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The role of mitogen-activated protein kinases in toxic shock syndrome toxin-1 mediated signalling in… Drews, Steven Jeffrey 1998

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THE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES IN TOXIC SHOCK SYNDROME TOXIN-1 MEDIATED SIGNALLING IN THP-1 CELLS. by STEVEN JEFFREY DREWS B.A., The University of British Columbia, 1992 B.Sc, The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF EXPERIMENTAL PATHOLOGY We accept this thesis as conforming to the required standard  THE UNIVERSITY OF^RTTTM^OtiuJ^AMAY 1998 © STEVEN JEFFREY DREWS  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or  her  for  Department  of  *?JfoUtiUC°7  The University of British Vancouver, Canada  Date  DE-6 (2/88)  Columbia  purposes  gain  requirements that  agree  may  representatives.  financial  permission.  I further  the  be  It  shall not  that  the  by  understood be  allowed  an  advanced  Library shall  permission  granted  is  for  for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  Abstract Toxic shock syndrome toxin-1 (TSST-1) is a 22 kDa superantigen produced by Staphylococcus aureus and is the primary cause of toxic shock syndrome (TSS) in humans. The purpose of this project was to determine the effect of mitogen-activated protein (MAP) kinase activity in THP-1 cells prior to the recruitment of T cells. M A P kinases interact with several different kinase pathways and play important roles in many physiological processes including growth and maturation, bacterial pathogen-mediated signalling and cytokine-induced events. THP-1 cells constitutively expresses the TSST-1 binding human leukocyte antigen-DR (HLADR) molecule which we have shown to be upregulated with 100 U/ml IFN-y. Initial experiments set pathophysiological conditions for TSST-1 (MN8) and lipolysaccharide (LPS) mediated signalling in pro-monocytic THP-1 cells. To induce tumor necrosis factor-a (TNF-a) release, TSST-1 (1 ng/ml) required the presence of both THP-1 and T cells in co-culture. The constitutive levels of H L A - D R on THP-1 cells were sufficient to bind to TSST-1 and present SAg to T cells in a manner which induced TNF-a release. Under these condition, TSST-1 induced the release of TNF-a within 12 hours (n=3). This TSST-1-induced TNF-a release was sustained for up to 84 hours after treatment. In contrast, LPS (1 ug/ml) was sufficient to induce the transient release (12 hours) of TNF-a from THP-1 cells alone (n=3). The addition of IFN-y (100 U/ml) and LPS to THP-1 culture induced the sustained release of TNF-a from these cells. To screen for early M A P kinase activation in THP-1 cells alone, THP-1 cells were then treated with 1 ng/ml TSST-1, 1 n M phorbol 12- myristate li-acetate (PMA) or RPMI 1640 and lysed within 30 minutes (n=3). Cellular proteins were separated by anion exchange chromatography and assayed for M A P kinase activity using myelin basic protein (MBP) kinase assays. Both TSST-1 and P M A induced a two-fold peak increase in THP-1 M B P kinase  ii  activity, at 30 minutes and 5 minutes respectively. The presence of other non-ERK-1/2 kinase activities in these anion exchange column eluents indicate that non-ERK-1/2 M B P kinases may also play an important role in TSST-1-induced signalling in monocytic cells. THP-1 cells were treated with TSST-1 (1 ng/ml), LPS (1 ug/ml), RPMI 1640 and a TSST-1 mutant (G31Rmut) (1 ng/ml) and lysed within 30 minutes (n=6). Extracellular-signal regulated kinase-1 (ERK-1) and ERK-2 isoforms of M A P kinase were immunoprecipitated and assayed with M B P kinase assays. When the median of areas under ERK-2 activation curves were compared, G31Rmut TSST-1 induced an ERK-2 activity profde that favored activation over deactivation (Wilcoxin signed rank test, P= 0.0313, n=6). In contrast, TSST-1 and LPS induced early ERK-2 activity profiles that favored neither activation nor deactivation. A l l three toxins also induced the early activation of ERK-1 but none of these profiles favored activation over deactivation. The differences between G31Rmut TSST-1 and TSST-1 induced ERK-2 activation patterns are probably due to the lowered binding affinity that G31Rmut TSST-1 has to M H C II and may reflect the physiological differences reported in cultures treated with both toxins. In comparison, the similarities in LPS and TSST-1-induced ERK-1/2 signalling may be due to the induction of common pathways in monocytic cells. In the future, it is hoped that this model can be used to study M A P kinase activation in THP-1 and T cell co-cultures.  iii  Table of Contents  Abstract List of Tables List of Figures List of Abbreviations Acknowledgements Chapter 1. Introduction 1.1. The epidemiology and history of toxic shock syndrome. 1.2. The microbiology of toxic shock syndrome. 1.3. A comparison of superantigens to conventional antigens. 1.3.1. Antigen binding to major histocompatability complex molecules on antigen presenting cells. 1.3.2. Binding of the MHC-Sag complex to T cell receptors. 1.3.3. Superantigen binding to mutiple M H C alleles. 1.4. The Staphylococcus aureus superantigen TSST-1. 1.4.1. Pathogenic mechanisms of TSST-1. 1.5. Lipopolysaccharide induced shock: A comparison to TSST-1 -induced pathogenesis. 1.5.1. Structure and function. 1.5.2. The CD14-dependent pathway. 1.5.3. The CD14-independent pathway.  iv  1.5.4. A comparison between LPS and TSST-1 mediated pathogenesis of septic shock models. 1.6. Mitogen activated protein kinase pathways. 1.6.1. M A P kinases: A n introduction.  15 16 16  1.6.2. MEK-1 and M E K - 2 : Direct upstream activators of ERK-1 and EPvK-2. 1.6.3. Ras-Raf-1 pathway activation of MEK-1/2.  17 18  1.6.4. M A P K or E R K kinase kinase activation pathways: A second Ras-dependent pathway.  21  1.6.5. Ras-independent activation of E R K pathways: A tangled web to unweave.  23  1.6.6. The role of ERK-1 and ERK-2 in transcriptional control. 1.6.7. Activation of downstream kinases.  24 25  1.6.8. Mitogen activated protein kinases: Upstream activation conditions.  26  1.6.8.1. Signalling via bacterial toxins.  26  1.6.8.2. Cell growth and maturation.  27  1.6.8.3. The role of M A P kinases in cytokine-induced signalling  28  1.6.9. Deactivation of M A P kinases: A role for M A P kinase phosphatase. 1.7. The focus of this thesis.  28 29  Chapter 2. Materials and Methods.  33  2.1. THP-1 cell cultures for H L A - D R mRNA, H L A - D R protein, and T N F - a release studies.  33  2.2. The use of reverse-transcriptase polymerase chain reaction (RT-PCR) to determine IFN-y-induced H L A - D R mRNA expression in THP-1 cells.  33  2.3. Fluorescence-activated cell scanning (FACS) of H L A - D R on THP-1 cells. 2.4. Isolation of donor T cells from whole human blood.  36 36  2.5. Treatments of THP-1 and monocyte-depleted T cells with RPMI 1640, TSST-1 and LPS. 2.6. TNF-a enzyme-linked immunosorbance assays.  37 37  2.7. THP-1 cell culturing, stimulation and lysis for M A P kinase studies. 2.8. The determination of total cell lysate protein concentration.  38 39  2.9. Anion exchange chromatography and fractionation of highspeed supernatants.  40  2.10. The myelin basic protein kinase phosphotransferase assay of Bio-Scale Q fractions.  40  2.11. ERK-1 /ERK-2 immunoprecipitation from THP-1 high-speed supernatants. 2.12. Polyacrylamide electrophoresis of THP-1 proteins.  40 42  2.13. Western blotting and chemiluminescent staining of THP-1 proteins separated by gel electrophoresis. Chapter 3. The Induction of TumOr Necrosis Factor-a Release from THP-1 and Human Donor T Cell Co-cultures Treated with TSST-1. 3.1. Introduction. 3.2. Results. 3.2.1. RT-PCR analysis of IFN-y-induced mRNA upregulation in THP-1 cells. 3.2.2. FACScan analysis of H L A - D R upregulation on THP-1 cells following treatment with IFN-y. 3.2.3. Determination of the sensitivity of the T N F - a enzymelinked immunosorbance assay (ELISA). 3.2.4. Release of T N F - a from THP-1 and T cell co-cultures treated with TSST-1 and IFN-y. 3.2.5. The effect of 1 ng/ml TSST-1 and 100 U/ml IFN-y on T N F - a release from donor T cells. 3.2.6. The effect of 1 ng/ml TSST-1 and 100 U/ml IFN-y on TNF-a release from THP-1 cells. 3.2.7. T N F - a release from THP-1 cells treated with LPS and IFN-y.  3.3. Discussion. vii  Chapter 4. The Effect of TSST-1 on Mitogen-Activated Protein Kinases in THP-1 Cells.  71  4.1. Introduction.  71  4.2. Results.  73  4.2.1. The effect of P M A on M B P kinase activity in THP-1 cells.  73  4.2.2. The effect of TSST-1 on M B P kinase activity in THP-1 cells.  73  4.2.3. The phosphorylation of ERK-1 and ERK-2 in M B P kinase Peaks I, II and III.  74  4.2.4. The effect of either P M A or TSST-1 on immunoprecipitated ERK-1/2 activity.  74  4.2.5. Anti-phosphotyrosine blotting of THP-1 whole cell lysates treated with TSST-1, G31Rmut TSST-1, LPS and RPMI 1640.  75  4.2.6. Immunoprecipitated ERK-2 activity from ultracentrifuged cell lysates of THP-1 cells treated with either TSST-1, G31Rmut TSST-1, LPS and control media.  84  4.2.7. Immunoprecipitation of ERK-1 activity from ultracentrifuged cell lysates of THP-1 cells treated with TSST-1, G31Rmut TSST-1, LPS and control media. 4.3. Discussion.  88 92  viii  Chapter 5. Conclusions and Future Research Directions.  101  5.1. Models of TSST-1 and LPS-mediated T N F - a release.  101  5.2. TSST-1-mediated activation of ERK-1 and ERK-2: A comparison with G31Rmut TSST-1-mediated signalling.  102  5.3. Future directions of research.  104  Bibliography.  106  ix  List of Tables.  Title  Table L Criteria for the diagnosis of toxic shock syndrome. Table II. The physical and chemical traits of TSST-1 staphylococcal enterotoxins, and VP specificity for human T cell receptors. Table III. The effect of different IFN-y doses on H L A - D R mRNA expression in THP-1 cells. Table IV. A time course for H L A - D R mRNA expression in THP-1 cells treated with a pre-optimized dose of IFN-y (100 U/ml).  List of Figures. Title  Page  Figure 1. A n A P C and T cell model of TSST-1 mediated pathogenesis. Figure 2. Different models for LPS binding to eukaryotic cells and the induction of TNF-a release from these cells. Figure 3. The multiple roles of M A P kinases in eukaryotic cell signalling. Figure 4. The M A P K activation pathway web in eukaryotic cells. Figure 5. The effect of different IFN-y doses on P-actin and H L A - D R mRNA expression in THP-1 cells. Figure 6. RT-PCR analysis of IFN-y (100 U/ml)-induced H L A - D R and P-actin mRNA expression in THP-1 cells. Figure 7. Relative H L A - D R staining intensity on THP-1 cells treated with IFN-y (100 U/ml). Figure 8. A standard curve for the determination of TNF-a from O.D. 490 readings. Figure 9. The release of TNF-a from THP-1 and T cell co-cultures treated with TSST-1 (1 ng/ml) in the presence or absence of 100 U/ml IFN-y (100 U/ml). Figure 10. The release of TNF-a from cultures treated with TSST-1 (1 ng/ml). Figure 11. The release of TNF-a from THP-1 cells treated with LPS (1 ug/ml) in the presence or absence of P M B (10 ug/ml) and IFN-y (100 U/ml). Figure 12. A summary of LPS, TSST-1 and IFN-y-induced events in THP-1 cells alone. Figure 13. Activation of M B P kinase in P M A treated cells. Figure 14. Activation of M B P kinases by TSST-1. Figure 15. Detection of ERK-112 by immunoblotting of Peak I, II and III eluent fractions with anti-ERK-1/2 antibodies following treatment of THP-1 cells with TSST-1, P M A and RPMI 1640. Figure 16. Detection of tyrosine-phosphorylation by immunoblotting of Peak I, II and III eluent fractions with an anti-tyrosine antibody following treatment of THP-1 cells with TSST-1, P M A and RPMI 1640. Figure 17. ERK-1/2 specific phosphotyrosine staining in Peak I, II and III eluent fractions following treatment with of TSST-1 and P M A . Figure 18. Relative ERK-1/2 activity in THP-1 cells after treatment with TSST-1 and P M A . Figure 19. Anti-phosphotyrosine staining of whole cell lysates treated from THP-1 cells treated with LPS and RPMI 1640. Figure 20. Anti-phosphotyrosine staining of whole cell lysates treated from THP-1 cells treated with TSST-1 and G3 IRmut TSST-1. Figure 21. Relative ERK-2 activity in THP-1 cells. Figure 22. Areas beneath gross ERK-2 activation and ERK-2 deactivation curves. xi  11 13 20 22 49 51 53 55  56 58  59 62 76 77  78  79 80 81 82 83 85 86  Title  Page  Figure 23. Relative ERK-1 activity in THP-1 cells. Figure 24. Areas beneath gross ERK-1 activation and ERK-1 deactivation curves. Figure 25. The effect of TSST-1, G31Rmut TSST-1 and LPS on ERK-1/2 in THP-1 cells. Figure 26. The roles of TSST-1, G31Rmut TSST-1 and LPS in pathophysiology.  