anti-CD8, followed by incubation with para-magnetic beads coated with sheep anti-mouse IgG (Dynal, Lake Success, NY) . To obtain co-cultures of CD4 + T cells and APCs from (CBA x C57BL/6) F, mice, spleens were depleted of CD8 + T cells and B cells as described above. Peritoneal exudate cells (PEC) were elicited by injecting 2 mL of 2 mg/mL thioglycollate broth (Difco, Detroit, MI) into the peritoneal cavity of C57BL/6 x C B A F, mice. Five days later, peritoneal cells were harvested by lavage and plated on tissue culture-treated plates for 4-6 22 hours. The wells were then washed three times with warm medium to remove non-adherent cells. B cells were obtained from the spleens of Balb/C nu/nu mice by dispersion into a single cell suspension and culture on tissue-culture treated plastic for 1 hour to remove adherent cells prior to use. B cells blasts were obtained by culturing dispersed Balb/C nu/nu spleen cells at a concentration of 2xl0 6 cells/mL in tissue culture-treated flasks with medium supplemented with 15 ng/mL LPS. At 48 hours, non-adherent cells were harvested, leaving behind adherent macrophages. 2.3 Cell culture: Cells were cultured at 37°C in humidified incubators gassed with 5% C 0 2 using RPMI 1640 medium (Stem Cell Technologies, Vancouver, BC), supplemented with 10% (v/v) FCS (Intergen, Purchase, NY), 50 \iM 2-ME and 1 mM sodium pyruvate in tissue culture-treated 96-well plates (A/S Nunc, Roskilde, Denmark). For co-cultures involving antigen-specific stimulation, AND CD4+ T cells (8 x 105per well) and either AND or B10.BR splenic APCs (2 x 105 per well) were stimulated as described. For polyclonal stimulations, purified AND CD4+ T cells (8 x 105 per well), or splenic CD4+ T cells and APCs (1.6 x 106 cells per well) from (CBA x C57BL/6) F, mice, or unfractionated spleen from C57BL/6 were cultured in media supplemented with 2% X063mIL-2 conditioned media (Karasuyama and Melchers, 1988) on plates pre-coated with anti-CD3 (2C11-145, ATCC, Rockville, MD), prepared by incubating 2C11 (1 ^g/mL, unless otherwise stated) in PBS for 2 hours at 37° C, then washing 3 times with PBS. 23 PEC were stimulated as described in the figure legends with LPS (10 ng/mL) (Difco, Detroit. MI), IFNy (400 u/raL) (Genzyme, Cambridge, MA), and anti-IL-10 (JES5-2A5) (10 H-g/mL), Pharmingen, San Diego, CA. 2.4 Cell stimulations, immunoprecipitaiiqns and immunoblotting: Cells were washed in Hank's buffered saline solution supplemented with 20 mM HEPES, pH 7.2, re-suspended in serum-free RPMI buffered with 20 mM HEPES and incubated at 37° C for 2-4 hours prior to stimulation. In experiments using T cells, unless otherwise stated, a secondary goat anti-hamster IgG antibody was included to maximize crosslinking of the primary antibody. T cell stimulations were done as follows: the primary antibody was added on ice for 5 minutes, the secondary antibody was added while the cells remained on ice, and cells were then incubated at 37° C for the time intervals indicated. Each experiment included two control samples, one treated identically except without antibodies, and one sample treated with the secondary antibody alone. A secondary antibody was not used for stimulations of B cells. The primary antibody was added to the cells which were incubated at 37° C for the time indicated. Antibodies were from the following sources: hamster anti-mouse 2C11-145 (ATCC, Rockville, MD); hamster anti-mouse CD28 and hamster anti-Fas (Jo2) from (Pharmingen, San Diego, Ca); goat anti-hamster IgG (KPL, Gaithersburg, MD); sheep anti-mouse IgM F(ab')2 |i-chain specific (Jackson ImmunoResearch, West Grove, PA); the anti-CD40 mAb IC10 (a gift from M . Howard, DNAX, Palo Alto CA). Synthetic mouse IL-4 was a gift from I. Clark-Lewis. Cells extracts were prepared from equivalent numbers of cells in solubilization buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40 (NP-40), 1 mM sodium molybdate, 2 mM sodium orthovanadate, 1 mM sodium fluoride, 50 mM p-glycerol phosphate, 10 |ag/mL aprotinin, 10 ^ / m L soybean trypsin inhibitor, 0.7 |ig/mL pepstatin, 2 (j,g/mL 24 leupeptin, and 40 jig/mL phenylmethylsulfonyl fluoride). A small aliquot of cell lysate was resolved by S D S - P A G E and then analysed by western immunoblotting with the anti-phosphotyrosine mAb, 4G10 (Upstate Biotechnology, Lake Placid, NY) to monitor whole cell protein tyrosine phosphorylation. The remainder of the cell lysate was subjected to immunoprecipitation. For immunoprecipitations, 107 cells were used per sample. p38 M A P kinase was precipitated with 2 ^ L of rabbit anti-p38 M A P kinase antisera raised against full-length CSBP-2 (Lee et al., 1994), followed by adsorption onto protein A Sepharose (Pharmacia, Uppsala, Sweden). Beads were washed extensively with solubilization buffer, boiled in SDS sample buffer, and the eluate was subjected to SDS-PAGE. A l l immunoblots were done by first blocking the membrane in Tris-buffered saline (TBS) with BSA (5%) and ovalbumin (1%) for 30 minutes, then diluting the primary antibody in TBS with B S A (1%) and ovalbumin (.2%) and incubating blots overnight at 4° C with shaking. The membranes were washed 3 times with T B S N (TBS with 0.05% NP40), and incubated with the appropriate secondary anti-immunoglobulin Ab coupled to horeradish peroxidase. The blots were then washed 3 times with T B S N and developed using enhanced chemiluminescence (Amersham, NY) accoring to the manufacturer's instructions. Immunoblotting was done first with 4G10 to detect tyrosine phosphorylation, and subsequently with an anti-p38 M A P kinase polyclonal antibody (1:50 dilution of Ab in Tris-buffered saline) (Santa Cruz Biotechnology, Santa Cruz, CA) to assess equivalency of p38 M A P kinase loading. To detect tyrosine phosphorylated p38 M A P kinase by direct immunoblotting, cells were stimulated and lysed as above, except that 1.5x 10 6 cells were used per sample and the amounts of stimulants reduced accordingly. Cell lysates were subjected to SDS-PAGE and immunoblotting using anti-sera specific for a peptide surrounding 25 phosphotyrosine 182 of p38 MAP kinase (1:50 dilution of Ab in Tris-buffered saline) (New England BioLabs, Beverly, MA). 2.5 Kinase assays: p38 MAP kinase assays were performed on p38 MAP kinase after immunoprecipitation as described above. Immune-complexes were washed twice in solubilization buffer, once in kinase assay buffer (25 mM HEPES pH 7.2, 25 mM MgCl2 , 2 mM dithiothreitol, 0.1 mM NaV04) and then re-suspended in 20 (j.1 kinase assay buffer. The kinase reaction was initiated by the addition of 1 ng/mL ATF-2 (Santa Cruz Biotechnology, Santa Cruz, CA), 50 nM (r)ATP and 10 nCi [y-3 2P] ATP and was stopped after 20 minutes at 30°C by the addition of SDS sample buffer. To determine the effect of SB 203580 on the in vivo kinase activity of p38 MAP kinase, cells were incubated with or without 1 ^ M SB 203580 (a gift from J. C. Lee, SmithKline Beecham Pharmaceuticals, King of Prussia, PA) in 1 mL for the times indicated prior to stimulation, unless otherwise indicated. Stimulations, preparation of cell lysates and immunoprecipitations for kinase assays were performed as described above using sheep anti-mouse MAPKAP kinase-2 antiserum (Upstate Biotechnology, Lake Placid, NY), and protein G Sepharose beads (Pharmacia, Uppsala, Sweden). Precipitates were washed twice with lysis buffer and once with kinase assay buffer (25 mM HEPES pH 7.2, 25 mM MgCh, 2 mM dithiothreitol, 0.5 mM NaV04). The kinase reaction was initiated by the addition of 2 u.g recombinant murine Hsp25 (StressGen Biotechnology, Victoria, B. C.) and 10 pCi L Y - ^ P ] ATP and was stopped after 20 minutes at 30°C by the addition of SDS sample buffer. Phosphorylated proteins were resolved by SDS-PAGE, and visualized by autoradiography. 26 2.6 Quantitation of Cell Death At the indicated times cells were fixed in 70% ethanol/ 1 M glycine, pH 2.5 for 20 minutes at -20°C, washed and re-suspended in PBS with 10 ng/mL propidium iodide (Calbiochem, LaJolla, Ca) and 100 ng/mL RNaseA. Stained cells were analyzed by flow cytometry and apoptotic cells were quantitated as the percentage of events which corresponded to a sub-diploid D N A content (Nicoletti et al., 1991). 2.7 Flow Cytometry: Cells were stained with the primary mAb (1 |ag/106 cells) in FACS buffer (RPMI 1640 with 2% FCS and 20 mM HEPES) for 15 minutes on ice. Cells were washed once with FACS buffer and in cases where the primary mAb was biotinylated, cells were incubated with streptavidin-FITC for a further 10 minutes on ice (Jackson ImmunoResearch, West Grove, PA) followed by washing. Stained cells were analyzed on FACScan IV flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest software. Antibodies used for FACS analysis were anti-CD4-phycoerytherin (PE) (H129.19), anti-CD69-FITC (H1.2F3) and anti-CD40L-biotin (MR-1), all obtained from Pharmingen (San Diego, CA). 2.8 ELISAs Plates (MaxiSorp, A/S Nunc, Roskilde, Denmark) were coated with capture antibodies overnight at 4° C, then washed three times to remove unbound Ab. A l l washes were done with 0.05% Tween 20/PBS using a pressurized garden sprayer. Plates were blocked for 2 hours at 37° C using 2% BSA (Boehringer Mannheim) in PBS. Supernatants and standards were added to the plates for 2-14 hours. The plates were washed 6 times and the detection antibody diluted in blocking buffer was added, followed by incubation for 1 hour. Plates were washed 6 times, and then streptavidin-HRP (Genzyme, Cambridge, M A ) diluted in blocking buffer was added for 15 minutes. The plates were washed 7 times and the substrate tetramethylbenzadine (TMB) (Sigma, St. Louis, MO) was added. After 30 minutes at room temperature, plates were read at the dual wavelengths of 405 and 490 nm. Capture and detection antibody pairs were 27 obtained from Pharmingen (San Diego, CA): anti-p35 IL-12 capture mAb cocktail (Red-T/G297-289), anti-IL-12 p40 capture mAb (C15.6), anti-p40 biotinylated detection mAb (C17.8), anti-IL-10 capture mAb (JES5-2A5), anti-IL-10 biotinylated detection mAb (SCX-1), anti-IFNy capture mAb (R4-6A2), and anti-IFNy biotinylated detection mAb (XMG1.2). 28 Chapter 3 Activation of the p38 MAP kinase pathway in lymphocytes. 3.1 Rationale Our hypothesis was that p38 MAP kinase would be involved not only in responses to stress, but also in the regulation of the immune response. The initial experiments described here were therefore aimed at determining whether p38 MAPK would be activated following ligation of antigen receptors or co-stimulatory molecules which play key roles in adaptive immune responses. The literature indicated a possible role for p38 MAPK in apoptosis and therefore the hypothesis that p38 MAPK could regulate apoptosis in lymphocytes was tested. It had been shown that over-expression of MKK3 or of apoptosis signal-regulating kinases-1 (ASK1), kinases that can activate p38 MAP kinase when over-expressed in cells, results in the induction of apoptosis (Ichijo et al., 1997; Xia et al., 1995). The p38 MAP kinase inhibitor had also been shown to protect fibroblasts from apoptosis induced by sodium salicylate (Schwenger et al., 1997), but did not protect fibroblasts from apoptosis induced by TNFa (Beyaert et al., 1996). In summary, crosslinking of the TCR or Fas on proliferating T cells, or crosslinking of the BCR or CD40 on freshly-isolated or LPS-activated splenic B cells, or on the B lymphoma, WEHI 231, all resulted in the rapid activation of p38 MAP kinase and activation of MAPKAP kinase-2. The regulation of apoptosis in two immunological models using T and B cells was also investigated. The results show that inhibition of p38 MAP kinase activity in vivo using a specific inhibitor, SB 203580, had no effect on either activation-induced cell death in T cells, or on BCR-induced apoptosis in WEHI 231 cells. Therefore, suppression of p38 MAP kinase activity by SB 203580 does not affect the regulation of apoptosis induced via antigen receptors on lymphocytes. 3.2 Results 29 3.2.1 p38 MAP kinase is rapidly activated by crosslinking of the TCR complex on proliferating T cells. T cells from lymph nodes or spleen were activated by culture on anti-CD3-coated plastic for 48 hours in the presence of IL-2, then removed from anti-CD3 and expanded in IL-2. Crosslinking of the CD3 chains of the TCR complex on proliferating T cells using an anti-CD3 mAb resulted in the rapid activation of p38 MAP kinase (Fig. 3.1). This was determined by subjecting immunoprecipitates of p38 MAP kinase from lysates of control or stimulated T cells to an in vitro kinase assay using ATF-2 as a substrate. Equal quantities of p38 MAP kinase were immunoprecipitated in each lane, as demonstrated by re-probing the immunoblot with polyclonal anti-p38 antisera (Fig. 3.1). Activation of p38 MAP kinase in freshly-isolated lymph node T cells was investigated, but it was repeatedly found that p38 MAP kinase was activated in unstimulated samples and that stimulation with anti-CD3 or anti-Fas did not result in significant induction of p38 MAP kinase activity above levels detected in unstimulated cells (data not shown). mm mm ATF-2 Figure 3.1: p38 MAP kinase is activated by crosslinking of the TCR on proliferating T cells. Proliferating splenic T cells were left unstimulated (Con), or stimulated with 10 ^g/mL anti-CD3 and 30 ug/mL anti-hamster Ab for 5 minutes (aCD3), 10 ug/mL anti-Fas mAbs and 30 ng/mL anti-hamster Ab for 3 minutes (aFas), or with 30 ug/mL anti-hamster Ab alone for 5 minutes (alg). p38 MAP kinase was immunoprecipitated from cell lysates and activation was detected by phosphorylation of ATF-2 in an in vitro kinase assay. Reaction products were resolved by SDS-PAGE, transferred to nitrocellulose and the levels of phosphorylation were assessed by autoradiography (ATF-2). The quantity of p38 MAP kinase protein immunoprecipitated in each lane was assessed by probing the immunoblot with anti-p38 MAP kinase polyclonal antibodies (ap38). 30 3.2.2 p38 MAP kinase is rapidly activated in response to crosslinking of Fas. Next, the effect of crosslinking of Fas on p38 MAP kinase activity was investigated. Fas was crosslinked on proliferating T cells using an anti-Fas mAb and p38 MAP kinase activity was assessed as before. The kinetics of p38 MAP kinase activation induced by ligation of Fas were very rapid. Activation was detected between 1 and 3 minutes following crosslinking (Fig. 3.1 and 3.2), but was undetectable after 5 minutes (data not shown). ATF-2 ap38 Figure 3.2: p38 MAP kinase is rapidly activated in response to crosslinking of Fas on proliferating T cells, a) Proliferating lymph node T cells were left untreated (Con), stimulated with 10 fig/mL anti-Fas mAb and 30 ng/mL anti-hamster for 1 minute ( a F a s 1'), or for 3 minutes ( aFas 3'), or with 30 ng/mL anti-hamster Ab alone for 3 minutes (alg). p38 MAP kinase was immunoprecipitated from cell lysates and activation was detected by phosphorylation of ATF-2 in an in vitro kinase assay. Reaction products were resolved by SDS-PAGE, transferred to nitrocellulose and the levels of phosphorylation were assessed by autoradiography (ATF -2) . The quantity of p38 MAP kinase protein immunoprecipitated in each lane was assessed by probing the immunoblot with anti-p38 MAP kinase polyclonal antibodies (ap38). 31 3.2.3 Crosslinking of the TCR or Fas activates MAPKAP kinase-2, and this activation can be suppressed by a specific inhibitor ofp38 MAP kinase activity. M A P K A P kinase-2 is a substrate of p38 M A P kinase in other cell types (Ben-Levy et al., 1995; Freshney et al., 1994; Rouse et al., 1994), and therefore it was necessary to determine whether it was activated in proliferating T cells stimulated by crosslinking of CD3 or Fas. M A P K A P kinase 2 was immunoprecipitated from lysates of stimulated or control T cells and in vitro kinase assays were performed using Hsp 25 as a substrate. As seen in Figure 3.3 and 3.4 M A P K A P kinase-2 was rapidly activated by crosslinking of either Fas or the TCR. M A P K A P kinase-2 can be activated in vitro by both ERKs or p38 M A P kinase (Foltz et al., 1997; Freshney et al., 1994). Crosslinking of the TCR also induces E R K activation (Robey and Allison, 1995), and therefore it was of interest to determine whether the activation of M A P K A P kinase-2 by the TCR was dependent upon p38 M A P kinase activity in vivo. The activity of p38 M A P kinase can be inhibited in vivo using SB 203580 (Foltz et al., 1997; Lee et al., 1994; McLaughlin et al., 1996). We have previously demonstrated that SB 203580 fails to block E R K activity in vivo at concentrations that completely block p38 M A P kinase activity (Foltz et al., 1997). Pre-incubation of proliferating T cells in SB 203580 resulted in suppression of p38 M A P kinase activity induced in vivo by crosslinking of the TCR or Fas. As the inhibitor was to be used in biological experiments which were to last up to 24 hours, it was necessary to determine whether the inhibitor could stably suppress p38 M A P kinase activity throughout this time in culture. Proliferating T cells were incubated for 24 hours with 1 or 5 u.M SB 203580, or without drug, followed by stimulation with anti-CD3 in the presence of the same culture media. Activation of M A P K A P kinase-2 was used as an index of p38 M A P kinase activity in vivo. As 32 shown in Fig. 3.3, when proliferating T cells were cultured in the presence of 5 nM SB 203580 for 24 hours, followed by crosslinking of CD3, activation of M A P K A P kinase-2 was suppressed by over 90%, compared to cells stimulated with anti-CD3 but not pre-incubated in SB 203580 (Fig. 3.3). This demonstrates that (/) activation of M A P K A P kinase-2 by the TCR was dependent on p38 M A P kinase activity and (ii) that 5 uM SB 203580 effectively suppressed p38 M A P kinase activity induced in vivo by crosslinking of the TCR to basal levels following a period of 24 hours incubation in the presence of the drug. + + + ocCD3 - 1 5 SBHxM) Hsp 25 Figure 3.3: In vivo activation of MAPKAP kinase-2 by crosslinking of the TCR on proliferating T cells is inhibited by SB 203580. Proliferating lymph node T cells were incubated at 37° C for 24 hours at a concentration of 2.5xl0 5 / mL with 1 or 5 uM SB 203580, or without inhibitor. Cells were then pelleted and re-suspended at 2xl0 7 cells/mL in the same media. Cells incubated without SB 203580 for 24 hours were left unstimulated (far left lane) or stimulated with 10 ng/mL anti-CD3 for 5 minutes (aCD3). Cells incubated in the presence of 1 or 5 nM SB 203580 for 24 hours were stimulated with 10 ng/mL anti-CD3 for 5 minutes as indicated. M A P K A P kinase-2 was immunoprecipitated from cell lysates and activation of M A P K A P kinase-2 was detected by an in vitro kinase assay using Hsp 25 as a substrate. Reaction products were resolved by S D S - P A G E , and the levels of phosphorylation were assessed by autoradiography (Hsp 25). 33 Crosslinking of Fas on proliferating lymph node T cells also induced the activation of MAPKAP kinase-2 (Fig. 3.4). This was blocked by a 30 minute pre-treatment of proliferating T cells with 1 uM SB 203580 (Fig. 3.4), demonstrating that MAPKAP kinase-2 activation induced by Fas crosslinking was dependent upon p38 MAP kinase activity. The activation of MAPKAP kinase-2 following crosslinking of Fas occurred within minutes, similar to the rapid activation of p38 MAP kinase. + + - aFas - + - S B — Hsp 25 Figure 3.4: In vivo activation of MAPKAP kinase-2 by crosslinking of Fas on proliferating T cells is inhibited by SB 203580. Proliferating lymph node T cells were incubated at 37° C for 30 minutes with or without 1 ^ M SB 203580. Cells were then left untreated (Con), stimulated with 10 ug/mL anti-Fas mAb and 30 ug/mL anti-hamster Ab (aFas) for 3 minutes, stimulated identically in the presence of 1 nM SB 203580 (aFas + SB), or stimulated with 30 ug/mL anti-hamster Ab alone (alg). MAPKAP kinase-2 was immunoprecipitated from cell lysates and activation of MAPKAP kinase-2 was detected by an in vitro kinase assay using Hsp 25 as a substrate. Reaction products were resolved by SDS-PAGE, and the levels of phosphorylation were assessed by autoradiography (Hsp 25). 34 We have consistently observed that when cells were pre-treated with SB 203580 for short periods, for instance, 30 minutes to one hour, 1 SB 203580 was sufficient to inhibit p38 MAP kinase activity (Fig. 3.4, 3.6 3.9a and (Foltz et al., 1997)). However, when cells were cultured with the inhibitor for prolonged periods, for example, 24 hours, 5 \iM SB 203580 was required to effectively suppress p38 MAP kinase activity (Figs. 3.3 and 3.9b and c). These observations could reflect instability of the inhibitor during culture. 3.2.4 Crosslinking of CD28 synergized with crosslinking of CD3 for activation of p38 MAP kinase and MAPKAP kinase-2. Crosslinking of CD28 alone using concentrations of anti-CD28 ranging from 0.5 ng/mL to 20 ng/mL failed to activate p38 MAP kinase or MAPKAP kinase-2 (data not shown). Therefore we asked whether crosslinking of CD28 synergized with lower doses of anti-CD3 (0.5-5 ng/mL) that activated p38 MAP kinase weakly. Titration of anti-CD3 in the presence of 10 ng/mL anti-CD28 demonstrated strong synergistic activation of p38 MAP kinase at lower concentrations of anti-CD3 (Fig. 3.5). As shown in Figure 3.5, proliferating T cells stimulated with 5 ng/mL anti-CD3 for 5 minutes exhibited a modest activation of p38 MAP kinase. However the combination of 10 ng/mL anti-CD28 and 5 ug/mL anti-CD3 resulted in strong synergistic activation of p38 MAP kinase, as determined both by phosphorylation of ATF-2 in an in vitro kinase assay, and by tyrosine phosphorylation of p38 MAP kinase detected by probing the immunoblot with anti-phosphotyrosine mAbs (Fig. 3.5). The immunoblot was then stripped and re-probed a second time with polyclonal anti-p38 antisera to confirm the equivalency of immunoprecipitated protein in each lane (Fig. 3.5). 