xii  89 90 99 103  List of Abbreviations. Ab AP-1 APC BSA °C CBP CHO CREB DNA EDTA ERK-1 ERK-2  E. coli ELISA FACS H7 HLA-DR IL-1 IL-2  ICAM-1/2 JAK KDa LFA-1 LBP LPS MAM mA mCD14 mRNA MAPK MAPKAPK MAPKK MBP MEK MEKK M H C II MKP Mnk ng/ml nM NF-KB  O.D. PBMC PCR  Antibody Activator protein-1 Antigen presenting cell Bovine serum albumin Degrees celcius CREB-binding protein Chinese hamster ovary c A M P response element-binding protein Deoxyribonucleic acid Ethylene diaminetetra acetic acid Extracellular-signal regulated kinase-1 Extracellular-signal regulated kinase-2 Escherichia coli Enzyme-linked immunosorbent assay Fluorescence-activated cell sorting/scanning [l-(5-isoquinolinylsulfonyl)-2-methylpiperazine Human leukocyte antigen isoform-DR Interleukin-1 Interleukin-2 Intercellular adhesion molecule-1 Janus kinase Kilodalton Leukocyte function-associated antigen-1 LPS-binding protein Lipopolysaccharide M . arthritidis mitogen Milliamperes Membrane-bound CD 14 Messenger R N A Mitogen-activated protein kinase MAPK-activated protein kinase M A P K kinase Myelin basic protein M A P K or E R K kinase M E K kinase Major histocompatability complex II M A P kinase phosphatase M A P K interacting kinase Nanograms per millilitre Nanomolar Nuclear factor K - B Optical density Peripheral blood monocuclear cells Polymerase chain reaction xiii  PI-3K PKC PMA RSK RT-PCR S. aureus SAg SAPK sCD14 SEA SEB SEC SED SEE SEF STAT TAK TCR TF TNF TPA TSS TSST-1 U/ml ug/ml V v p  Phosphatidyl inositol-3 kinase Protein kinase C Phorbol-12-myristate-13-acetate Ribosomal S6 kinase Reverse transcriptase-polymerase chain reaction Staphylococcus aureus Superantigen Stress-activated protein kinase Soluble CD 14 Staphylococcal enterotoxin A Staphylococcal enterotoxin B Staphylococcal enterotoxin C Staphylococcal enterotoxin D Staphylococcal enterotoxin E Staphylococcal enterotoxin F Signal transducers and activators of transcription Tumor-associated kinase T cell receptor Transcription factor Tumor necrosis factor 12-O-tetradecanoylphorbol-13 -acetate Toxic shock syndrome Toxic shock syndrome toxin-1 Enzymatic units per millilitre micrograms per millilitre Volts p chain of the variable region of the T cell receptor  xiv  Acknowledgements I would like to thank my supervisor, Dr. Anthony Chow, for his support and guidance during my graduate studies. I would also like to thank the members of my thesis committee, Drs. Steven Pelech, Herman Ziltener, John O'Kusky and Gary Quamme for their input during the course of my experiments. I am especially grateful to Drs. Steven Pelech, Bill Sahl and Jasbinder Sanghera at Kinetek Pharmaceuticals Inc. for their help in understanding signal transduction and putting up with some very novice questions. For their technical assistance, I would like to thank Drs. Keith Knutson, Winnie Kum and Devki Nandan in the Division of Infectious Diseases, UBC. Finally, I would like to dedicate this thesis to Tara, Buttons and my parents who have put up with all my graduate school tribulations and the requisite poverty.  xv  Chapter 1. Introduction.  1.1.  The epidemiology and history of toxic shock syndrome. Over the last seven decades, clinicians have noted symptoms such as fever, shock and  rash which were associated with Staphylococcus aureus infection in patients. The presence of these symptoms plus swelling of the skin, desquamation and multiple organ involvement were later used as criteria for toxic shock syndrome (TSS) (Table I) (1). For a patient, the most serious manifestation of TSS is hypotension leading to hypoperfusion of the body organs and lethal shock i f left untreated (Table I). The general public became acquainted with TSS during the early 1980's when clusters of menstruating women in the Northeastern United States became ill with TSS symptoms. These clusters of illness correlated with the use of rayon-based Rely™ tampons and the subsequent colonization of the female reproductive tract with Staphylococcus aureus (1). In vitro experiments have shown that rayon-based tampons create an ideal environment for staphylococcal colonization and growth (2). Studies at the United States Centers for Disease Control and Prevention showed that nearly 90% of TSS associated S. aureus isolates produced a 22 kDa exoprotein (3,4). This 22 kDa protein is a superantigen that was initialy called staphylococcal enterotoxin F (SEF) by Bergdoll's group and staphylococcal pyrogenic exotoxin type C by Schlievert's group. In an attempt to simplify classification, this novel toxin was later renamed as toxic shock syndrome toxin-1 (TSST-1) (3). Recent in vitro work has shown that S.aureus produces TSST-1 in either the stationary phase or the logarithmic phase of growth (5). S. aureus also produces several different hydrophilic molecules which migrate on sodium dodecyl sulphate polyacrylamide gel 1  electrophoresis (SDS-PAGE) within a mass range of 26 kDa and 30 kDa (6). These exotoxins known as staphylococcal enterotoxins A (SEA), B (SEB), C (SEC), D (SED) and E (SEE) are also superantigens and are involved in food poisoning and shock in humans and animals but play minor roles in toxic shock syndrome (TSS) in humans (Table II) (7,8). Today, we know that TSS affects both sexes and individuals of all ages, races and socioeconomic status. Surprisingly, TSS affects both females and males in a ratio of 4:1 (female:male) with non-menstrual TSS (NMTSS) cases occurring equally in both sexes (7). TSS is classified into menstrual toxic shock syndrome (MTSS) which occurs in 46 % of all TSS cases and non- NMTSS which occurs in 54 % of all TSS cases (9). Non-menstrual TSS describes cases where shock-inducing S. aureus colonizes in post-traumatic or post-surgical wounds (7) and compromised patients (10-12). The majority of menstrual TSS and nonmenstrual TSS cases involve TSST-1-producing strains of S. aureus (i.e. 62 % of NMTSS cases and 84 % of MTSS cases) (13). Work by Marples et al. (7) has shown that MTSS occurs in individuals between 15 and 60 years of age with a peak incidence occurring in patients between 15 and 20 years of age. In comparison, NMTSS occurs in patients between the ages of zero and 70+ years with no distinct age peak (7).  1.2. The microbiology of toxic shock syndrome. S. aureus is a gram-positive bacteria which acts in a persistant and opportunistic fashion and produces several proteins involved in TSS (Table II) (14). This bacteria is considered a persistent hospital pathogen (12,15) because it cannot be cleared from the hospital environment regardless of the steps taken in disinfection (16). Bacterial growth is optimal in aerobic conditions, but can occur anaerobically with some strains. Due to its aerobic requirements, S. aureus colonizes the skin and mucous membranes of the host, including the respiratory tract,  2  Table 1. 2. 3. 4. 5.  6.  I: Criteria for a Diagnosis of Toxic Shock Syndrome Fever Rash Desquamation of palms and soles within 1-2 weeks of onset. Hypotension (systolic blood pressure < or = 90 mm Hg for adults) Involvement of three ormore of the following organ systems: A . Gastrointestinal B. Muscular C. Mucous membrane D. Renal E. Hepatic F. Hematological G. Central nervous system Negative results of the following, i f obtained: A . Blood, throat, or cerebrospinal fluid cultures B. Serological tests for Rocky Mountain spotted fever, leptospirosis, or measles.  From Chesney et al., 1984.  3  Table II. Physical and chemical traits of TSST-1 and staphylococcal enterotoxins, and VP specificity for human T cell receptors.  pi  Disulfide Bond  VP Specificity  SEA  Molecular Mass (kDa) 27.8  7.3  Yes  SEB  28.3  8.6  Yes  SEC  26.0  8.6  Yes  SED SEE  27.3 29.3  7.4 7.0  Yes Yes  TSST-1  22.0  7.2  No  1.1,5.3,6.3, 6.4, 6.9, 7.3 3, 12, 14, 15, 17, 20 12, 13.2, 14, 15, 17, 20 5.1,6.1, 12 5.1,6.1-6.4, 6.9, 8,8.1, 18 2,2.1  Toxin  From Bell et al., 1995, Blank et al., 1993, Choi et al., 1990, Hewitt et al., 1992, Irwin et al., 1993, Johnson et al., 1991, Leung et al., 1995 and Marrack et al., 1990.  4  the reproductive tract, the peritoneum and the gastrointestinal tract. Since S. aureus is an opportunistic pathogen, damage to non-specific host barriers during surgery, trauma or invasive procedures creates favourable conditions for infection (10). Upon release, TSST-1 interacts locally with host cells (17) or becomes systemic by crossing epithelial barriers (18). The mobility of TSST-1 was shown in studies which showed that TSST-1 can cross  human  intestinal epithelial cell layers (Caco-2) by transcytosis (18).  1.3. A comparison of superantigens to conventional antigens. 1.3.1. Antigen binding to major histocompatibility complex molecules on antigen presenting cells. Superantigens (SAgs) are proteins, usually produced by bacteria or viruses, which differ from conventional antigens (Ags) in several ways. SAgs bind outside the peptide binding groove on major histocompatibility complex I (MHC I) or II (MHC II) molecules expressed on the cell membrane of A g presenting cells (APCs) (19). This means that the SAg does not need to be 'digested' by the A g presenting cell and that there is no specific size of SAg required for optimal activation of T cells (20). In comparison, conventional Ags are either endocytosed or phagocytosed by antigen presenting cells (APCs), digested by intracellular proteases and then presented to T cells within the peptide binding groove of the M H C I or M H C II molecule (21). Work by Srinivasan and co-workers shows that the M H C II peptide-binding groove can accommodate peptides between 13 and 25 amino acid residues in length (22). In comparison, the optimal size of the peptide epitope within the M H C I molecule is between 8 to 10 amino acid range (22). Peptide fragments of greater or less than optimal size induce a less efficient cytotoxic specific lysis of M H C I bearing APCs (21).  5  1.3.2. Binding of the M H C - S A g complex to T cell receptors. The second difference between SAgs and conventional Ags is that SAgs only need to bind to the V P element of the T cell receptor (TCR) in order to activate T cells (23,24). The exact nature of the MHC-SAg-TCR interaction is not known (25), but preliminary work by Labreque has indicated that some SAgs may link TCRs to M H C II molecules in a direct manner (26). This interaction has been shown to rely largely on the V P specificity of T cell receptors. For example, Karp has shown that VP2 T cells were disproportionally utilized and clonally expanded in peripheral blood mononuclear cells (PBMCs) treated with TSST-1 in the presence of HLA-DR1 transfected human fibroblasts (25). In comparison, the conventional A g - M H C II complex requires an interaction with the V a , Ja, V p , Dp and jp elements of the T cell receptor (TCR) (27). The third difference between SAgs and conventional Ags is related to the role of the Vp2 determinant on T cell receptors. Since SAgs are required only to bind to a specific V P element on T cells, the proportion of T cells activated by SAgs is higher than those activated by conventional Ags. Some workers have suggested that the level of T cell activation by SAgs may be as high as one in five (28) or between 10 % and 40 % of the T cell population (29). In contrast, the level of T cell activation by conventional Ags may be as low as one in 10,000 (27). SAgs also induce what Miethke describes as a "paradox" on the T cell population (30). Relatively low doses of the SAg activate T cells and cause them to release cytokines and enter mitosis. Higher doses of SAg concentrations lead to functional but clonally anergic T cells (31) that release shock inducing cytokines but do not enter the cell cycle (32).  1.3.3. Superantigen binding to multiple M H C alleles.  6  Unlike conventional antigens, SAgs are not restricted to one single M H C isoform and may bind to a variety of M H C II isotypes. This binding variability to M H C molecules may be due to the lack of processing of SAgs by the A P C , the similarity of binding sites on the various M H C molecules or a combination of both factors. For example, SEB is not processed within the A P C and binds to both the H L A - D R and H L A - D Q alleles of the M H C II molecule on the A P C membrane (33). In contrast, peptide binding grooves of either M H C class I or M H C class II molecules are specifically designed for conventional Ags (21,22). The small size of these peptides, the requirement for their processing within the A P C and their presentation within the peptide binding groove predicates greater specificity for a M H C molecule that can present that molecule. Other cell surface molecules may play a role in assisting SAg binding to a M H C molecule. Although H L A - D R is the primary binding site for TSST-1, recent studies now suggest that TSST-1 binding to H L A - D R can be enhanced by co-expression of the invariant chain and H L A - D M (34).  1.4. The Staphylococcus aureus superantigen TSST-1. Binding of TSST-1 to both T cells and APCs is known to induce both T cell mitogenesis, as measured by tritiated thymidine incorporation (35) and cytokine release from both T cells and APCs (36,37). These released cytokines include interleukin-l(3 (IL-ip), interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-ct (TNF-a), tumor necrosis factor-p ( T N F - P ) and interferon-y (IFN-y) (38-42). See and co-workers used formaldehyde fixation and trypan blue exclusion staining to show that this TSST-1-induced cytokine release and T cell mitogenesis requires viable T cell and APCs as well as the interaction of costimulatory molecule interactions on both cell types (Figure 1) (43).  7  TSST-1 is divided into a T cell binding domain known as domain A (35) and an A P C binding domain known as domain B (44). Structurally, domain A forms an N-terminal central a-helix as well as a five stranded P sheet and consists of amino acid residues 1 to 17 and 90 to 194 (27). Distinct regions of the long central a-helix near the carboxy-terminus of TSST-1 have been shown to play a role in T cell activation and mitogenesis. Substitution of histidine-135 to alanine-135, in domain A , does not prevent TSST-1 binding to M H C II but decrease T N F - a release and T cell mitogenesis in mouse splenic cells (45). Other workers have shown that mutations of glutamate-132 to lysine-132 and glutamine-136 to lysine-136 decreased T cell mitogenesis in mouse splenocytes when measured by tritiated thymidine incorporation (46). It is now known that the TSST-1 domain A binds preferentially to T cells which express the Vp2 isoform of the T C R (47,48). Choi used reverse transcriptase-polymerase chain reaction (RTPCR) (49,50) techniques to show that Vp2 genotypic T cells were clonally expanded in human PBMCs treated with TSST-1 (49). The TSST-1 domain B consists of amino acid residues 18 to 189 which form a barrel of five N-terminal P strands (35) which is required for TSST-1 binding to the M H C II molecule. Work by Kum and co-workers showed that the mutation of glycine-31 to arginine-31. decreases TSST-1 binding to human PBMCs, TNF-a release from PBMCs and T cell mitogenesis in P B M C culture (44). Various workers used anti-HLA DR antibodies (51), transfection (25,52) :  and mutation studies (19) to show that TSST-1 binds primarily to the HLA-DR1 isoform of the M H C II molecule on human antigen presenting cells. However, TSST-1 also binds to other H L A - D R isoforms including H L A - DR2, -DR4, -DR6 and -DR7 (52). Co-stimulatory molecules present on T cells and monocytes play an important role in TSST-1 mediated cytokine secretion in donor monocyte and T cell co-cultures (43,53). Antibodies against the a and P chain of the lymphocyte function antigen-1 (LFA-1) on T cells 8  prevented LFA-1 binding to intercellular adhesion molecules-1/2 (ICAM-1/2) on APCs and decreased TSST-1 mediated T N F - a and IL-1 P release into cell culture supernatants (43). Other cell adhesion molecules such as CD28 mediate TSST-1 induced lethality in D-galactosamine (D-gal) sensitized C57BL/6 mice (53). Work by Leung and co-workers indicates that TSST-1 upregulates cell surface antigens including cutaneous lymphocyte-associated antigen (CLA) on T cells. This upregulation of C L A indicates that cell surface markers on both T cells and antigen presenting cells play a wide variety of roles that have not yet been fully elucidated (54). These cell adhesion molecules may control TNF-a secretion by modulating the signal transduction cascades in both APCs and T cells (55-57).  1.4.1. Pathogenic mechanisms of TSST-1.  TSST-1 binds to the H L A - D R molecule on APCs (58) and the Vp2 TCR molecule on T cells (50) and results in the release of pro-inflammatory cytokines (Figure 1) (36). Both T N F - a and TNF-P play the central role in TSST-1 mediated shock and lethality in animal models. Miethke and co-workers used the monoclonal antibody V l q to neutralize murine TNF-a and TNF-p cytokines in vivo and protect D-galactosamine sensitized mice against TSST-1 induced death (59). P B M C culture and cell line studies suggest that monocytic cells are a primary source of TNF-a after treatment with superantigens (42,60). In contrast, T cells appear to be the primary source of TNF-P and a secondary source of TNF-a after treatment with TSST-1 (38,61) and may also play an important role in the pathogenesis of TSS through clonal proliferation (17,59). Systemic and localized TNF-a  and TNF-P induce multi-organ  manifestations such as myocardial suppression, capillary leakage, respiratory distress, hypoxia, diarrhea, renal failure, suppressed erythropoiesis and disseminated intravascular coagulation  9  which can lead to shock and death (62,63). The injection of a bolus of recombinant T N F - a decreases mean arterial pressure within two days in canines (64) and leads to fatal shock in primates (65).  1.5. Lipopolysaccharide induced shock: A comparison to TSST-1 induced pathogenesis. 1.5.1. Structure and function. Bacterial lipopolysaccharide (LPS) is the functional molecule in septic shock and induces the release of cytokines such as TNF-a, TNF-p and IFN-y from monocytes (36), 1  PBMCs (66) and THP-1 cells (67) (Figure 2). LPS is an important component of the outer membrane of gram-negative bacteria and is required for cell growth and division (68). This molecule consists of 4 distinct domains including a lipid A domain (69,70), a 2-keto-3 deoxyoctonic acid (KDO) domain (70), an O-specific chain (71) and the outer core (71). Of the four distinct LPS domains, the lipid A and the K D O domains are the most pathogenic (70). The lipid A domain is embedded in the outer membrane of the bacteria (69) and plays the most important role in LPS activation of monocytic cells (68,72). When gram-negative bacteria are lysed, the lipid A portion is exposed to the external environment and interacts with the host immune system by binding to membrane-bound CD 14 (mCD14) (73,74) or soluble CD 14 (sCD14) (75,76) molecules or intercalating within the eukaryotic cell membrane (49) (Figure 2). The lipid A domain may also play a role in binding the 60 kDa LPS-binding protein (LBP) found in serum (77,78). The inner core domain of LPS contains one or more 2-keto-3deoxyoctonic acid (KDO) residues and plays an important role in the activation of monocytes and macrophages and the release of TNF-a from these cells (79). The other two (regions of LPS are the O-specific chain that is attached to lipid A and the outer core (79). (regions of LPS are the O-specific chain that is attached to lipid A and the outer core (79). The O-specific domain  10  Figure 1. A n A P C and T cell model of TSST-1 mediated pathogenesis. In the presence of both APCs and T cells, TSST-1 is able to induce shock-inducing cytokine release from both cells and the clonal proliferation of Vp2+ T cells.  11  of Legionella pneumophila LPS has been suggested to play a role in mediating virulence but has not been reported to directly induce shock (80). 1.5.2. The CD14-dependent pathway.  A survey of the current literature has revealled two possible mechanisms for the LPSinduced release of TNF-a from monocytic cells . The first is the CD14-mediated binding of low concentrations  (less than 0.1 ug/ml) (75) of LPS (81,82) to either a glycosyl  phosphatidylinositol (GPI) anchored 55 kDa mCD14 (72) in myeloid cell culture (74) or sCD14 in non-myeloid cell culture (i.e. endothelial cells) (76) (Figure 2). The importance of CD 14 in LPS binding was shown in CD 14 competition studies. In murine models, the addition of excess sCD14 competed with mCD14 and prevented LPS mediated lethal shock (72). The binding of LPS to CD 14 and the subsequent activation of signal transduction pathways induces the expression of TNF-a mRNA through different transcription factors including N F - K B (83). Mutation of the CD 14 molecule shows that LPS binds to an unknown domain on C D 14 but induces nuclear factor-KB ( N F - K B ) signalling in U373 cells (ATCC HTB17) through a signalling domain that includes amino acids 7-16 (84). Several workers have suggested that LPS-binding protein (LBP) (77) is involved, but not required, in both mCD14 and sCD14 mediated signalling. For some non-monocytic cells, CD 14 mediates LPS binding (75) and may (76) or may not require the presence of a 60 kDa LPS binding protein (LBP) (77) for cytotoxic activity to occur. Serum LBP (78) may enhance the binding of LPS to mCD14 and the subsequent induction of TNF-a release from monocytic cells (68). For example, LPS and L B P co-treatment of rabbit peritoneal exudate macrophages induced TNF-a production that was 1000-fold over LPS cell treatments alone (78). This  12  LPS Non-CD 14  /^ZZ.  