35 - 0.5 2 5 0.5 2 5 - ccCD3 (ug/mL) + - - - + + + - aCD28 mm* ATF-2 aPY WB ap38 WB Figure 3.5: Synergistic activation of p38 MAP kinase by simultaneous crosslinking of CD3 and CD28. Proliferating T cells were stimulated with the antibodies indicated for 5 minutes at 37° C at the following concentrations: anti-CD3 - as indicated (ocCD3); anti-CD28 - 10 ug/mL (aCD28); anti-hamster Ab was added to samples at a 3:1 ratio of the primary antibody. Other cell samples were left untreated (far left lane) or stimulated with anti-hamster Ab alone (far right lane). p38 MAP kinase was immunoprecipitated from each cell lysate and activity was assessed by phosphorylation of ATF-2 in an in vitro kinase assay. Reaction products were resolved by SDS-PAGE, transferred to nitrocellulose, and the levels of phosphorylation were assessed by autoradiography (ATF-2). The immunoblot was then probed with the anti-phosphotyrosine mAb, 4G10 (aPY), then stripped and re-probed with anti-p38 MAP kinase polyclonal antibodies to assess the quantity of protein immunoprecipitated in each lane (ap38). These results were confirmed by kinase assays of MAPKAP kinase-2, which was also activated in a synergistic manner by simultaneous crosslinking of CD3 and CD28 (Fig. 3.6). As observed with activation of p38 MAP kinase, 10 |xg/mL anti-CD3 induced a stronger activation of MAPKAP kinase-2 (Fig. 3.3) than that induced by 5 ug/mL anti-CD3 (Fig. 3.6). Likewise, crosslinking of anti-CD28 alone did not result in activation of MAPKAP kinase-2 (Fig 3.6). However, co-stimulation with 10 ug/mL anti-CD28 plus 5 ug/mL anti-CD3 resulted in synergistic activation of MAPKAP kinase-2 (Fig. 3.6). This was dependent on p38 MAP kinase activity, as activation of MAPKAP kinase-2 was inhibited by pre-treatment of cells with 1 |iM SB 203580 (Fig.3.6). 36 + - + + - aCD3(5ug/mL) - + + + - aCD28 + - - + - luMSB mm Hsp 25 Figure 3.6: Synergistic activation of MAPKAP kinase-2 by simultaneous crosslinking of CD3 and CD28. Cell samples which had been pre-incubated at 37° C for 30 minutes with or without 1 nM SB 203580 (1 SB) were stimulated with antibodies as follows: anti-CD3 - 5 ^g/mL, anti-CD28 - 10 ug/mL for 5 minutes, anti-hamster Ab was added to samples at a 3:1 ratio of the primary antibody. Other cell samples were left untreated (far left lane) or stimulated with anti-hamster Ab alone (far right lane). M A P K A P kinase-2 was immunoprecipitated from cell lysates and activation of M A P K A P kinase-2 was detected by an in vitro kinase assay using Hsp 25 as a substrate. Reaction products were resolved by SDS-PAGE, and the levels of phosphorylation were assessed by autoradiography (Hsp 25). 3.2.5 Suppression of p38 MAP kinase activity does not inhibit activation-induced cell death. The specific p38 M A P kinase inhibitor, SB 203580, was used to determine whether signaling through p38 M A P kinase by the TCR or Fas was necessary for AICD. T cells isolated from lymph node or spleen were initially activated by culturing on anti-CD3-coated plastic, and after 48 hours were removed from the antibody-coated dishes and cultured in the presence of IL-2. Four days later, AICD was induced by re-exposing the proliferating T cells to anti-CD3-coated plastic, in the presence or absence of SB 203580 (1 or 5 uM) for 24 hours. As shown in Fig. 3.7, proliferating T cells that were re-stimulated for 24 hours with anti-CD3 underwent AICD. Incubation of T cells with SB 203580 (1 or 5 jiM) had no effect on the induction of cell death (Fig. 3.7). A similar lack of effect of SB 203580 on AICD was seen in independent experiments on T cells derived from either the spleen or lymph nodes of mice cultured in IL-2 for 3 days to 8 days after the initial activation by anti-CD3 (data not shown). 37 Finally, SB 203580 did not affect AICD induced by crosslinking of CD3 on the T cell hybridoma, A l . l at concentrations up to 10 yM (data not shown). These results demonstrate that concentrations of SB 203580 which suppressed p38 M A P kinase activity induced by crosslinking of anti-CD3 (Fig. 3.3) failed to inhibit AICD. 50 40 A 30 A % sub-diploid events 20 A wA SB 203580 (uM) Figure 3.7: p38 MAP kinase activity is not essential for activation-induced cell death. Lymph nodes T cells were isolated as described and incubated on dishes coated with 1 ug/mL anti-CD3 mAb in media supplemented with IL-2 for 48 hours, then removed from anti-CD3 cultured in media supplemented with IL-2 for 4 days. Proliferating T cells (2.5xl0 5 / mL, plated in triplicate) were then incubated for 24 hours on dishes coated with 1 ug/mL anti-CD3 mAb (gray bars) or on dishes without mAb (black bars), with or without SB 203580 added at the indicated concentrations. Triplicate samples were pooled and harvested, stained with propidium iodide and the percentages of events with sub-diploid DNA content were quantitated by flow cytometry. 38 3 . 2 . 6 ' p38 MAP kinase is activated by crosslinking oflgM or CD40 on freshly-isolated or LPS-activated mature B cells, and in the immature B lymphoma, WEHI231. Crosslinking of the BCR of mature, peripheral B cells results in proliferation (Goodnow et al., 1995), whereas crosslinking of the BCR on the immature B lymphoma, WEHI 231, results in apoptosis (Benhamou et al., 1990; Hasbold and Klaus, 1990). Crosslinking of CD40 contributes positively to proliferation in both of these cell types (Durie et al., 1994; Foy et al., 1996), and suppresses the effects of anti-IgM-induced apoptosis in WEHI 231 cells (Tsubata et al., 1993). We therefore sought to determine whether the activation of p38 MAP kinase in response to BCR or CD40 crosslinking would differ in different B cell populations. B cells from athymic nude mice spleens were used immediately, or after being activated to undergo blastogenesis by LPS for 2 days. Crosslinking of IgM or CD40 on freshly-isolated splenic B cells resulted in activation of .p38 MAP kinase, as shown by the ability of immunoprecipitated p38 MAP kinase to phosphorylate ATF-2 in an in vitro kinase assay (Fig. 3.8a). Crosslinking of IgM or CD40 also resulted in activation of p38 MAP kinase in LPS-stimulated B cell blasts (Fig. 3.8b) and in WEHI 231 cells (Fig. 3.8c). The kinetics of p38 MAP kinase tyrosine phosphorylation in response to BCR or CD40 crosslinking were investigated in LPS-activated B cells. In Figure 3.8d, rabbit polyclonal antibodies that specifically recognize phosphorylation of tyrosine 182 of the p38 MAP kinase TGY activation motif were used to monitor activation of p38 MAP kinase. Tyrosine phosphorylation of p38 MAP kinase was apparent five minutes after crosslinking of the BCR, and ten minutes after crosslinking of CD40. Both stimuli induced sustained phosphorylation of p38 MAP kinase for approximately 35 minutes (Fig. 3.8d). 39 c ATF-2 A * ^ * C cj>v c>v <•> ^ — - — aPY cl C 5 10 20 30 40 60 80 minutes , _ ^ « . . . . - - algM aCD40 40 Figure 3.8: p38 MAP kinase is activated by crosslinking of the BCR or CD40 in freshly-isolated or LPS-activated mature B cells, and in the immature B lymphoma, WEHI 231. a) Freshly-isolated splenic B cells were left unstimulated (C), stimulated with 40 |ig/mL anti-IgM for 5 minutes (algM), 10 |ag/mL anti-CD40 for 10 minutes (aCD40), or with 10 ng/mL synthetic murine IL-4 for 10 minutes (IL-4). p38 M A P kinase was immunoprecipitated from cell lysates and activity was assessed by phosphorylation of ATF-2 in an in vitro kinase assay. Reaction products were resolved by SDS-PAGE, and the levels of phosphorylation were assessed by autoradiography (ATF-2). b) LPS-stimulated splenic B cells were left unstimulated (C), stimulated with 40 ng/mL anti-IgM for 3 minutes (algM 3'), or for 10 minutes (algM 10'), with 10 jxg/mL anti-CD40 for 10 minutes (ccCD40), with 10 ng/mL synthetic murine IL-4 for 10 minutes (IL-4 10'), or for 20 minutes (IL-4 20'). c) WEHI 231 cells were left unstimulated (C) or stimulated with 40 ng/mL anti-IgM (algM) or 10 |ag/mL anti-CD40 (aCD40) for 10 minutes. p38 M A P kinase was immunoprecipitated from cell lysates, samples were resolved by SDS-P A G E and activation was detected by tyrosine phosphorylation of p38 M A P kinase by probing the immunoblot with the anti-phosphotyrosine mAb, 4G10 (aPY). The immunoblot was then stripped and re-probed with anti-p38 M A P kinase polyclonal antibodies to assess the quantity of protein immunoprecipitated in each lane (ap38). In a - c, the differences in the intensity of phosphorylation between different stimuli cannot be compared directly due to differences in the times and doses of stimuli used, d) LPS-stimulated splenic B cells were left unstimulated (C) or stimulated for the indicated times with 40 ng/mL anti-IgM (algM) or 10 ng/mL anti-CD40 (aCD40). Cell extracts were resolved by SDS-PAGE and immunoblots were probed with an anti-p38 M A P kinase phospho-specific polyclonal antibodies which specifically recognize phosphorylation of tyrosine 182. 41 3.2.7 MAPKAP kinase-2 activation is induced by crosslinking of the BCR or CD40 and is dependent on p38 MAP kinase activity. The activation of M A P K A P kinase-2 was induced by crosslinking of either the BCR or CD40 on LPS-activated splenic B-cells (Fig. 3.9 a). In both cases, in vivo activation of M A P K A P kinase-2 was demonstrated to be dependent on p38 M A P kinase activity, as pre-treatment of the cells with 1 u.M SB 203580 for 1 hour prior to stimulation inhibited the activation of M A P K A P kinase-2 (Fig. 3.9 a). The p38 M A P kinase inhibitor was also used on the immature B lymphoma, WEHI 231, in biological assays which lasted up to 24 hours. To determine if the inhibitor could stably suppress the induction of p38 M A P kinase activity, WEHI 231 cells were incubated with 1 or 5 yM SB 203580 for 24 hours followed by crosslinking of IgM (Fig. 3.9 b) or CD40 (Fig. 3.9 c). M A P K A P kinase-2 activation was then assayed as an index of p38 M A P kinase activity in vivo. Following 24 hours of culture, 5 \xM SB 203580 inhibited 90% of M A P K A P kinase-2 activity induced by anti-IgM (Fig. 3.9 b), and induction of kinase activity stimulated by anti-CD40 was reduced to below detectable levels (Fig. 3.9 c). These data indicate that the activation of M A P K A P kinase-2 induced by crosslinking of either IgM or CD40 depended on p38 M A P kinase activity. 3.2.8 Failure of IL-4 to activate p38 MAP kinase. IL-4, like CD40 and the BCR, is also an important regulator of B cell growth and differentiation (Clark and Ledbetter, 1994), and we were therefore interested to determine whether treatment of cells with IL-4 would activate p38 M A P kinase. However, treatment of freshly isolated B cells or LPS-activated B cell blasts with IL-4 failed to induce either p38 M A P kinase or M A P K A P kinase-2 activity (Fig. 3.8 and 3.9), consistent with observations we have made in myeloid cell lines (Foltz et al., 1997). IL-4 was active on both splenic B lymphocytes and WEHI 231 cells, as demonstrated by the IL-4-induced tyrosine phosphorylation of IRS-2, detected in aliquots of whole cell lysates resolved by SDS-PAGE and immunoblotted with the anti-phosphotyrosine mAb, 4G10 (data not shown). 42 Hsp25 + + + algM - 0 1 5 SB(uM) *m~~ Hsp 25 + + aCD40 1 5 SB (uM) Hsp 25 Figure 3.9: In vivo activation of MAPKAP kinase-2 by crosslinking of the BCR or CD40 on LPS-activated or WEHI 231 cells is inhibited by SB 203580. a) LPS-activated splenic B cells were left untreated (C), or stimulated with 40 ug/mL anti-IgM (algM), pre-incubated for 30 minutes with 1 uM SB 203580 and stimulated with 40 ug/mL anti-IgM (algM + SB), stimulated with 10 ug/mL anti-CD40 (aCD40), pre-incubated for 30 minutes with 1 uM SB 203580 and stimulated with 10 ug/mL anti-CD40 for 10 minutes (aCD40 + SB), or stimulated with 10 ug/mL IL-4 for 10 minutes (IL-4). b) WEHI 231 cells were incubated at 37° C for 24 hours at a concentration of 2.5xl0 5 / mL with 1 or 5 uM SB 203580, or without inhibitor. Cells were then pelleted and re-suspended at 2xl0 7 cells/mL in the same media. Cells incubated without SB 203580 for 24 hours were left unstimulated (far left lane) or stimulated with 40 ug/mL anti-IgM for 10 minutes (algM). Cells incubated with 1 or 5 uM SB 203580 were stimulated with anti-IgM in the same way. c) WEHI 231 cells were incubated with or without SB 203580 as above then left unstimulated (far left lane), or stimulated with 10 ug/mL anti-CD40 for 15 minutes (aCD40), M A P K A P kinase-2 was immunoprecipitated from cell lysates and activation of M A P K A P kinase-2 was detected by an in vitro kinase assay using Hsp 25 as a substrate. Reaction products were resolved by SDS-PAGE, and the levels of phosphorylation were assessed by autoradiography (Hsp 25). 43 3.2.9 BCR-induced apoptosis of the immature B lymphoma, WEHI 231, is not affected by suppression of p38 MAP kinase activity. SB 203580 was used to determine whether suppression of p38 M A P kinase activity inhibited apoptosis in the immature B lymphoma WEHI 231 induced by anti-IgM antibodies. Apoptosis of WEHI 231 cells was induced by crosslinking the BCR with anti-IgM in the presence or absence of SB 203580 (1 or 5 nM), and no difference in the amount of cell death was observed (Fig. 3.10). As previously reported, anti-IgM-induced apoptosis was suppressed in WEHI 231 cells through co-stimulation delivered by crosslinking CD40 (20). The presence of 1 or 5 uM SB 203580 had no effect on the ability of crosslinking of CD40 to suppress apoptosis (Fig. 3.10). Similar results in WEHI 231 cells were obtained using SB 203580 at a concentration of 25 uM (data not shown). We therefore conclude that inhibition of p38 M A P kinase activity does not affect either BCR-induced apoptosis or CD40-mediated suppression of apoptosis in these cells. % sub-diploid events 40 30 20 10 I no mAb • algM • algM aCD40 0 5 SB 203580 (uM) Figure 3.10: SB 203580 does not affect anti-IgM-induced apoptosis or anti-CD40-mediated suppression of apoptosis in WEHI 231 cells. WEHI 231 cells were cultured for 24 hours (triplicate cultures of 2x l0 5 cells/mL) without antibodies (black bars), with 12 |xg/mL anti-IgM (gray bars) or with 5 |a.g/mL anti-CD40 and 12 ng/mL anti-IgM (white bars), in the presence and absence of the indicated concentrations of SB 203580. Triplicate samples were pooled and harvested, stained with propidium iodide and the percentage of events with sub-diploid D N A content was quantitated by flow cytometry. 44 3.3 Discussion These experiments have demonstrated that the p38 M A P kinase, and its substrate, M A P K A P kinase-2 are rapidly activated in T or B cells by crosslinking of the TCR or BCR, or by crosslinking of TNF receptor family members, Fas or CD40. Each of these molecules have roles in the control of apoptosis in the immune system. The data presented here, however, demonstrate that suppression of p38 M A P kinase activity does not inhibit the induction of apoptosis in two immunological models, activation-induced cell death in T cells and BCR-induced apoptosis in an immature B lymphoma. Crosslinking of the CD3 complex on proliferating T cells derived from mouse spleen or lymph node resulted in the rapid activation of p38 M A P kinase (Fig. 3.1 and 3.2). The effects of crosslinking of CD3 on activation of p38 M A P kinase in freshly-isolated peripheral T cells obtained from lymph nodes were also investigated. The activity of p38 M A P kinase was high in unstimulated cells, and no significant increase above control levels was seen upon crosslinking of CD3 or Fas. p38 M A P kinase was also reported to be active in unstimulated, freshly-isolated thymocytes, and it proposed that this was due to signals present in the thymic micro-environment (Sen et al., 1996). However, p38 M A P kinase is strongly activated by stress and therefore it is difficult to exclude the possibility that the activation of p38 M A P kinase seen in the experiments described here using freshly-isolated lymph node cells, or in those of Sen et al (1996) using thymocytes, was induced as a result of stress during the death of the animal or in preparation of single cell suspensions. Crosslinking of CD3 also resulted in activation of M A P K A P kinase-2 (Fig. 3.3), known to be a substrate of p38 M A P kinase in other cell types (Ben-Levy et al., 1995; Freshney et al., 1994; Rouse et al., 1994). Activation of M A P K A P kinase-2 stimulated by crosslinking of the 45 TCR (alone or together with crosslinking of CD28), or of the BCR, Fas or CD40 was dependent on p38 M A P kinase activity, as shown by inhibition of M A P K A P kinase-2 activity when cells were pre-treated with the p38 M A P kinase inhibitor, SB 203580 (Fig. 3.3, 3.4, 3.6 and 3.9). In vitro experiments indicate that M A P K A P kinase-2 is a substrate of both p38 M A P kinases and the ERKs (Freshney et al., 1994; Stokoe et al., 1992). Ligation of either the TCR or the BCR activate E R K (Gold and Matsuuchi, 1995; Weiss and Littman, 1994), as well as p38 M A P kinase and therefore it was of interest to use the p38 M A P kinase inhibitor to dissect the downstream effects of these two M A P kinases. The data indicate that the in vivo activation of M A P K A P kinase-2 via the TCR (Fig. 3.3) or via the B C R (Fig. 3.9), was dependent upon p38 M A P kinase activity, as activation of M A P K A P kinase-2 could be inhibited by SB 203580, which does not inhibit activation of the ERKs (Cuenda et al., 1995; Foltz et al., 1997). We have made similar observations in myeloid cells. The hemopoietic growth factors SLF, IL-3 and G M -CSF activate both ERKs and p38 M A P kinase, however, the subsequent activation of M A P K A P kinase-2 by these factors is dependent on p38 M A P kinase activity (Foltz et al., 1997). A synergistic activation of p38 M A P kinase and M A P K A P kinase-2 was observed when CD28 and CD3 were simultaneously crosslinked (Fig. 3.5 and 3.6). A similar synergy between CD3 and CD28 signaling was reported for activation of JNK in Jurkat cells (Su et al., 1994). Some molecules acting upstream of JNKs have been identified in Jurkat T cells. Lck is required for activation of JNK by CD3 and CD28, as demonstrated using Jurkat T cells deficient in expression of Lck. Over-expression of Syk enhanced the activation of JNKs by CD3 and CD28, and expression of a dominant-negative form of Rac inhibited activation of JNKs by CD3 and CD28 (Jacinto et al., 1998). It is likely that the components of the signaling pathway upstream of p38 M A P K in T cells would be similar to those involved in activation of JNK. 46 Signals delivered through CD28 on proliferating T cells do not appear to influence the decision of the cell to undergo AICD (Van Parijs et al., 1996). Although crosslinking of CD28 on proliferating T cells also synergizes with anti-CD3 to upregulate bcl-xL expression and IL-2 production (Van Parijs et al., 1996), the increases in levels of Bcl-xL mRNA and IL-2 do not correlate with survival in proliferating T cells as they do in naive T cells (Van Parijs et al., 1996). Therefore, the observation that crosslinking of CD28 synergized with crosslinking of CD3 to activate p38 M A P kinase, provides further evidence against a correlation between the levels of p38 M A P kinase activity and the induction of apoptosis. It is possible that the synergistic activation of p38 M A P kinase by simultaneous crosslinking of CD3 and CD28 could function to enhance the production of cytokines from T cells, through mechanisms similar to those through which p38 M A P kinase activity enhances the production of TNFa, IL-1, GM-CSF and IL-6 from other cells types (Beyaert et al., 1996; Lee et al., 1994). Crosslinking of Fas also induced activation of p38 M A P kinase and M A P K A P kinase-2 in proliferating T cells. The kinetics of p38 M A P kinase activation were rapid, as kinase activity was detectable between 1 and 3 minutes following crosslinking of Fas (Fig. 3.1 and 3.2), suggesting that activation of p38 M A P kinase by Fas crosslinking is a direct, membrane-proximal effect of Fas signaling. These rapid kinetics are consistent with the rapid activation of tyrosine kinase activity that follows crosslinking of Fas in the Jurkat T cells (Eischen et al., 1994). The data presented here showing a rapid and transient activation of p38 M A P kinase following ligation of Fas, contrast with those of Jou et al (1997), who recently reported that p38 M A P kinase activation occurred two to four hours after crosslinking of Fas in Jurkat T cells. They noted that the delayed kinetics of p38 M A P kinase activation correlated with the induction of Fas-mediated apoptosis. It is possible, however, that the delayed activation of p38 M A P 47 kinase was a response to the stress associated with the onset of the death process, rather than a direct effect of membrane-proximal Fas signals (Fig. 3.1, 3.2 and 3.4). Their observation that activation of p38 M A P kinase was inhibited by protease inhibitors which blocked Fas-induced apoptosis, is more consistent with the notion that the delayed activation of p38 M A P kinase they observed was secondary to the onset of apoptosis, and was not a direct consequence of Fas-mediated signaling. In a report that was published after the results described above, the authors show that the TNFa- or Fas-induced activation of caspases is not blocked by SB 203580 (3 or 30 uM). Interestingly, they observe a biphasic activation of JNKs and conclude that the later activation observed was not necessary for apoptosis and probably occurs as a consequence of apoptosis (Roulston et al., 1998). Signals transduced via both the TCR and Fas are necessary for AICD (Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995). TCR-induced apoptosis was not affected by concentrations (5 uM) of the p38 M A P kinase inhibitor (Fig. 3.7), which stably suppressed p38 M A P kinase activity in vivo throughout the duration of the apoptosis assay (Fig. 3.3 and 3.4). AICD is a Fas-dependent process, and therefore these data agree with the report that Fas-induced apoptosis of Jurkat T cells was not affected by SB 203580 (Juo et al., 1997). The findings in this thesis are also consistent with reports which demonstrate that inhibiting TNF-induced p38 M A P kinase activity using SB 203580 does not block TNF-mediated apoptosis (Beyaert et al., 1996). The functional significance of p38 M A P kinase activation in response to ligation of Fas is unclear. There are reports that Fas could contribute positively to growth signals for lymphocytes and it is possible that p38 M A P kinase could play a role in these functions. For example, B cells from Fas-deficient animals proliferate