TNF-a  Figure 2. Different models for LPS binding to eukaryotic cells and the induction of TNF-i from these cells. This model takes into account CD14-independent and CD14-dependent mechanisms of LPS binding to eukaryotic cells.  13  increased activation of macrophages and the subsequent release of TNF-a into the culture supernatant may be mediated by the Lipid A region of LPS since LBP binds to this region on both complete and fragmented LPS molecules (78). LPS binding to a specific domain on CD 14, in the presence or absence of LBP, induces the activation of several distinct signalling pathways, including M A P kinases (84) and P K C (85). Earlier workers treated murine peritoneal macrophages with LPS and found increases in tyrosine phosphorylation of 41, 42 and 52 kDa proteins (86). This LPS binding activates the 44 kDa extracellular-regulated kinase-1 (ERK-1) isoform, the 42 kDa extracellular-regulated kinase-2 (ERK-2) (87) isoform in CD 14 positive human monocytes (87) and the p56 stressactivated protein kinase (SAPK) isoform of the mitogen activated protein kinase (MAPK) family in CD14 positive murine macrophages (88). LPS treatment of human monocytes also upregulates phosphorylation of 36-kDa and 38-kDa proteins (89) which are within the molecular mass range of a set of stress activated protein kinases known as p38 or HOG-1 (90).  1.5.3. The CD14-independent pathway. The second mechanism of LPS binding to monocytic cells is independent of binding to the CD14 molecule and usually involves large doses of LPS between 0.1 ug/ml and 10 ug/ml (75) (Figure 2). The presence of this CD14-independent pathway was demonstrated by using macrophages from CD 14 knockout mice, CD14i  ow  cells and anti-CD 14 antibody inhibition  studies. Macrophages from CD 14 knockout mice treated with LPS showed upregulated levels of TNF-a mRNA when compared to basal treatments (81). LPS also activates TNF-a mediating signal transduction cascades in cell lines which express low levels of cell surface CD14 (i.e. the undifferentiated promonocytic cell line THP-1) (91). In undifferentiated THP-1 cells, high doses of LPS led to activation of an enhancer, ( N F - K B ) , which binds to transcription promoters 14  (  in TNF-a and IL-ip genes (92). Weinstein and co-workers showed that LPS induces M A P kinase signalling in GDI4 positive and differentiated THP-1 macrophages treated with an excess of anti-CD 14 antibodies. Even after treatment with an excess of anti-CD 14 molecules, high concentrations of LPS induced tyrosine phosphorylation, within the molecular mass range of ERK-1 and ERK-2 (93). There are several possible mechanisms for the CD14-independent binding of LPS to eukaryotic cells and the subsequent induction of signals within these cells. LPS binds either to a cell membrane receptor (94,95), reacts directly with the cell membrane (75) or uses a combination of both mechanisms (96) (Figure 2). These alternate receptors are proteins since LPS binding to human leukocytes, in the presence or excess doses of anti-CD 14 antibodies, was trypsin sensitive (95). The integrin C D l l c / C D 1 8 has been proposed as a alternative LPS binding site and can induce N F - K B signalling within CD1 lc/CD18-transfected Chinese hamster ovary (CHO) cells (94). Integration of the LPS molecule into the eukaryotic cell membrane is an alternative to LPS binding to membrane bound proteins (Figure 2). Resonance energy transfer (RET) was used to show that LPS can intercalate non-specifically into phospholipid bilayers and that this intercalation can be enhanced in the presence of LBP (75).  1.5.4. A comparison between LPS and TSST-1 mediated pathogenesis of septic shock models. Regardless of its binding mechanism to monocytic cells, LPS mediated TNF-a release differs from the TSST-1 model of TNF-a release. TSST-1-induced TNF-a release requires the presence of both viable APCs and T cells (Figure 1). In comparison, LPS induces the release of TNF-a from monocytic cells alone, through a variety of potential mechanisms (75,81,94), and does not require the input of signals from other cells (67) (Figure 2). Current knowledge indicates that T cell interactions with APCs and TSST-1 provide several  15  important  requirements for TNF-a release from both monocytes and T cells in the peripheral blood (59,61) (Figure 2). TSST-1 contains both H L A - D R (44) and T C R (45) binding domains that must interact with their ligands for TNF-a release to occur. The interaction between SAg-bound T cells and APCs is now thought to involve a complex system of membrane bound receptors (36,53,54), the release of cytokines from either APCs (36,44) or T cells (36,45) and the clonal expansion of Vp2 T cells (17,37). LPS and TSST-1 can interact synergistically within the human body and modify each other's activity with immune cells (97). Low doses of TSST-1 synergistically enhance the release of cytokines (i.e. IFN-y, IL-1 P ) in LPS treated PBMCs (97) and markedly enhances lethality in animal models.  1.6. Mitogen activated protein (MAP) kinase control pathways. 1.6.1. M A P kinases: A n introduction. M A P kinases are a family of protein-serine/threonine kinases that include the 44 kDa extracellular signal-regulated kinase-1 (ERK-1), the 42 kDa (ERK-2) (Figure 3), the 38 kDa HOG-1 (90,96) and a group of c-Jun kinases (INK) (96,98) also called stress activated protein (SAP) kinase (99). It was decided that ERK-1 and ERK-2 would be the focus of this thesis, because of the pleiotrophic roles they play (100) (Figure 4) and the multiple upstream pathways that mediate ERK-1 and ERK-2 activity. Like other protein kinases, ERK-1 and ERK-2 feature 11 distinct catalytic subdomains. These proteins are activated as a result of autophosphorylation (101,102) or targeted phosphorylation by upstream M A P kinase kinases (MAPKKs) (103,104) at specific tyrosine and threonine residues between subdomains VII and VIII in the C-terminus of each molecule (99,105,106) (Figure 3). Upon activation, the ERK-1/2 molecules can translocate to the nucleus (105,107), can remain in the cytoplasm and phosphorylate cytoplasmic proteins (108-110), can interact with cytoskeletal kinases (100) or interact with  16  plasma membrane proteins (111). Deactivation of both ERK-1 and ERK-2 is accomplished by dephosphorylation of specific tyrosine and threonine residues by M A P kinase phosphatases (112-115). These M A P kinase phosphatases can be found in the cytoplamic and plasma membrane fractions (116) as well as in the nucleus (117). The second ERK-1/2 domain of interest is a Myc substrate recognition domain which is C-terminal of domain V. The creation of HOG-1 fusion proteins with this domain allowed the phosphorylation of Myc under stress conditions that would normally activate HOG-1 substrates (106). Although this fusion-protein model has some weaknesses, the loss of this C-terminal domain on ERK-2 decreased Myc phosphorylation under conditions shown to activate ERK-2 (106).  1.6.2. MEK-1 and M E K - 2 : Direct upstream activators of ERK-1 and ERK-2. Both M A P kinase kinase-1 (MAPKK-1) (103,118) and M A P K K - 2 (104,118-120), also known as M A P K / E R K kinase-1/2 (MEK-1/2) (121) are specific for both ERK-1 and ERK-2 (Figure 4). The specificity of MEK-1 and M E K - 2 for either ERK-1, ERK-2 (122) or both isoforms (104,123) depends on the cells studied and the stimuli used in activation (114). This variability may involve activation of multiple upstream pathways that can differentially control M A P K K activity (124). For example, angiotensin and platelet derived growth factor signal from MEK-1-»ERK-1 in rat aortic vascular smooth muscle culture (125). In comparison, the transfection of constitutive ly active M E K - 1 into the megakaryocyte line K562 induced increased ERK-2 activation when compared to non-transfected cells (126). Both MEK-1 and M E K - 2 can activate either of the E R K isoforms. The treatment of mouse Swiss 3T3 fibroblasts with PD 098059, at inhibitory concentrations for both M A P K K - 1 and M A P K K - 2 , decreased  17  ERK-2 activity (118). The phosphorylation of M E K s at specific serine residues between subdomains VII and Vfll of both MEK-1 and M E K - 2 has been shown to be required for M E K activity  (124,127).  For example,  mutations  of serine-218—>alanine-218  and  serine-  222—»threonine-222 on MEK-1 decreased M A P kinase phosphorylation (124). Mutations of serine-218—»alanine-218 and serine-222—>alanine-222 decreased MEK-1 activity in N I H 3T3 cells when compared to basal activity (127). The fact that M E K - 2 shares a sequence homology with MEK-1 in this regulatory region (124) indicates that this molecule is also regulated by phosphorylation at serine^ 18 and serine-222.  1.6.3. Ras-Raf-1 pathway activation of MEK-1/2. The Ras-Raf-l pathway is one of several pathways that can potentially activate MEK-1 and M E K - 2 in eukaryotic cells (101,104,128,129) (Figure 4). There are 2 main activators of the Ras  family of protein-serine/threonine kinase (119,130): GTPases/GTP-binding proteins  (119,131) and non-receptor tyrosine kinases in eukaryotic cells. Ras is activated by an increased GTP/GDP ratio (132) that occurs after Ras binds to the (5y subunits of GTP-binding proteins (G-proteins) that are coupled to membrane receptors (133). Oncogenic mutants of Ras, found in many human cancers, are molecules with chronic GTP binding that leads to sustained Ras activity and activation of downstream pathways (131,134) (Figure 4). Transient over-expression of oncogenic Ras in Swiss 3T3 cells has been shown to increase M A P kinase activity (134) and activates the N F - K B transcription factor (131). The activated Ras protein then interacts with a Ras-binding domain (RBD) on a 74 kDa (135) protein-tyrosine kinase known as Raf-1 (130). The binding of Ras to Raf-1 and the subsequent serine-phosphorylation of Raf-1 are thought to be two important steps in Raf-1 activation (130). Mutations of amino acids 32-»38 in this  18  PvBD was shown to prevent Ras binding to the serine/threonine kinase Raf-1 (134) and decreased M A P kinase activity when compared to non-transfected cells (134). Ras can also be activated by interacting with non-receptor protein-tyrosine kinases (101) (Figure 4). In some physiological models, the activation of this Raf-1 dependent E R K pathway requires the involvement of non-receptor protein-tyrosine kinases of the Src family. In THP-1 cells, disruption of microtubules has been shown to involve both E R K and Src in a Raf-1 dependent pathway (136). Inhibition of Src with the inhibitor CP 118556 prevents E R K activation and microtubule disruption in THP-1 cells (136). Raf-1 has been shown to be an activator of MEK-1 and M E K - 2 and downstream pathways in eukaryotic cells by transfection studies, enzyme/substrate studies and enzyme kinetics studies. Constitutively active Ras was shown to be sufficient to upregulate downstream M A P kinase pathways. NIH 3T3 cells transfected with oncogneic forms of c-Raf-1 (p35EC12) have levels of M B P kinase activity that is higher than non-transfected cells (135). The creation of an oncogenic Raf-1:estrogen receptor (Raf-1 :ER) fusion protein induced increases in recombinant MEK-1 (rMEK-1) and recombinant ERK-2 (rERK-2) phosphorylation (121). In comparison, the transfection of a dominant-negative Raf-1 mutant into human 293 embryonic kidney cells followed by treatment with the phorbol ester TP A resulted in decreased ERK-1 activity when compared to treatment by untransfected cells (137). Enzyme/substrate studies show that immunoprecipitated Raf-1, from PMA-treated Jurkat cells, are able to phosphorylate recombinant M E K molecules in vitro (132). Kinetic studies have shown the early sequential activation of Raf-1 ->MEKs->MAP kinases in Madin-Darby canine kidney (MDCK) epithelial cells subjected to hyperosmolar conditions (119).  19  Cytokines  Cell Stresses  MKP  ERK-1 PEHDTGFLT*EY*VATRWYRAPE ERK-2 PDHDTGFLT*EY*VATRWYRAPE  Parallel Signalling Pathways  Transcription Factor Mediation  Figure 3. The multiple roles of M A P kinases in eukaryotic cell signalling. Multiple pathways lead to the activation of ERK-1/2 by M A P K K s and deactivation of ERK-1/2 by M K P s . E R K 1/2 plays important roles in cell growth and maturation, transcription factor control and cytokine-mediated signalling and bacterial pathogenesis.  20  1.6.4. M A P K or E R K kinase kinase activation pathways: A second Ras-dependent pathway. A review of the literature indicates that Ras—»Raf dependent pathways are not the sole activators of M E K - 1 / 2 - * ERK-1/2 pathways (101) (Figure 4). M E K kinase - 1 , -2 and -3 are protein-serine/threonine kinases capable of activating MEK-1/2 —»ERK-l/2 pathways through another Ras-dependent pathway (Figure 4) (138,139). Earlier work by Minden and co-workers indicates that E R K activities were increased in PC 12 cells transfected with excess Raf-1 and M E K K but not with Raf-1 or M E K K alone (140). Some workers have suggested that MEKK-»MEK activation differs physiologically from Raf-1 - * M E K activation due to the differential phosphorylation of specific serine residues on M E K serine (124). Transfection studies of CV1 monkey kidney cells with Raf-1 and M E K K indicate that M E K K preferentially phosphorylated M E K serine-218 while Raf-1 phosphorylates serine-218 and serine-222 with equal affinity (124). There are a wide variety of potential activators of Raf-1 other than Ras that will need to be studied to clarify this E R K activation pathway. For example, some workers have shown that janus kinase-2 (JAK-2) is capable of phosphorylating Raf-1 in a signal transducer and activator of transcription (STAT)-dependent manner (125). In contrast to the Ras-»Raf-1 pathway M E K can also be phosphorylated and activated (141) by a 93-95 kDa Raf isozyme (142) known as RafB (142) (Figure 4). Investigators have indicated that RafB and Raf-1 are s controlled by different upstream regulators (143). Upon treatment of PC 12 cells with nerve growth factor (NGF) (144), RafB M E K kinase activity and levels of RafB/ heat shock protein 90 (HSP90) complexes are higher than those seen with Raf-1  21  c CD  0)  co  CL  a «  U  co JD  ft  S3  •s o  >H  —  CO  300 .s G CD  co" M O (U  co  o 13  u+  h  4  g  6 fe  fe CD  b  °  cd  o  3  O  S s  . cn  *  3  =  £ CDc ~CD  CD  § .23 o  1 & S  c  /  / /  w  O fD S3 O o _  * -3 3  o° 5  CO  C  CD  ^ ^£ i  d  2 2  C  l i  M  rL_I 43 +3 co3 25  . co co «J  rt  8 ^ £ 3 43 43 °0  .^H  + CL,  u  22  fe  +3 CU  &  CCJ  ft  (144). RafB has been shown to bind and become activated (145,146) by a member of the Ras family of GTP-binding proteins known as Rap (147)(Figure 4). As a member of the Ras family, it is possible that Rap is activated by similar upstream factors including non-receptor PTKs. However, these upstream pathways are still in doubt and need to be futher elucidated (143).  1.6.5. Ras-independent activation of E R K pathways: A tangled web to unweave.  There is a growing body of evidence for activation of the MEK—»ERK pathway by Rasindependent upstream activators (123,148). For example, binding of integrins on N I H 3T3 fibroblasts activated ERKs in a Ras-independent fashion (123). Kashiwada and co-workers showed that binding of the cytoplasmic domain of the CD40 molecule on the human embryonic kidney cell line 293 with the TNF receptor-associated factor-6 (TRAF-6) also activated E R K in a Ras-independent manner (148). There are a wide number of upstream pathways that can activate the M E K - > E R K pathway in eukaryotic cells. Phosphatidylinositol-3 (PI-3) kinase has been suggested to be a Ras-independent activator of M A P kinases (115,149-151) (Figure 4). Inhibition of PI-3 kinase with wortmannin decreased IL-2 mediated E R K activation in the murine T cell line CTLL-2 (120). Protein kinase C (PKC) has also been implicated as an upstream activator or mediator of the E R K activation pathway (152,153) (Figure 4). Inhibition of P K C with Ro 318220 and GF 109203X has been shown to inhibit M A P K A P kinase l-(3 and p70 ribosomal S6 kinase in rabbit skeletal muscle (153) (Figure 4). A Ca -independent P K C 2+  isoform, P K C - ^ , has been implicated in angiotensin II mediated ERK-1/2 activation in rat vascular smooth muscle cell culture (154). It is also possible that ERK-1/2 activity can be mediated by C a -fluxes in eukaryotic cells (114,127,155) (Figure 4). 2+  23  1.6.6. The role of ERK-1 and ERK-2 in eukaryotic transcription control.  M A P kinases help mediate transcription by controlling both the activity and the levels of transcription factors in eukaryotic cells. These kinases have been shown to activate transcription factors involved in controlling cytokine expression (156) and the levels of chromosomal structural proteins. In cell cultures, ERK-1 stimulation induces transactivation and D N A binding of transfected Elk-1 and Sap l a (157), which are members of the Ets transcription factor family and control the expression of the transcription factor c-Fos (114,150). Both ERK-1 and ERK-2 may play an inhibitory role in regulating c-Jun binding to D N A by phosphorylating tyrosine residues within a C-terminal region of c-Jun (158). This binding inhibition could affect cytokine production, because TNF-a mRNA upregulation in the murine T cell line Ar-5 requires the c-Jun molecule (159): Finally, M A P kinases and M A P K K s have been shown to be required for the expression of a non-histone chromosomal architectural protein HMGI-C in salivary epithelial cells (121). It is also possible that M A P kinases play a more indirect role in human cytokine production by regulating the production of other transcription factors. For example, recombinant ERK-2 phosphorylates the C-terminal region of the ternary complex factor (TCF) (160) which controls U V induced production of the transcription factor c-fos in NIH 3T3 cells (160). In another example, the transfection of kinase-defective M A P / E R K Kinase 1 (MEK1) into human Jurkat T cells inhibited the transcription of a chloramphenicol acetyltransferase (CAT) reporter system linked to an IL-2 promoter (93).  24  1.6.7. Activation of down-stream kinases. ERK-1 and ERK-2 can activate a wide variety of M A P K activated protein ( M A P K A P ) kinases including M A P K A P K - l p , p70 S6 kinase (101,104,119,128,153,161) and M A P kinase interacting kinase-1/2 (Mnk-1) (108) (Figure 4). These downstream kinases may play physiological roles such as control of motility (i.e. myosin light chain kinase) (100) or may control other events such as transcription (i.e. Mnk-1) (108). For example, Mnk-1 and Mnk-2 act as protein-serine kinases on eukaryotic initiation factor-4E (eIF-4E). These downstream pathways may also play indirect roles in transcriptional control. Other pathways downstream of M A P kinases mediate cellular responses to physiological perturbations. For example, healthy Wistar rats treated with insulin showed an increase in M A P kinases and 90 kDa ribosomal S6 kinase activity (p90 Rsk) in skeletal muscle preparations (162) (Figure 4). M A P K K s have also been suggested to activate ribosomal S6 kinase (Rsk) in renal cells cultured in hyperosmolar conditions (119). The p90 Rsk molecule binds to the CREB-binding protein (CBP) molecule and forms a complex that binds to the c A M P response element-binding (CREB) protein and controls c-AMP mediated transcription (163). Work by Tsai and co-workers indicates that mutations in the c-AMP response element genome and subsequent decreases in the inducibility of this gene reduced cyclosporin A (CsA) mediated TNF-transcription in the mouse T cell line Ar-5 (159).  25  1.6.8. Mitogen activated protein kinases: Upstream activation conditions. 1.6.8.1. Signalling via bacterial toxins. LPS induced increased levels of myelin basic protein kinase activity and increased levels of M A P kinase tyrosine phosphorylation in C D 14 positive human monocytes (87,164). This LPS-induced ERK-1/2 activation has also been shown to be correlated with TNF-a release in monocytic cells (164). Pre-treatment of differentiated THP-1 macrophages with a protein tyrosine kinase inhibitor, herbimycin A , prior to LPS reduced both the tyrosine phosphorylation of 40-44 kDa proteins (ERK-1/2) and LPS mediated TNF-a release into the cell culture supernatant (93). This LPS-induced ERK-1/2 activation has been suggested to involve the Src family of protein-tyrosine kinases (165,166), the Raf-1—>ERK-1/2 pathway (130) and other potential activators of M A P K s (165,167). Human monocytes treated with LPS, and induced to release TNF-a, were shown to utilize, but not require (130), the Src family kinases Lyn and Hck (166). Other workers have shown that the treatment of human monocytes with LPS, induces the interaction of p53/56 Lyn with the potential M A P K activator PI 3-kinase (165). Similarily, LPS activates PKC-c^ in THP-1 cells (137) and this isoform has been implicated in angiotensin-mediated ERK-1/2 control in smooth muscle cells (154). TSST-1 induces events that are simlar to those involved in LPS-induced ERK-1/2 activation. For example, the inhibition of protein-tyrosine kinase (PTK) activity with genistein, in T cells and monocytes treated with TSST-1 prevented the release of TNF-a from these cells (168). TSST-1 induces protein-tyrosine kinases because it induces cycling of early tyrosine phosphorylation and dephosphorylation in the M A P kinase molecular mass range in human  26  monoyctes (169). Early increases  in tyrosine phosphorylation of proteins within the M A P  kinase mobility range were visible upon treatment of monocytes with biotin-avidin cross-linked TSST-1 (170). Some of this PTK activity in monocytes may be due to the Src family of proteintyrosine kinases. SEE and SED have been shown to induce activation of the non-receptor tyrosine kinase Fyn in Jurkat T cell culture (171) and this family of kinases has been implicated in M H C II-mediated events (172). P K C has also been shown to be required for T N F - a release from T cell and monocyte co-cultures (168) and is activated in monocyte cultures treated with TSST-1 (173). TSST-1 shares another similarity with LPS signalling (174) in that it also activates members of the AP-1 transcription regulation family. This SAg activates N F - K B in THP-1 cells and promotes its binding to a transfected DNA-binding motif (175).  1.6.8.2. Cell growth and maturation.  