less well than B cells expressing Fas in response to acute stimulation with antigen in vivo (Rathmell et al., 1996). Similarly, when human T cells are 48 cultured in vitro, crosslinking of Fas using a monoclonal antibody enhances proliferation in response to crosslinking of anti-CD3 (Alderson et al., 1993). Crosslinking of the BCR or CD40 in freshly-isolated and LPS-activated splenic B cells or WEHI 231 cells also resulted in activation of p38 M A P kinase (Fig. 3.8) and of M A P K A P kinase-2 (Fig. 3.9). The data on these three B cell populations agree with results which have been reported using WEHI 231 cells (Sutherland et al., 1996). The use of the specific inhibitor of p38 M A P kinase demonstrated additionally that activation of M A P K A P kinase-2 was dependent on p38 M A P kinase activity (Fig. 3.9). Crosslinking of the BCR results in different fates in either mature peripheral B cells, where it induces proliferation (Goodnow et al., 1995) and in the immature B lymphoma, WEHI 231, where it results in apoptosis (Benhamou et al., 1990; Hasbold and Klaus, 1990). A number of gene products have been implicated in the control of apoptosis in WEHI 231 cells. Crosslinking of IgM on WEHI 231 cells results in decreased activity of N F K B which in turn results in decreased expression of c-myc. Ectopic expression of c-myc in WEHI 231 cells blocks apoptosis induced by crosslinking of anti-IgM. Furthermore, crosslinking of CD40 stabilizes the levels of c-myc expression (Sonenshein, 1997). Therefore, c-myc may be a key regulator of survival in these cells. Crosslinking of IgM also increases the levels of p53, a protein implicated in apoptosis, and the cyclin-dependent kinase inhibitor, p21 W A F I / C I P I 5 which is a target gene of p53. Blocking the function of these proteins, either by expressing a dominant-negative form of p53 or an antisense form of p21, or by microinjection of neutralizing antibodies to p21 all lead to a decrease in anti-IgM-induced apoptosis in WEHI 231 cells (Wu et al., 1998). 49 Activation of p38 M A P kinase by ligation of the B C R did not correlate with the subsequent fate of the B cell. Furthermore, crosslinking of CD40, which promotes survival in each of the B cell populations studied, also activated p38 M A P kinase (Fig. 3.8). In WEHI 231 cells, activation of p38 M A P kinase was induced by crosslinking of both the BCR and CD40, two stimuli with opposing influences on cell survival, again, demonstrating a lack of correlation of p38 M A P kinase activity with induction of apoptosis. Finally, concentrations of SB 203580 that showed inhibited p38 M A P kinase activity in these cells (Fig. 3.9) failed to affect BCR-induced apoptosis or CD40-mediated suppression of apoptosis (Fig. 3.10). The demonstration that activation of p38 M A P kinase minutes after crosslinking of IgM in naive or LPS-activated B cells or in WEHI 231 cells (Fig. 3.8), contrast with those obtained in human B lymphoma, B104, where anti-IgM-induced activation of p38 M A P kinase was shown to occur only 4-8 hours after crosslinking (Graves et al., 1996). This delayed activation was thought to correlate with apoptosis, because the kinetics of activation of p38 M A P kinase were similar to the kinetics of induction of apoptosis by anti-IgM. However, delayed activation of p38 M A P kinase following crosslinking of the BCR in these cells is likely to be a consequence of the cell undergoing apoptosis, rather then as a direct result of BCR signaling, and thus resembles the delayed activation of p38 M A P kinase seen hours after Fas crosslinking (Juo et al., 1997). In another study using human B cell lines, Graves et al (1998) observed that the late activation of both p38 M A P K and JNK that occurred 8-10 hours following crosslinking of either the BCR or Fas was blocked by inhibitors of caspase activity however, they found that BCR but not Fas-induced apoptosis was blocked by SB 203580. The manner in which they used the p38 M A P K inhibitor differed from our technique of using 1-5 uM SB 203580 throughout the assay, as they 50 pre-treated the cells with SB 203580 (20 \iM) before inducing apoptosis in the continued presence of titrated doses of SB 203580 (Graves et a l , 1998). This concentration of SB 203580 (20 uM) has been shown to inhibit JNK kinases as well as p38 M A P K (Chen et al., 1998; Whitmarsh et al., 1997) and therefore their observations could reflect the inhibition of enzymes other than p38 M A P K . Irrespective of when p38 M A P kinase is activated following receptor ligation in T and B cells, kinase activity induced by crosslinking of antigen receptors can be suppressed using SB 203580 throughout the course of a biological assay (Figs. 3.3 and 3.9). These data demonstrate the lack of effect of SB 203580 on apoptosis. Taken together with the data that incubation of cells with 5 uM SB 203580 suppressed the activity of p38 M A P kinase, this indicates that regardless of the activation patterns of p38 M A P kinase over the period during which apoptosis is being induced, kinase activity was suppressed throughout the assay. 51 Chapter 4 A role for p38 MAP kinase in the production of cytokines by T cells and APCs. 4.1 Rationale Given that ligation of the TCR and CD28 on T cells resulted in synergistic activation of p38 M A P K and M A P K A P kinase-2, (Fig. 3.5 and 3.6) (DeSilva et al., 1997; Salmon et al., 1997) and that p38 M A P K is involved in the production of cytokines by macrophages (Lee et al., 1994), we wanted to test the hypothesis that this enzyme was also involved in the production of cytokines during the activation of T cells by antigen. Therefore, the role of p38 M A P K in the production of IL-12 and IFNy during the priming of CD4 + T cells by APCs and cognate antigen was investigated. Inhibition of p38 M A P K activity using the specific inhibitor, SB 203580, resulted in decreased production of antigen-initiated IL-12 by APCs as well as decreased production of IFNy by T cells. We then asked whether the production of IL-12 induced by LPS was also regulated by p38 M A P K . When macrophages were stimulated with LPS, the production of IL-12 was actually increased in the presence of SB 203580. The production of IL-10 stimulated by LPS was inhibited by SB 203580, providing a likely explanation for the increased production of IL-12. 4.2 Results 52 4.2.1 Inhibition of p38 MAPK activity suppressed the antigen-initiated, CD40-dependent production of IL-12 by APCs. To investigate the role of p38 M A P K in the production of cytokines by APCs and T cells, the specific inhibitor of p38 M A P K activity, SB 203580 was used at a concentration of 5 JJ.M. It was previously demonstrated that incubation of T cells or B cells with this concentration of SB 203580 is sufficient to suppress the in vivo activity of p38 M A P K triggered by ligation of the TCR on T cells or CD40 on B cells over a period of 24 hours in culture ( Fig. 3.3) (Salmon et al., 1997). To investigate whether inhibition of p38 M A P K activity would affect the antigen-initiated, CD40-dependent production of IL-12 by APCs, co-cultures of A N D CD4 + T cells and splenic APCs were activated with PCC. CD4 + T cells were obtained from the lymph nodes of A N D transgenic mice expressing an ap TCR specific for PCC in the context of I-EK(Kaye et al., 1989). In some experiments, the APCs were derived from congenic B10.BR spleens by negative selection as described, and in others from A N D spleens by positive selection for C D l l b 7 C D l l c + . Similar results were obtained in both cases. Supernatants from these co-cultures were collected 12 hours later and assayed for the production of IL-12 p70 by ELISA using anti-p35 mAbs as a capture reagent and anti-p40 mAbs as a detection reagent. This strategy ensured that an excess of free IL-12 p40 subunits would not interfere with detection of the IL-12 p70 heterodimer. As shown in Figure 4.1a, the production of IL-12 p70 was induced by the addition of antigen and was dependent on CD40L as addition of a neutralizing mAb to CD40L inhibited the antigen-initiated production of IL-12 p70. The inclusion of SB 203580 (5 \iM) resulted in a significant (80%) inhibition in the antigen-stimulated production of IL-12 p70 (Fig 4.1a). 53 The effects of SB 203580 on the antigen-stimulated production of IL-12 p40 were also analyzed. As expected, the IL-12 p40 subunit was produced at much higher levels in response to antigenic stimulation than the IL-12 p70 heterodimer (Fig. 4.1b). Consistent with the results seen in Figure la, the antigen-initiated production of IL-12 p40 was reduced 80-90% by the presence of SB 203580 (5 uM) (Fig. 4.1b). A similar decrease in the production of IL-12 p70 was observed when co-cultures of CD4 + T cells and splenic APCs from (C57BL/6 x CBA) F, mice were polyclonally activated with immobilized anti-CD3 mAb (Fig. 4.1c). a. p70 IL-12 (pg/mL) Con PCC PCC PCC P C C SB SB (XCD40L (1 uM) (5 uM) Figure 4.1: Inhibition of p38 MAP kinase activity results in decreased production of IL-12 by APCs. a) CD4 + T cells were negatively selected from the lymph nodes of AND mice and cultured with APCs isolated by negative selection from B10.BR mice as described in medium alone (Con) or with PCC, 5 uM (PCC), PCC, 5 uM and SB 203580, 1 uM (PCC 1 uM SB) or PCC, 5 uM and SB 203580, 5 uM (PCC 5 uM SB), or PCC and anti-CD40L, 10 ug/mL (PCC aCD40L). Supernatants were harvested after 12 hours and assayed for the production of IL-12 p70. 54 Con PCC PCC (0.5 uM) (5 uM) b) CD4 + T cells isolated by positive selection from the lymph nodes of A N D mice were cultured with C D l l b 7 C D l l c + APCs isolated as described from the spleens of A N D mice in media alone (Con) or with PCC (0.5 or 5 (JVI) in the presence or absence of SB 203580 (5 \iM), as indicated. Supernatants were harvested after 12 hours and assayed for the production of IL-12 p40. c) Spleen cells form (CBA x C57BL/6) F, mice depleted of B cells and CD8 + T cells were cultured in media alone or polyclonally activated by culturing on anti-CD3- (2C11) coated plastic with IL-2 in the presence or absence of SB 203580 (5 |a.M) as described. Supernatants were harvested after 12 hours and assayed for the production of IL-12 p70 by ELISA. A l l results shown are the mean of triplicate cultures +/- S E M and are representative of at least three independent experiments. 55 4.2.2 The mechanism of inhibition of CD40L-dependent, antigen-initiated IL-12 production by SB 203580. We wished to determine whether the inhibition of IL-12 production by SB 203580 in T cell/APC co-cultures reflected actions on the T cells or on the APCs. There was no evidence that decreased production of IL-12 reflected a defect in antigen presentation as the use of anti-CD3 mAb (Fig. 4.1c) to activate T cells resulted in an inhibition of IL-12 production in the presence of SB 203580 (Fig. 4.1c) similar to that seen following antigenic stimulation (Fig. 4.1a and b). The activation of T cells by antigen did not appear to be compromised by the presence of SB 203580, as the CD4 + T cell/APC co-cultures were observed to undergo antigen-induced blastogenesis and proliferation in the presence or absence of SB 203580 (data not shown). To further assess the activation of T cells, the up-regulation of CD69, a hallmark of T cell activation (Yokoyama et al., 1988), was monitored by flow cytometry 24 hours after the addition of PCC (5 uM). The upregulation of CD69 induced by stimulation with antigen was unaffected by the presence of SB 203580 (Fig. 4.2). The up-regulation of CD69 was also unaffected by inhibition of p38 M A P K activity when T cells were stimulated with 10-fold less antigen (0.5 uM PCC) (data not shown). Con PCC PCC + 5 LIM SB CD69 > Figure 4.2: SB 203580 does not affect the antigen-initiated up-regulation of CD69 on CD4+ T cells. C D 4 + T cells negatively selected from lymph nodes of A N D mice were cultured with splenic APCs isolated by positive selection from A N D mice in medium alone (Con) or with PCC, 5 uM (PCC), PCC, 5 uM and SB 203580, 5 uM (PCC 5 uM SB). After 24 hours, expression of CD4 and CD69 were analyzed by flow cytometry. Both axes represent mean fluorescence intensity plotted on a logarithmic scale and similar results were obtained in four independent experiments. 56 The antigen-induced expression of CD40L by T cells (Foy et al., 1996) is critical to the production of IL-12 by APCs and was therefore another candidate for regulation by p38 M A P K . CD40L is a member of the TNF family of cytokines (Foy et al., 1996) and as SB 203580 was originally identified as an inhibitor of TNFa (Lee et al., 1994), we hypothesized that p38 M A P K could play a role in controlling antigen-initiated up-regulation of CD40L on T cells. However, the expression of CD40L induced on CD4 + T cells by PCC was unaffected by treatment with SB 203580 (5 j i M ) (Fig. 4.3). Similar results were obtained using immobilized anti-CD3 in co-cultures of splenic APCs and CD4 + T cells from (C57BL/6 x CBA) F[ mice (data not shown). Con PCC P C C + 5 ixM SB Figure 4.3: SB 203580 does not affect the up-regulation of CD40L on T cells. CD4 + T cells negatively selected from lymph nodes of A N D mice were cultured with splenic APCs isolated by positive selection from A N D mice in medium alone (no Ag) or with PCC, 5 piM (PCC), PCC, 5 nM and SB 203580, 5 uM (PCC 5 uM SB). After 6 hours, the cells were stained to detect expression of CD4 and CD40L and analyzed by flow cytometry. Both axes represent mean fluorescence intensity plotted on a logarithmic scale. 5 7 Next, the possibility that p38 M A P K was involved in the production of IL-12 by splenic APCs was investigated. Ligation of CD40 on B cells results in activation of p38 M A P K (Fig. 3.8) (Salmon et al., 1997; Sutherland et al., 1996). It is possible that a similar signaling pathway exists downstream of CD40 in APCs and if so, p38 M A P K activity could play a role in cytokine production in these cells. To test this hypothesis, splenic APC were positively selected using anti-CD l i b and anti-CD 11c mAb as described and CD40 was crosslinked using anti-CD40 mAbs. Macrophages express FcyR, and to eliminate possible effects of anti-CD40 mAbs on FcyR, APCs were coated with anti-FcyR II/III (2.4G2, rat IgG 2 a) during the purification process. As seen in Figure 3.5, crosslinking of CD40 on APCs resulted in the production of large amounts of IL-12 p40, which were reduced by 50 % by the presence of SB 203580 (5 uM). The APCs were capable of responding to other stimuli in the presence of SB 203580 as production of IL-12 from splenic APCs stimulated by LPS and IFNy was not inhibited (data not shown). p40 IL-12 (ng/mL) Con ocCD40 ccCD40 SB Figure 4.4: Inhibition of p38 MAPK activity results in decreased production of IL-12 by APCs stimulated by crosslinking of CD40. C D l l b 7 C D l l c + APCs isolated as described from the spleens of A N D mice were left unstimulated (Con) or stimulated with anti-CD40, 10 ug/mL (aCD40) in the presence or absence of SB 203580 (5 uM), as indicated. Supernatants were harvested 24 hours later and assayed for IL-12 p40 by ELISA. Results shown are the mean of triplicate cultures +/- S E M and are representative of three independent experiments. 58 4.2.3 Inhibition of p38 MAPK activity results in decreased production oflFNyby naive CD4+ T cells. IL-12 is a potent regulator of IFNy production by N K cells and greatly enhances the antigen-initiated production of IFNy by T cells (Fig. 1.6) (Trinchieri, 1997). Therefore, the effects of inhibition of p38 M A P K activity on the antigen-initiated production of IFNy by CD4 + T cells were investigated. Supernatants from the T cell/APC co-cultures described in Fig. 4.1b above were harvested 48 hours after the initial antigenic stimulation and assayed by ELISA for the production of IFNy. As shown in Figure 4.5, the presence of SB 203580 (5uM) resulted in a dramatic inhibition of the antigen-initiated production of IFNy. IFNy (ng/mL) Con PCC PCC (0.5 L I M ) (5uM) Figure 4.5: SB 203580 inhibits the antigen-initiated production of IFNy by naive CD4+ T cells. C D 4 + T cells isolated by positive selection from the lymph nodes of A N D mice were cultured with CD1 l b V C D l l c + APCs isolated as described from the spleens of A N D mice in medium alone (Con) or with PCC, 0.5 or 5 uM, in the presence or absence of SB 203580 (5 uM), as indicated. Supernatants were harvested after 48 hours and assayed for the production of IFNy by ELISA. Results shown are the mean of triplicate cultures +/- S E M and are representative of five independent experiments. 59 We next asked whether the reduced production of IFNy in cultures treated with SB 203580 reflected a direct effect of the p38 M A P K inhibitor on the synthesis of IFNy by T cells or whether it occurred as a consequence of decreased levels of IL-12 in these cultures. CD4 + T cells were isolated by positive selection and stimulated with immobilized anti-CD3 mAb and IL-2 in the absence of additional APCs capable of producing IL-12. In these cultures, the production of IFNy induced by crosslinking of anti-CD3 was decreased by 80% in the presence of 5 uM SB 203580 (Fig. 4.6a). In a second set of experiments, exogenous IL-12 was added to co-cultures of CD4 + T cells and APCs stimulated with PCC, resulting in a large increase in IFNy production over stimulation with PCC alone. However, the enhanced production of antigen-induced IFNy was still decreased approximately 80% by the presence of SB 203580 (5 uM) (Fig. 4.6b). Taken together, these results suggest that SB 203580 acted directly on the T cell to result in decreased production of antigen-initiated IFNy. Figure 4.6: SB 203580 acts directly on T cells to result in decreased production of IFNy. a) CD4 + T cells isolated by positive selection from the lymph nodes of A N D mice were cultured in media alone (Con) or stimulated polyclonally using plate-bound anti-CD3 mAbs and IL-2, in the presence or absence of SB 203580 (5 uM), as described. Supernatants were harvested after 48 hours and assayed for the production of IFNy by ELISA. Results shown are the mean of triplicate cultures +/- S E M and are representative of three independent experiments. 60 b. ». 25 ; 20 • IFNY 1 5 ; (ng/mL) 10 • 0 5 Con P C C P C C P C C IL-12 IL-12 SB b) CD4 + T cells isolated by positive selection from the lymph nodes of AND mice were cultured with CD1 lb7CDl lc + APCs isolated from the spleens of AND mice and in medium alone (Con) or with PCC (5 uM) in the presence or absence of SB 203580 (5 uM) and IL-12 p70 (1 ng/mL) as described. Supernatants were harvested after 48 hours and assayed for the production of IFNy by ELISA. Results shown are the mean of triplicate cultures +/- SEM. 4.2.4 Inhibition of p38 MAPK activity affects the production of IFNy to a lesser degree in activated T cells than in naive T cells. Naive T cells differ from activated T cells in many respects. For example, activation of naive T cells requires higher concentrations of antigen and more potent co-stimulatory signals than is the case for activated or memory T cells (Croft et al., 1994; Croft et al., 1992; Dubey et al., 1996; Ericsson et al., 1996). We asked whether there would be a differential requirement for p38 M A P K activity for the production of IFNy by activated versus naive T cells. Naive CD4 + T cells were activated with antigen as described in Figure 4.1 and allowed to proliferate in the presence of IL-2 for 6 days. The cells were then re-stimulated with immobilized anti-CD3 in the presence or absence of 5 \iM SB 203580 for 24 hours. A polyclonal activator was used in this experiment to eliminate any influence of APCs. Supernatants were harvested from these cultures and IFNy was analyzed by ELISA. In contrast to the results obtained with naive T cells in Figs. 4.5 and 4.6, little, if any inhibition of IFNy by SB 203580 in previously activated T cells was seen (Fig. 4.7). 61 60 • no SB ED 5 u M i IFNy (ng/mL) 40 20 0 Con 2C11 2C11 (0.1 ug/mL) (1 ug/mL) Figure 4.7: Inhibition of p38 MAPK activity has minimal effects on the production of IFNy by activated T cells. CD4 + T cells were isolated by positive selection from the lymph nodes of AND mice and cultured with C D l l b 7 C D l l c + APCs isolated as described from the spleens of AND mice and PCC (5 uM) and IL-2 for 48 hours, and then expanded and grown in the continued presence of IL-2 for 6 days. Cells were then washed and cultured at 5 x 105/ mL in media alone (Con) or re-stimulated with plate-bound anti-CD3 mAb (2C11) at the concentrations indicated. Supernatants were harvested after 24 hours and assayed for the production of IFNy by ELISA. All results shown are the mean of triplicate cultures +/- SEM and are representative of three independent experiments. 4.2.5 The production of IL-12 induced by LPS from PECs is enhanced by SB 203580. The production of IL-12 can be induced at two distinct stages during the immune response. As discussed above, IL-12 is produced by APCs in a CD40-dependent antigen-initiated manner. However, during the innate immune response, which precedes the induction of antigen-specific immunity, the production of IL-12 by APCs is triggered by infectious agents such as bacterial LPS. Given the finding that SB 203580 inhibited the production of antigen-initiated IL-12 (Fig. 4.1) we wished to see if inhibition of p38 MAPK would have a similar effect on the production of IL-12 stimulated by LPS. 62 Thioglycollate-elicited PECs and resident peritoneal macrophages were used to study the effect of SB 203580 on the production of IL-12 induced by LPS and IFNy. Thioglycollate-elicited PECs were stimulated with LPS, IFNy or both and IL-12 p70 was measured by ELISA after 24 hours. As seen in Fig. 4.8a, the production of IL-12 p70 elicited by LPS and IFNy was significantly enhanced when SB 203580 (5 ixM) was added. Similar observations were made using resident peritoneal macrophages stimulated with LPS and IFNy in the presence of SB 203580 (1 or 5 uM) (Fig. 4.8b). The enhancement of IL-12 by SB 203580 (1 uM) as seen in Figure 4.8b was consistently higher than that induced by SB 203580 (5 \iM) in both PECs (data not shown) and resident peritoneal macrophages. 