ERK-1 and ERK-2 have been implicated in a wide variety of cell cultures treated with tissue specific growth factors (Figure 3). For example, ERK-1 and ERK-2 activation occurs in fibroblasts treated with basic-fibroblast growth factor (176) and in PC 12 neural cells treated with nerve growth factor (177). The treatment of murine macrophages with the mitogen P M A was shown to activate ERK-1/2 phosphorylation (119). However, growth factor-induced signalling is not restricted to ERK-1/2 and can occur through other signal transduction pathways. For example, Welham and co-workers have shown that the lymphocyte cell line FDMACII requires IL-4 for cell proliferation but that IL-4 does not signal through ERK-1 or ERK-2 (178). M A P kinases are also involved in maturation-associated events of cells in tissue culture conditions. Integrins have been implicated in cell adhesion, growth and differentiation. In the 27  NIH 3T3 cell, the adhesion of integrins to fibronectin activates M A P kinase activity within 1 hour of adhesion (123). Maturation events may involve the sustained activation of ERK-1 and ERK-2 molecules in eukaryotic cells. For example, the treatment of megakaryocytes with 12-0tetradecanoylphorbol-13-acetate (TPA) induces both cell maturation and an activation of M A P kinases that is sustained for 24 hours (103). These maturation events require M A P kinases and can be inhibited by treating megakaryocytes with the M A P P K inhibitor PD98059 (103).  1.6.8.3. The role of M A P kinases in cytokine-induced signalling. M A P kinases are activated in cell culture upon treatment with cytokines and may play a role in mediating feedback inhibition of cytokine expression (179) (Figure 3). IL-lp\ another cytokine implicated in TSS, was shown to induce early ERK-1 and ERK-2 phosphorylation and activation in human mesanglial cells (180). In some cases, cytokine-induced activation of M A P kinases requires pre-activation of cells with P M A or is increased with pre-activation. For example, IL-12 can induce the activation of ERK-1 and ERK-2 only in phorbol-12 myristate 13-acetate (PMA) activated primary T cell culture (179). In comparison, the IL-2 activation of M A P kinases in the murine T cell line CTLL-2 does not require pre-activation but is increased 3-fold with P M A pre-treatment (120).  1.6.9. De-activation of M A P kinase: A role for M A P kinase phosphatase. M A P kinase phosphatases have been suggested as the primary feedback inhibition response for ERK-1 and ERK-2 activation in eukaryotic cells (114,115,127). ERK-1/2 activity can be inhibited by the activation of M A P kinase tyrosine phosphatases (116,181) throughout the cell (116) or dual specificity tyrosine/threonine phosphatases in the nucleus (112) (Figure 3). These dual specificity phosphatases which are able to target either ERK-1 or ERK-2 include the ERK-1 specific M A P kinase phosphatase-1 (MAPK-1) (113) and the ERK-2 specific M K P 28  2 and PAC-1 (113). The control mechanisms of M K P control are still being elucidated and may require the presence of Ca  -dependent signal transduction pathways (114). It is also known  that E R K activation induces early production of MPK-1 in the oncogenic human clone Rat-1 (114). In a rat hepatic cell line, Hire B, insulin-induced M A P kinase activity was attenuated following activation of MKP-1 (115). However, the role of MKP-1 in controlling ERK-1/2 activity is quite complex and may even include the activation of MEK-1 and M E K - 2 as part of the feedback mechanism (127).  1,7. The focus of this thesis. TSST-1 is a 22 kDa superantigen that has been implicated in lethal cases of toxic shock in humans. Several workers have suggested that TSST-1 can induce signals within monocytic cells alone (182,183). Unfortunately, these workers gave no justification for the doses of TSST-1 used and performed signal transduction studies under conditions where TSST-1 may be acting in a non-superantigenic fashion. The experiments in this thesis first required the determination of the conditions, including toxin doses and culture conditions, whereby TSST-1 could act in a superantigenic fashion. TNF-a release was used as a pathophysiological endpoint for TSST-1 signalling due to its critical role in the pathogenesis of TSS (59,184). It is known that this TSST-1 mediated TNF-a release requires the presence of both APCs and T cells (168). LPS mediated TNF-a release from THP-1 cells (67) was chosen as a comparison to the two-cell model of TSST-1 mediated TNF-a release. It was thought that LPS mediated events in THP-1 cells would also act as a suitable control for M A P kinase activation (81). M A P kinases were selected as the enzymes to study because of the multiple roles they play in eukaryotic physiology. At the beginning of this thesis project, it was thought that M A P kinases were an attractive downstream focal point for events leading to TSS including multiple  29  transcription factors, mitogenesis and cell maturation. The purpose of the thesis research was not to determine the role of M A P kinases in TSST-1 mediated events leading to TNF-a release from THP-1 cells. Instead, the purpose of this study was to determine the early TSST-1 mediated M A P kinase signalling events in monocytic cells prior to the recruitment of T cells. This TSST-1 mediated M A P kinase signalling could be compared to signalling induced by the MHCII binding mutant, G31Rmut TSST-1 (44) and LPS mediated signalling (87). After determining the role of M A P kinases in TSST-1 induced signalling, it will be possible to study the signal transduction events in THP-1 and T cell co-cultures in the future. There are several reasons why the THP-1 cell line, rather than human donor cells, was chosen to study TSST-1 mediated pathogenesis. The use of cell lines decreases variability usually seen in primary tissue culture. A previously observed variability in TSST-1-induced monocyte protein-tyrosine phosphorylation (169) could be due to donor-dependent factors including levels of H L A - D R expression and concentrations or activities of protein kinases or phosphatases. Secondly, THP-1 is capable of expressing the TSST-1 ligand H L A - D R in a more consistent manner than donor monocytes. Third, THP-1 expresses and releases T N F - a in response to treatment with several molecules including LPS (67), other bacterial superantigens (60) and phorbol esters (185). There was also a strong chance that mitogen activated protein (MAP) kinases will act in a uniform fashion when a cell line is treated with TSST-1. There are several drawbacks to using THP-1 cells as a monocytic model of TSST-1 pathogenesis experiments. The constitutive level of H L A - D R may not be high enough to bind to TSST-1 and treatment with IFN-y may be required to up-regulate H L A - D R expression on the cell surface. Al-Daccak and co-workers showed that THP-1 expresses very low levels of H L A DR which require up-regulation with IFN-y in order to bind to the Mycoplasma arthritidis-T cell mitogen ( M A M ) SAg (60). Previous workers have shown that 100 U/ml of recombinant 30  human IFN-y can up-regulate both H L A - D R mRNA (60,186) and cell surface proteins (187) in THP-1 cells within 24 hours of treatment. Although IFN-y up-regulates H L A - D R on the surface of monocytic cells, it may also affect other physiological processes in these cells. Interferon-y activates cells and causes changes in the levels of monocytic proteins including up-regulation of cell membrane FcyRI, FcyRIII (CD 16) (188), soluble TNF-a receptor (sTNF-R) release from monocytes (189) and down-regulation of CD l i b , CD 18 and C D 14 on monocyte membranes (190). IFN-y also activates signal transduction cascades involving Janus kinases (Jak) (191) signal transducers and activators of transcription (STAT) molecules (192,193) and protein kinase C (PKC) isoforms (187). There are some concerns that should be addressed when using a tumor cell line to describe possible pathogenic mechanisms in humans. THP-1 is a leukemia cell line that may not represent normal human donor monocytes in signal transduction or the release of cytokines following treatment with TSST-1. Due to high rates of division (194), the basal level of M A P kinase activity in tumor cell lines may be quite high when compared to M A P kinase activity levels in healthy normal primary cell cultures (194). Tumor cells may also express novel kinases that produce unique signal transduction profdes not found in normal healthy tissue. For example, human squamous cell carcinomas and adenocarcinomas of the lung contain a 40 kDa tumor-associated protein kinase (p40TAK) that has myelin basic protein kinase activity which might be mistaken for ERK-1 and ERK-2 activity (195).  31  This thesis will attempt to study several questions that will lead to a better understanding of TSST-1 induced M A P kinase signalling: 1. What are the conditions required for TSST-1 induced and pathophysiologically relevant signalling, measured by TNF-a release, in a THP-1 cell and T cell co-culture ? 2. How do these TSST-1 induced events differ from LPS induced events as a function of TNFa release ? 3. Are M A P kinases involved in early TSST-1 induced signalling in THP-1 cells alone ? 4. How does TSST-1 induced M A P kinase signalling differ from LPS, G31Rmut and P M A induced early signalling in THP-1 cells ?  32  Chapter 2. Materials and Methods. 2.1. THP-1 cell cultures for H L A - D R mRNA, H L A - D R protein, and TNF-a release studies.  THP-1 cells (ATCC TIB 202) (91,196) were utilized between 6 and 12 passages while earlier passages were stored at -70°C in liquid nitrogen. Cells were grown at 37°C, 5% CO2 in a culture media containing 10% fetal calf serum (Hyclone/VWR Canlab, Mississauga, Ont.), 1% penicillin/streptomycin/amphotericin B antibiotic (Sigma Chemical Co., St. Louis, Mo) and 89% RPMI 1640 media with L-glutamine (Stem Cell, Vancouver, B.C.) (91,196).  Cultures  were maintained between 10 and 10 cells/ml in culture plates and flasks (Becton-Dickenson, 5  6  Lincoln Park, NJ) and counted using trypan blue exclusion and hemocytometry. For H L A - D R protein studies, THP-1 cells were grown under the same conditions in 25 ml Teflon culture flasks (Norton Performance Plastics, Akron, OH).  2.2. The use of reverse-transcriptase polyacrylamide chain reaction (RT-PCR) to determine IFN-y induced H L A - D R mRNA expression in THP-1. THP-1 cells were seeded in 24 well plates (Beckton Dickenson, Lincoln Park, NJ) at a concentration of 5 x 10 cells/ml in 1 ml culture media. Cells were treated with 0 U/ml to 400 5  U/ml IFN-y (Gezyme Corp., Boston, M A ) and incubated for 0 to 36 hours at 37°C, 5% C 0 , for 2  6 hours. Cells were aspirated with Pasteur pipettes (Elkay Products Inc., Worcester, M A ) and placed into 1.5 ml microcentrifuge tubes (Elkay Products Inc., Worcester, M A ) . THP-1 cells were centrifuged for 10 minutes at 300 x g in a microcentrifuge (Biofuge 15, Baxter Diagnostics Corp., Mississauga, Ont.). A l l fluid was removed from the THP-1 cell pellets which were then treated with 300 ul of Trizol reagent (Gibco B R L Products, Gaithersburg, M D ) (197-199). A Pasteur pipette was used to lyse the THP-1 cells and the Trizol preparations were frozen at -70°C until use (199). 33  Prior to experimentation all glassware was baked and aqueous reagents were treated with diethyl pyrocarbonate (DEPC) (0.1% in ddH20) (Gibco B R L , Gaithersburg, MD). Sixty ul chloroform (Gibco B R L , Gaithersburg, M D ) were added to each lysed-cell preparation and shaken by hand at room temperature for 15 seconds. Chloroform preparations were then incubated at room temperature for 3 minutes and centrifuged at 12,000 x g and 4°C (Biofuge 15, Baxter Diagnostic Corp., Mississauga, Ont.) for 15 minutes. The upper aqueous phase was removed and used for further steps while the lower organic phase was stored at -70°C. The aqueous phase was placed into 1.5 ml microcentrifuge tubes (Elkay Products Inc., Worcester, M A ) and treated with 150 ul isopropanol (Gibco B R L Products, Gaithersburg, MD). Isopropanol preparation were incubated at room temperature for 10 minutes and then centrifuged at 12,000 x g, 4°C (Baxter Diagnostic Corp., Mississaugua, Ont.) for 10 minutes. Three hundred ul of 75% ethanol were added to each R N A pellet and the pellets were centrifuged at 7,500 x g, 4°C for 5 minutes. R N A was air-dried at room temperature and A260/A280  readings  were  measured  by  spectrophotometry  (Beckman  DU  640  spectrophotometer, Beckman Industries, Mississauga, Ont.) (197-199). R N A stability was tested by running 0.5 ug R N A preparations in loading buffer IV (0.25% bromphenol blue, 40% (w/v) sucrose) on a 1.5% ultrapure agarose gel (Gibco B R L , Gaithersburg, M D ) , at 60-90 V , for 2 hours on a horizontal gel apparatus (Gibco B R L , Gaithersburg, MD). For RT-PCR (198,200,201), 0.04 ug total R N A were placed on ice in RT solution (1 x 1 strand buffer, 0.1 M DTT, 1 n M dGTP, 1 n M dATP, 1 n M dCTP, 1 n M dTTP, st  5 u M oligo(dT) 12-18, 2.5 U/ml M - M L V - R T ) (Gibco B R L , Gaithersburg, M D ) (202). The reverse transcriptase (RT) solutions sat at room temperature for 10 minutes and underwent one cycle in the theromocycler (DNA fhermocycler 480) (Perkins Elmer Cetus Instruments, Norwalk, CT) at 42°C for 15 minutes, 99°C for 5 minutes, 5°C for 5 minutes and stored at 5°C 34  until use. During the P C R step, 9 ul of cDNA were placed into 38 ul P C R solution (2 m M M g C l , 1.2 x P C R buffer and 0.024 U/ml Taq polymerase) (Gibco B R L , Gaithersburg, MD). 2  For each P C R reaction (203), 20 u M of either H L A - D R a (Exon 2) sense (5'-CGA G T T C T A TCT G A A T C C T G A CCA-3') or (Exon 4) antisense (5'- GTT C T G C T G C A T T G C TTT TGC GCA-3') primers were used to amplify H L A - D R cDNA (197). To amplify human cytoplasmic p-actin cDNA , (Exon E) sense (5'-CAC C C C GTC C T G C T G A C C G A G  Gee-  s ' ) and (Exon E) anti-sense (5'- C C A C A C G G A G T A CTT G C C CTC AGG-3') primers (197) were used. These primers were a gift from Dr. D. Nandan. PCR cycling was programmed for 30 cycles at 94°C for 1 minute, 55°C for 1 minute and 72°C for 3 minutes. Amplified H L A - D R and P-actin cDNA sequences were resolved on a 1.5 % agarose gel (3:1 wide range standard) (Sigma Chemical Co., St. Louis, MO) containing 1 ng/ml EtBr at 80-100 V for 1 hours. To visualize D N A staining each gel was placed on an ultraviolet light box (Sigma Chemical Co., St. Louis, MO) and black-and-white instant photos were taken (Eastman Kodak Co., Rochester, N Y ) . The molecular mass of each amplified D N A band was estimated by comparison with D N A digest ladders (Gibco BRL, Gaithersburg, MD) and the masses were compared to cDNA sequences listed in Genbank (Genbank, NIH, Bethesda, MD). This procedure was done for 2 separate experiments. Intensities of bands for amplified P-actin and H L A - D R mRNA were measured by densitometry (Trimage Version 2.19, 1996, TN-Image Analysis Software, Rockville, MD). The average of 3 density readings for each band was calculated and the average H L A - D R mRNA band density for each time point was divided by the density of its corresponding P-actin mRNA band.  35  2.3. Fluorescence-activated antibody cell scanning (FACS) staining of H L A - D R on THP-1 cells. THP-1 cells (1 x 10 ) were treated with 100 U/ml IFN-y (Genzyme Corp. Boston, M A ) 6  for 0, 12, 36 and 48 hours. Cells were counted and viability was determined by trypan blue exclusion. Approximately 5 x 10 THP-1 cells were loaded into 1.5 ml microcentrifuge tubes 5  (Elkay Products, Worchester, M A ) and centrifuged at 4°C for 30 minutes (Baxter Diagnostic Corp, Mississauga, Ont.). Cell pellets were resuspended in 20 ul fluorescein isothiocyante (FITC)- conjugated mouse IgG2b anti-HLA-DR (HHLDR01) or mouse IgG2b isotypic control (MG2b01) (Cedarlane Laboratories, Ltd., Hornby, Ont.) in wash buffer (3% fetal calf serum, 0.1% (w/v) NaN3, in Hank's buffered saline solution). THP-1 cells were incubated in the dark at 4°C for 30 minutes. Cells were centrifuged at 300 x g, 4°C for 10 minutes (Biofuge 15, Baxter Diagnostics Corp, Mississauga, Ont.) and washed in 20 ul of antibody wash buffer with the same centrifuge conditions. Stained THP-1 cells were resuspended in 200 ul of antibody wash buffer and treated with 100 ul of 2% paraformaldehyde. H L A - D R levels on THP-1 cells were assayed as a measure of fluorescence intensity on a flourescence antibody cell scanner (Becton-Dickinson FACScan™, Becton-Dickinson, Mississauga, Ont.) at an absorption wavelength of 495 nm and an emission wavelength of 530 nm (202,204-207). A population of 5000 cells were gated for both control and experimental stainings at each IFN-y treatment time point, at each time point.  2.4. Isolation of donor T cells from whole human blood. Y  T cells were isolated from three healthy donors as originally described by See et al. (168). Briefly, human peripheral blood cells were isolated from whole blood by centrifuging plateletphoresis buffy coats over Histopaque 1.077 (Sigma Chemical Co., St Louis, Mo.). 36  Interface cells were washed five times with Hank's buffered salt solution (Stem Cell, Vancouver, B.C.) and T cells were separated from non-T cells by rosetting with activated sheep red blood cells (SRBC's). Sheep red blood cells were lysed with NH4 C l and washed three times with Hank's buffered salt solution. Partially purified human T cells were further purified from monocytes and B cells expressing M H C II (HLA-DR) using complement lysis with antiCD l i b (OKM1, ATCC), anti-HLA-DR (L243, A T C C ) monoclonal antibodies and pooled rabbit complement (Cedarlane Laboratories Ltd., Hornby, Ont.) (168,205). Monocyte depleted T cells were then counted using trypan blue exclusion and were used immediately after purification.  2.5. Treatments of THP-1 and monocyte-depleted T cells with RPMI 1640, TSST-1 and LPS.  One million THP-1 cells, monocyte-depleted donor T cells and THP-1/T cell (1:1) cocultures were seeded into 96 well plates (Becton Dickinson, Lincoln Park, NJ) at a concentration of 1 x 10 cells/ml respectively. The cultures were then treated with 10 ug/ml 6  polymyxin B sulphate (PMB) (Sigma Chemical Co., St. Louis, Mo.) except for cultures that were to be treated with LPS. Experimental THP-1/T cell co-cultures, T cells and THP-1 cells alone were then treated with 1 ng/ml TSST-1 (MN 8; a gift from Dr. W. Kum) or culture media (RPMI 1640) alone for 12, 26, 60 and 84 hours at 37°C, 5% C 0 in the presence or absence of 2  100 U/ml recombinant human IFN-y (Genzyme Corp., Boston, M A ) . Control THP-1 cells were treated with 1 ug/ml LPS in the presence or absence of both 10 ug/ml P M B (168) and 100 U/ml IFN-y (187) and cultured at 37°C, 5% CO2 for the same time course. At the appropriate times, culture supernatants were aspirated and frozen at -70°C until use.  37  2.6. TNF-a enzyme linked immunosorbance assay (ELISA).  The ELISA for TNF-a followed the protocol established by See et al. (168) in our laboratory. Briefly, 96 well Immulon-1 (VWR Canlab, Mississaugua, Ont.) ELISA plates were coated with goat anti-human TNF-a polyclonal antibodies at 20°C for 20 hours, and were washed three times with phosphate buffered saline- Triton X-100 (PBS-T). One hundred ul of harvested supernatant and known human recombinant controls were incubated at 37°C for 1.5 hours and washed three times with PBS-T. Plates were incubated with 100 ul/well of Streptavidin-alkaline phosphatase for 20 minutes at 37°C and were washed five times with 100 ul/well of tris-buffered saline (TBS). Fifty ul of B R L substrate were then pipetted into each well and incubated in the dark at room temperature for 15 minutes. Fifty ul of B R L amplifier reagent (Life Technologies, Gaithersburg, MD) were then added to each well and incubated in the dark for 20 minutes. The reactions were stopped with 0.3 M H 2 S O 4 and the plates were read at 495 nm on a Dynatech MR5000 ELISA reader (Dynatech Laboratories, Chantily, V A ) (168).  2.7. THP-1 cell culturing, stimulation and lysis for M A P kinase studies. THP-1 cells were cultured for 6-12 passages as described earlier and were counted by trypan blue exclusion and hemocytometry. These cells were washed 2 times at 300 x g in RPMI 1640 (208). Twenty million cells were then cultured in 1.5 ml ultracentrifuge tubes (Beckman Instruments, Palo Alto, C A ) in a total volume of 500 ul for each manipulation. Cells were treated with specific activators for the indicated time frame. As a control for constitutive M A P kinase activity, 2 x 10 THP-1 cells were left untreated in RPMI 1640 containing 10 ug/ml 7  polymyxin B sulphate (Sigma Chemical Co., St. Louis, MO). As a positive control for M A P 38  kinase activity, 2 x IO THP-1 cells were treated for 1 n M phorbol 12-myristate 13-acetate 7  (PMA) (Sigma Chemical Co., St. Louis, MO) (88) for 2 and 5 minutes in RPMI 1640 containing 10 ug/ml polymyxin B sulphate (PMB) (Sigma Chemical Co., St. Louis, MO) experimental groups consisted of 2 x 10 cells treated for 2, 5, 10, 15 and 30 minutes with 1 7  ng/ml TSST-1 in RPMI-1640 containing 10 ug/ml polymyxin B sulphate (168). At the appropriate time points following treatment, THP-1 cells were lysed at 4°C with sonication (Sonifier disruptor, Branson Sonic Power Co., Danbury, CT) and the addition of 500 ul lysis buffer. The lyses buffer contained 20 m M MOPS, pH 7.2, 1% Nonident P-40, 5 m M EGTA, 75 m M p-glycerol phosphate, 1 m M dithiothreitol (DTT), 1 m M PMSF and 1 m M N a V 0 3  (85,96,168). Whole cell  4  lysates were then ultracentrifuged at 100,000 x g (Optima T L X  ultracentrifuge, Beckman Industries, Palo Alto, C A ) to remove detergent insoluble cellular components (96). High-speed supernatants were frozen at -70°C until further use (96).  2.8. The determination of total cell lysate protein concentration. The total cellular protein concentration from high speed supernatants were determined by the Lowry/DC assay (Bio-Rad Laboratories Ltd., Mississaugua, Ont.) (209). Bovine serum albumin (BSA) (Hyclone/VWR Canlab, Mississaugua, Ont.) was used as standard for total cell protein at concentrations between 16 pg/ml 2000 pg/ml protein (85). Neat and serial dilutions of high-speed supernatants and triplicates of the B S A serial dilution standards were loaded as 5 ul volumes into 96-well ELISA plates (Immulon-1). DC/Lowry reagents were added and the reaction was allowed to develop for 15 minutes. The optical density for each control and treatment was read at 490 nm on an automated plate reader (Dynatech MR5000, Dynatech Laboratories, Chantily, V A ) . Unknown high-speed supernatant protein concentrations were determined from the B S A standard curve (85).  