600 p70 IL-12 400 (pg/mL) 2001 0 no SB 5p:MSB Con LPS IFNy LPS IFNy Figure 4.8: Inhibition of p38 MAPK activity enhances the production of LPS-stimulated IL-12. a. C57BL/6 x C B A F, mice were injected with thioglycollate broth. 5 days later, PEC were harvested, non-adherent cells were removed and macrophages were stimulated as described above. After 24 hours, supernatants were harvested and IL-12 was quantitated by ELISA. 63 p70 IL-12 (pg/mL) 300 200 100 0 Con no SB 1 LIMSB 5uMSB LPS IFNy LPS IFNy b. Resident peritoneal macrophages were harvested from C57BL/6 x C B A F, mice. Non-adherent cells were removed and macrophages were stimulated as described. After 24 hours, supernatants were harvested and IL-12 was quantitated by ELISA. 4.2.6 SB 203580 inhibits the production of IL-10 by PECs. Given the unexpected result that SB 203580 enhanced the LPS-stimulated production of IL-12, we hypothesized that inhibition of p38 MAPK activity must reduce the production of another cytokine that would normally exert negative effects on IL-12. The most potent inhibitor of IL-12 production induced in response to LPS is IL-10, and therefore we asked what the effect of SB 203580 would be on the production of IL-10. As seen in Figure 4.9, SB 203580 significantly reduced the production of IL-10 by PECs, and this correlated with the increase in the production of IL-12 observed in these same supernatants. 64 IL-10 (pg/mL) Con LPS IFNy LPS I F N Y Figure 4.9: SB 203580 inhibits the production of IL-10. C 5 7 B L / 6 x C B A F, mice were injected with thioglycollate broth. 5 days later, PEC were harvested, non-adherent cells were removed and macrophages were stimulated as described above. After 24 hours, supernatants were harvested and IL-10 was quantitated by ELISA. These are the same supernatants from which IL-12 was assayed in Figure 4.8a. We next asked what the effect of SB 203580 would be on the production of IL-12 in the absence of IL-10. PECs were stimulated as described above with LPS and IFNy in the presence and absence of neutralizing antibodies to IL-10 with or without SB 203580, and the supernatants were harvested after 24 hours. As expected, anti-IL-10 mAb greatly enhanced the production of IL-12 elicited by LPS and IFNy (Fig. 4.10), suggesting that under normal circumstances, IL-10 does in fact limit the production of IL-12 in these cultures. As before, SB 203580 (5 |oM) enhanced the production of IL-12, but not to the same degree as anti-IL-10. However, when SB 203580 (5 | iM) was added to cultures stimulated with LPS and IFNy and containing anti-IL-10 mAbs, the production of IL-12 was greatly enhanced above the levels seen in cultures stimulated with LPS, IFNy, and SB 203580 (5uM), but was decreased by 50% compared the cultures with 65 LPS, IFNy and anti-IL-10 mAbs. This suggests that inhibition of p38 MAPK activity resulted in a modest inhibition of IL-12 production that could not be detected in the presence of IL-10. 1000 800 p70 IL-12 600 ( P g / m L ) 400 200 0 QE Con no SB 5uM SB aIL-10 aIL-10 5pM SB LPS IFNy LPS IFNy Figure 4.10: The effect of SB 203580 on the production of IL-12 in the absence of IL-10. C57BL/6 x C B A F, mice were injected with thioglycollate broth. 5 days later, PEC were harvested, non-adherent cells were removed and macrophages were stimulated as described above. After 24 hours, supernatants were harvested and IL-12 was quantitated by ELISA. 4.3 Discussion 66 The inhibition of p38 M A P K activity during the priming of naive T cells by antigen reduces the production of two cytokines, IL-12 and IFNy- This thesis has presented evidence that the decreased production of IL-12 in the presence of SB 203580 was due to interference with CD40-mediated signaling in APCs, whereas the decrease in the production of IFNy was due to a direct effect on the T cell. In contrast, the production of LPS-elicited IL-12 was enhanced by SB 203580, and that this correlated to an inhibition of IL-10 in these cultures. The reduced production of antigen-initiated IL-12 in the presence of SB 203580 was not due to a defect in antigen presentation as the production of IL-12 by APCs was still inhibited when T cells were activated using anti-CD3 (Fig. 4.1c); nor was it due to impaired activation of T cells by antigen, as the up-regulation of CD69 and CD40L still occurred normally in the presence of SB 203580 (Fig. 4.2 and 4.3). The expression of CD40L on T cells is critical to the production of antigen-initiated IL-12 (Campbell et al., 1996; Kamanaka et al., 1996; Soong et al., 1996), but clearly, this process was not dependent on p38 M A P K activity (Fig. 4.3). The data showing that the production of IL-12 from APCs stimulated by ligation of CD40 was regulated by p38 M A P K (Fig. 4.4) suggest that the decreased production of antigen-initiated IL-12 in the presence of SB 203580 reflected a defect in signaling in APCs downstream of CD40. Ligation of CD40 on B cells results in the activation of p38 M A P K (Fig. 3.8) (Salmon et al., 1997; Sutherland et al., 1996) and it is likely that this also occurs in macrophages and dendritic cells expressing CD40. Ligation of CD40 in macrophages also induces the production of TNFa, IL-1, IFNy and nitric oxide (Grewal and Flavell, 1996). It is conceivable that p38 M A P K is involved in these processes as SB 203580 has been shown to inhibit the production of IL-6 in response to signaling via the TNFR (Beyaert et al., 1996). 67 The reduction in IFNy production by antigen-stimulated T cells in the presence of SB 203580 did not appear to be secondary to the reduced levels of IL-12 secreted by APCs. These conclusions were supported by the experiments showing that although antigen-initiated production of IFNy was greatly enhanced by the addition of exogenous IL-12, this enhanced production of IFNy was still inhibited by SB 203580 (Fig. 4.6a). Furthermore, SB 203580 inhibited the production of IFNy by purified CD4 + T cells that were polyclonally activated in the absence of additional APCs (Fig. 4.6b). Further evidence for a role for p38 M A P K in the production of IFNy by CD4 + T cells has recently come from transgenic mice expressing mutant molecules of the p38 M A P K pathway (Rincon et al., 1998). T cells expressing a transgene encoding a dominant negative mutant form of p38 M A P K exhibited a 50-90% decrease in the production of IFNy by Th l cells stimulated by Con A or antigen. A complimentary result was obtained with T cells that expressed a constitutively active mutant form of the upstream activating kinase of p38 M A P K , M K K 6 . These cells exhibited enhanced production of IFNy in response to Con A and IL-12. These data, together with ours (Fig. 4.5 and 4.6), clearly indicate that p38 M A P kinase plays a role in the synthesis of IFNy and therefore, could play a role in the regulation of Th l responses. We have also demonstrated that p38 M A P K is important for the CD40-dependent production of IL-12 by APCs and may therefore act prior to the synthesis of IFNy by T cells to influence the development of Thl cells. In contrast to its effects on naive T cells (Fig. 4.5 and 4.6) SB 203580, did not dramatically affect the production of IFNy by re-stimulated T cells (Fig. 4.7). Activated T cells differ from naive T cells in many respects. For example, following an encounter with antigen, 68 naive T cells undergo proliferation while activated T cells undergo activation-induced cell death (Nagata and Golstein, 1995). One possible explanation of the failure of SB 203580 to reduce the secretion of IFNy by activated T cells is that inhibition of p38 M A P K activity might block AICD, thereby resulting in an increased survival of IFNy-producing T cells and no net effect of SB 203580 on the production of IFNy. However, this possibility can be excluded as it was demonstrated previously that inhibition of p38 M A P kinase activity does not affect AICD (Fig. 3.7) (Salmon et al., 1997). Naive T cells require higher concentrations of antigen and are more dependent on co-stimulatory signals than activated T cells (Croft et al., 1994; Croft et al., 1992; Dubey et al., 1996). For example, in naive T cells the optimal production of IL-2 (Dubey et al., 1996) or IFNy (Fraser and Weiss, 1992; Teh and Teh, 1997) in response to ligation of CD3 is dependent upon co-ligation of CD28, whereas in previously activated T cells that have been re-stimulated with anti-CD3, the production of IL-2 or IFNy is independent of co-stimulatory signals (Dubey et al., 1996). Furthermore, signaling proteins such as phospholipase C-yl and M A P K are known to be differentially activated by ligation of the TCR in naive vs. activated T cells (Ericsson et al., 1996). The greater dependence of the production of IFNy on p38 M A P K activity in naive (Fig. 4.5) vs. activated (Fig. 4.7) T cells may reflect differences in such signaling molecules with alternatives to the p38 M A P K pathway being present in activated T cells. The finding that the production of IFNy by re-stimulated T cells was only modestly inhibited in the presence of SB 203580 (5 uM) (Fig. 4.7) differed from the observations of Rincon et al (1998) who observed a 50% decrease in the production of IFNy by re-stimulated Th, cells in the presence of SB 203580 (5 uM). However, this difference may reflect the 69 differences between the two studies in the cytokines in which the cells were cultured and the stimuli used to induce the production of IFNy. As discussed above, SB 203580 decreases the production of the pro-inflammatory cytokines IL-12 and IFNy that are involved in the development of Th l cells during an antigen-specific immune response. However, following stimulation with LPS, SB 203580 increases the production of IL-12 and decreases IL-10, a potent anti-inflammatory cytokine. Cells such as macrophages would be among the first cells to respond to an infection, and the spectrum of cytokines produced during this initial encounter would influence the nature of the acquired immune response. IL-10 is important in directing the development of Th2 cells, as the development of Th l cells is impaired in the presence of IL-10 (Hsieh et al., 1992; Hsieh et al., 1993). In mice deficient in IL-10, unchecked inflammatory responses result in colitis (Kuhn et al., 1993). However, these animals are more resistant to intracellular pathogens such as Listeria monocytogenes (Dai et al., 1997). Therefore, if p38 M A P K activity was inhibited during an infection, the decrease in IL-10 and increase in IL-12 could result in an enhancement of inflammatory responses. LPS-induced IL-10 was also shown to be inhibited by SB 203580, but not by an inhibitor of the E R K pathway, in human monocytes (Foey et al., 1998). We hypothesize that the enhancement of IL-12 (Fig. 4.8) by SB 203580 occurred as a result of inhibition of IL-10 production (Fig. 4.9). However, the involvement of other molecules which down-regulate IL-12 production cannot be excluded. For instance, prostaglandin E 2 is a product of activated macrophages and a potent inhibitor of IL-12 (van der Pouw Kraan et al., 1995). Inhibition of p38 M A P K activity has been shown to suppress the I L - l p - or TNFcc-induced expression of prostaglandin endoperoxide synthase-2 (PGHS-2) an enzyme that metabolizes arachadonic acid into prostaglandin H 2 , which in turn is processed into P G E 2 70 (Pouliot et al., 1997) and in another study, the production of PGE 2 induced by IL-ip shown to be inhibited by SB 203580 (Guan et al., 1997). The regulation of P G E 2 by p38 M A P K could therefore also influence the production of IL-12 by macrophages. IL-4 is also known to inhibit the production of IL-12, however, it is not thought to be a product of activated macrophages (D'Andrea et al., 1995; Koch et al., 1996). The molecular mechanism of the inhibition of antigen-initiated IL-12 and IFNy and LPS-induced IL-10 by SB 203580 remains to be elucidated. As noted above, SB 203580 acts on p38a and p38p, and given that p38p is expressed at low or undetectable levels in lymphoid tissues (Wang et al., 1997), the results may reflect inhibition of p38a, thereby implicating it in the production of IL-12 and IFNy. As described in the introduction, members of the p38 M A P K family act on two classes of substrates which could be involved in the transcriptional or translational control of cytokine production: kinases and transcription factors. There is some evidence implicating kinases downstream of p38 M A P K in the regulation of protein synthesis. For example, M N K 1 phosphorylates regulatory sites on eIF-4E, a protein involved in the initiation of translation and enhances its affinity for the 5' cap structure of mRNA (Minich et al., 1994). Indeed, SB 203580 has been shown to inhibit the production of TNFa and IL-lp at a translational level which may involve an inhibition of translational initiation regulated through A U U U A motifs present in the 3' untranslated region of the mRNAs (Lee et al., 1994; Prichett et al., 1995). SB 203580 has also been shown to exert its effects on cytokine production at a transcriptional level, in the case of IFNy (Rincon et al., 1998) and IL-6 (Beyaert et al., 1996). These data indicate that p38 M A P K s may influence cytokine production at multiple levels. Thus, it will be important to identify which of its substrates are important for the synthesis of IL-12, IFNy, and IL-10. 71 Chapter 5 Conclusions This thesis has focused on an investigation of the hypothesis that p38 M A P K would be activated in lymphocytes and play a role in the regulation of immune functions in lymphocytes and A P C s . We conclude that p38 M A P K is activated by a variety of stimuli including the ligation of antigen receptors on T or B cells and the ligation of Fas or CD40 . A role for p38 M A P K in apoptosis that was triggered by ligation of antigen receptors on T or B cells was excluded. However, p38 M A P K did play a role in the antigen-initiated production of IL-12 by A P C s the production of IFNy by T cells, and in the LPS-induced production of IL-10 by macrophages. Each of these three cytokines is important in regulating immune responses. A role for p38 M A P K in immune responses appears to be highly conserved in evolution, as recent work has demonstrated that the Drosophila p38 M A P K homologue is involved in the regulation of the of expression of immunity genes (Han et al., 1998). The function of p38 M A P K in cells of the immune system was analyzed using a loss-of-function approach by suppressing the activity of p38 M A P K in vitro with the drug, SB 203580. Inferences on the function of p38 M A P K drawn from observations of inhibitory effects of SB 203580 on the production of cytokines must be tempered by the reality that additional interactions of S B 203580 with other molecules can never be absolutely excluded. Complementary loss-of-function approaches to identify a requirement for p38 M A P K activity in a given biological function such as the production of cytokines would include the use of dominant inhibitory mutants of M K K 3 , M K K 6 or p38 M A P K . However, similar caveats apply to the use of dominant inhibitory mutant proteins as to SB 203580, as it is very difficult to rule out interactions of mutant proteins with molecules involved in related signal transduction pathways such as the J N K pathway or as yet uncharacterized molecules. The combined use of these two approaches would be the best strategy, providing a greater degree of confidence that p38 M A P K is involved in this process being examined. For instance, in independent 72 experiments, SB 203580 and a dominant inhibitory mutant of p38 MAPK both indicated a role for p38 MAPK in the production of IFNy (Fig. 4.5 and 4.6 and (Rincon et al., 1998)). The use of mice with targeted disruptions of p38 MAPK genes or M K K genes may be useful. However, as these enzymes belong to multigenic families, it is possible that more than one member would have to be disrupted to obtain a phenotype. This approach has been used to analyze the function of the closely related JNK pathway. Like the p38 MAPKs, the JNKs also form a multigenic family. However, mice deficient in one of three genes, JNK3, had a defect in apoptosis in neurons (Yang et al., 1997), indicating that not all the functions of members of this family were redundant. It is plausible that results similar to those obtained with SB 203580 could be seen in mice deficient in the a and p drug-sensitive isoforms of p38 MAPK. Indeed, given that the expression of p38p is not high in cells of lymphoid origin (Wang et al., 1997), it is possible that disruption of the p38a isoform alone would be informative. An upstream activating kinase for JNK, MKK4, has also been disrupted and this results in early lethality (Ganiatsas et al., 1998; Nishina et al., 1997; Swat et al., 1998; Yang et al., 1997). It is possible that enzymes in the p38 M A P K pathway may also be important for embryogenesis, in which case tissue-specific deletion of genes such as MKK3, MKK6 or isoforms of p38 MAPK may be more useful than null animals. Gain-of-function experiments could also be undertaken to analyze the role of p38 MAPK using cells transfected with cDNAs encoding constitutively active mutants of MKK3 or MKK6. Interestingly, inhibition of p38 MAPK activity using SB 203580 had no effect on Fas-induced apoptosis in Jurkat T cells, however, over-expression of a constitutively active mutant of MKK6 induced apoptosis and expression of a dominant negative version of MKK6 enhanced the survival of cells following ligation of Fas (Huang et al., 1997). MKK6 also activates the drug-resistant Y and 8 isoforms of p38 MAPK and thus it is possible that these isoforms and not the drug-sensitive a or p isoforms are involved in apoptosis. Alternatively, ectopic expression of this mutant protein could also activate other pathways which may not be activated by endogenous 73 MKK6 under physiological conditions. Yet another approach to analyzing the role of p38 MAPK would be the use of mutants of the a or (3 p38 MAPK drug-sensitive isoforms that have been rendered drug-resistant by substituting three residues in the ATP binding pocket for those found in the p38y isoform (Gum et al., 1998). If this cDNA was expressed in cells, any effects of the inhibitor in the presence of a drug-resistant version of p38 MAPK would indicate non-specific effects. Our method for analyzing activation of p38 MAPK by various stimuli involves immunoprecipitation of p38 MAPK using antisera that had be raised by immunizing rabbits with full-length p38cc MAPK (Lee et al., 1994). The four members of the p38 MAPK family are highly related (Wang et al., 1997), and it is possible that this antisera would immunoprecipitate all forms, and therefore, we cannot distinguish which of the p38 MAPKs are activated by the stimuli used here. It will be important to determine which of the p38 MAPKs are expressed in lymphocytes. The activation of MAPKAP kinase-2 observed was inhibitable by SB 203580, thus indicating that the a or (3 drug-sensitive forms of p38 MAPK are the only isoforms involved in the activation of MAPKAP kinase-2 in the cells studied here. This finding is consistent with reports that p38 MAPK a and 3 preferentially activate MAPKAP kinase-2, while p38 MAPK y and 5 do not (Kumar et al., 1997; Wang et al, 1997). This thesis demonstrates that p38 MAP kinase is activated by the engagement of receptors which send apoptotic signals, specifically the TCR and Fas on activated T cells, and the BCR on immature B cells. However, p38 MAP kinase was also activated by engagement of the BCR on mature B cells as well as by CD40, signals that contribute to proliferation. Thus, a simple correlation cannot be drawn between the activation of p38 MAP kinase and apoptosis or 74 survival in lymphocytes. Furthermore, suppression of p38 MAP kinase activity did not inhibit AICD in activated T cells or BCR-induced apoptosis in WEHI 231 cells. Our conclusions with regards to apoptosis in lymphocytes are based on observing no effect of the drug on the induction of apoptosis, despite the demonstration that the SB 203580 had effectively suppressed p38 MAP kinase activity induced by crosslinking of antigen receptors. Therefore, suppression of p38 MAP kinase activity does not inhibit apoptosis in two immunological models in lymphocytes. The hypothesis that p38 M A P K was involved in the synthesis of cytokines by lymphocytes and APCs was examined. The observations described here using antigen specific T cells and APCs presenting cognate antigen suggested that p38 M A P K plays two roles in regulating the cytokine environment during T cell priming, one in the regulation of the production of IL-12 by APCs and another in production of IFNy by T cells. These findings are consistent with the evidence that p38 MAPK is necessary for the production of pro-inflammatory cytokines (Lee et al., 1994). However, when the production of cytokines induced by LPS in macrophages was investigated, we found that p38 MAPK actually played a pivotal role in the production of the anti-inflammatory cytokine, IL-10. Furthermore, in cultures where the production of IL-10 was suppressed by SB 203580, the production of IL-12 was enhanced. These results suggest that inhibition of p38 MAPK activity could, in some circumstances, result in an increased production of pro-inflammatory cytokines. The results obtained here using antigen-specific T cells APCs and cognate antigen raised the possibility that the differentiation of Thl cells could be inhibited in the presence of SB 203580, a conclusion consistent with finding obtained using dominant-negative MKK6 (Rincon 75 et al., 1998). However, our observations that the inhibition of p38 M A P K activity during the LPS-induced production of cytokines could enhance the production of IL-12 by suppressing the production of IL-10 raises the possibility that the role of p38 M A P K in the development of Thl cells may be complex. It will be important to investigate the effects of inhibition of p38 M A P K activity during the course of an infection in vivo. It has been demonstrated in vivo that SB 203580 reduced the mortality induced by endotoxic shock initiated by injection of LPS (Badger et al., 1996). 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