39  2.9. Anion exchange chromatography and fractionation of high-speed supernatants. A 2-ml Bio-Scale Q anion exchange column (Bio-Rad Laboratories, Ltd., Mississagua, Ont.) attached to a fast protein liquid chromatography (FPLC) system (Pharmacia Biotech, Uppsala, Sweden) was equilibrated with Buffer A (12.5 m M MOPS, pH 7.2, 12.5 m M Bglycerol phosphate, 0.5 m M EGTA, 7.5 m M M g C l , and 1 m M DTT) (85,88). Approximately 2 2  mg of THP-1 high speed supernatant protein were loaded onto the Bio-Scale Q anion exchange column and were eluted in a 10 ml linear gradient between 0 m M NaCl to 0.8 m M NaCl. Forty fractions were collected for each sample in 250 ul volumes and were frozen at -70°C until further analysis (85,88).  2.10. The myelin basic protein kinase phosphotransferase assay of Bioscale-Q fractions. Bio-Scale Q eluent fractions were assayed for M A P kinase activity by the myelin basic protein (MBP) phosphotransferase activity assay. In this assay, 10 ul of each Bio-Scale Q fraction were incubated with 10 ug M B P in a buffer containing 10 m M MgCb, 1 m M M n C b and 50 u M [y- P]ATP (Mandel Scientific Co. Ltd., Guelph, Ont.) at room temperature for 20 32  minutes (87). Reactions were stopped by spotting 10 ul of the reaction mixture onto a 1.5 cm piece of p81 phosphocellulose paper. Dried papers were then washed six times, for 10 minutes each, in 1% phosphoric acid. After one hour of washing, papers were loaded into 20 ml scintillation vials containing 2 ml of scintillation fluid and the phosphotransferase activity was determined by scintillation counting (Beckman LS 1800, Beckman Industries, Palo Alto, C A ) (87,88).  •  2.11. ERK-1/ERK-2 immunoprecipitation from THP-1 high speed supernatants. Following lysis and high speed centrifugation of THP-1 cell lysates (100, OOOx g, Optoma T L X ultracentrifuge, Beckman Industries, Palo Alto, C A ) , 300 ug of protein were 40  diluted with homogenization buffer (HB) (12.5 m M MOPS, 12.5 m M P-glycerophosphate, 0.5 m M E G T A , 7.5 m M M g C l , 0.05 m M NaF, 1.5 u M aprotinin, 20 u M leupeptin, I m M DTT, 0.1 2  % PMSF/bezamide, 1 % Triton X-100) (Sigma Chemical Co., St. Louis, MO) to a final volume of 500 ul in Eppendorf tubes. The samples were pre-cleared with 20 ul protein A-Sepharose (PAS) beads (Pharmacia Biotech, Uppsala, Sweden) for 20 minutes at 4°C (96). The preclearing beads were removed from the supernatants by centrifugation (Biofuge 15, Baxter Diagnostics, Missisauga, Ont.) at 300 x g, 4°C, 2 M i n (96). The samples were then incubated with 30 ul PAS beads and 5 ul of either anti-ERK-1, anti-ERK-2 (Santa Cruz Biotechnology, Inc, Santa Cruz, C A ) (96) or anti-ERK-1 CT antibodies (Kinetek Pharmaceuticals Inc, Vancouver, B.C.) for 4 hours at 4°C (88). Beads were then washed with homogenization buffer and centrifuged at 10,000 rpm, 4°C for 2 minutes. Two final washes with KII buffer were completed and the pellets were then suspended in 20 ul KII buffer (pH 7.2, 12.5 m M MOPS, 12.5 m M p-glycerophosphate, 0.5 m M EGTA, 7.5 m M M g C l , 0.05 m M NaF, 0.01 m M p2  methyl aspartic acid), 10 ul M g C l (200 mM) and 10 ul myelin basic protein (MBP) (Kinetek 2  Pharmecuticals Inc., Vancouver, B.C.) (1.66 ug/ul) (88,96). At room temperature, 10 ul of radiolabeled [y- P] A T P (10 mM) were added to each sample and vortexed every five minutes 32  until the 20 minute incubation time was complete. Reactions were stopped by spotting 15 ul of the supernatant on p81 (Whatman/VWR Canlab, Mississauga, Ont.) filter paper and adding 20 ul of 3x sample buffer to the remaining beads and boiling for 5 minutes (88,96). M B P kinase activities were expressed as a percentage of corrected ERK-1 or ERK-2 activity over basal activity at each time point (Equation 1).  41  Equation 1. Relative E R K activity in THP-1 cells. % E R K activity over basal = 100 % (Treatment E R K activity per time-RPMI 1640 induced E R K activity per time point)/ (RPMI 1640 induced E R K activity per time point) The time zero treatments contained no reagent and were averaged and subtracted to give an absolute zero value with no error. Areas under the curve for both E R K activation and E R K deactivation were calculated by GraphPad Prism™ (Version 2.01, 1996, GraphPad Software, Inc., San Diego, CA). Portions of the curve were reported as positive values while areas under deactivation portions of the curve were reported as negative values. Areas under the activation and deactivation curves were compared by the Wilcoxin signed rank test (two-tailed) (169).  2.12. Polyacrylamide gel electrophoresis of THP-1 proteins.  THP-1 proteins (300 ug whole cell lysate or 250 ul eluent) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as first described by Laemmli (210). Samples were added to 3x sample buffer (187.5 m M Tris-HCl, pH 6.8, 30% glycerol, 6% SDS, 15% (3-mercaptoethanol) and were boiled for five minutes prior to loading onto a 4% stacking gel and a 11% resolving gel in a Bio-Rad gel apparatus (Bio-Rad Laboratories, Mississaugua, Ont.). Rainbow molecular markers (Amersham, Arlington Heights, Ont.) or house markers between 200 kDa and 14.3 kDa molecular mass (Kinetek Pharmaceuticals, Inc., Vancouver, B.C.) were used to determine mass of loaded proteins. Samples were run overnight at 8 mA for each gel and then amperage was increased to 15 mA until the dye front ran off the gel.  42  2.13. Western blotting and chemiluminescent staining of THP-1 proteins separated by gel electrophoresis.  Gels were disassembled from the gel apparatus and were soaked in 50 ml transfer buffer (40 m M glycerol, 20 % methanol, 4 m M SDS, 50 m M Tris) for 5 minutes. THP-1 proteins were transferred to 0.45 um nitrocellulose (Bio-Rad Laboratories, Mississagua, Ont.) with a semi-dry electroblotter (Ancos, Denmark). Effective protein transfer was ensured with Ponceau S staining (Sigma Chemical Corp., St. Louis, MO) (88). Blots were then blocked overnight with 3% B S A in tris buffered saline (TBS) (pH 7.4, 20 n M Tris, 0.3 M NaCl ). Following a quick TBS rinse, blots were incubated in 1/1000 (antibody/TBS) primary rabbit anti-ERKl-CT (Kinetek Pharmacuticals Inc., Vancouver, B.C.) or 1/3000 rabbit anti-Phosphotyrosine (4G10) (Santa Cruz Biotechnology Inc., Santa Cruz,CA) for 4 hours at room temperature. For the determination of both anti-ERK-1/2 and anti-phosphotyrosine staining from the same blot, nitrocellulose blots were first probed with 4G10 antibodies, stripped with stripping solution (100 m M p-mercapto ethanol, 2% SDS, 63 n M Tris-HCL, pH 6.7), blocked and reprobed with anti-ERK-1/2 antibodies. The blots were then washed with 0.05% NP40 (Sigma Chemical Corp., St. Louis, MO)-TBS (NTBS) and incubated in a 1/3000 (antibody/TBS) dilution of horse-radish peroxidase (HRP) conjugated anti-Rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, C A ) for 90 minutes. Blots were washed with NTBS and TBS prior to exposure with a 1:1 enzymatic chemiluminescence developing solution (Amersham Life Science, England). Proteins were visualized by exposing ECL-hyperfilm (Amersham Life Science, England) to each treated blot (96). The intensity of phosphotyrosine staining (4G10) and anti-ERK-1/2 staining were measured by densitometry (Trimage, Version 2.19, 1996, TN-Image Analysis Software, Rockville, MD). Raw densitometry counts were measured by selecting an area including the 43  ERK-1 and ERK-2 staining regions on Western blots. For determination of tyrosine phosphorylation and ERK-1/2 protein levels, background readings were subtracted from each reading, repeated three times, and the counts were transformed into a ratio of counts/area. Variations for the amount of ERK-1/2 loaded on the gel were corrected by transforming the data with Equation 2 (96). Equation 2. Tyrosine phosphorylation of ERK-1 and ERK-2. Relative phosphorylation = (Tyrosine intensity/area scanned)/(MAP kinase intensity/area scanned) Values from three separate experiments were combined and expressed as a ratio of standardized ERK-1/2  tyrosine  phosphorylation  for  each  phosphotyrosine staining at time zero.  44  treatment  over  standardized  ERK-1/2  Chapter 3. The Induction of Tumor Necrosis Factor-a Release from THP-1 and Human Donor T cell Co-cultures Treated with TSST-1. 3.1. Introduction. As described in Chapter 1, previous workers have shown that human donor monocyte and T cell co-cultures treated with TSST-1 are able to release TNF-a into the cell culture supernatant (36). This 2-cell model of TSST-1 induced T N F - a release requires multiple types of interaction between APCs and human T cells (36) (Figure 1) and contrasts to the LPSinduced model of TNF-a release from monocytic cells alone (36,67). Both cells must also be viable in order for TSST-1-induced TNF-a release to occur (36) and the utilization of either paraformaldehyde-fixed T cells or monocytes prevented the TSST-1-induced release of TNF-a and IL-ip (36). Some workers have questioned whether T cells are absolutely required for TSST-1-induced TNF-a release from monocytic cells and have suggested that certain T cell factors such as IFN-y may replace the T cell component of the TSST-1 -APC-T cell model (182). These workers used TSST-1 to induce TNF-a and IL-ip  mRNA expression in  monocytic cells co-treated with SAgs and IFN-y or T cell supernatants (60). Other investigators have replaced the T cell component of the 2-cell model with biotin-avidin crosslinked superantigens in order to study signal transduction (211). There are several important interactions that occur between T cells and APCs bound to SAgs. The most important interaction occurs between the SAg and its H L A - D R (19) and Vp2 TCR binding domains (49,212). It is not known i f the H L A - D R molecule physically interacts with the TCR molecule or i f TSST-1 links the TCR and H L A - D R molecules without receptorreceptor interaction. However, our laboratory has determined that the glycine-31 residue and the aspartic acid-135 residue on TSST-1 are required for binding to the M H C II and T cell receptor  45  molecules respectively (44). Accessory signals are also required for the optimal release of TNFa from this APC-SAg-T cell system. Workers in our laboratory have previously shown that an interaction between the intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and lymphocyte function antigen-1 (LFA-1) on both monocytes and T cells are required for the optimal release of TNF-a from a donor T cell-monocyte co-culture treated with TSST-1 (36). The purpose of this series of experiments was to determine the conditions required for the TSST-1 induced release of TNF-a from a THP-1 and donor T cell co-culture. THP-1 was chosen for several reasons, including the fact that it is an acute monocytic leukemia derived cell line. First, the use of THP-1 may reduce the variability seen in cell surface antigens, cytokine release and signal transduction pathways normally enountered using freshly isolated human monocytes (91). Second, this cell line is known to produce and release the shock-inducing cytokine TNF-a in other models of bacterial pathogenesis such as LPS induced septic shock (67). Third, other workers have shown that THP-1 is capable of expressing the H L A - D R isoform of the M H C II molecule and can bind to other superantigens including the Mycoplasma arthritidis T cell mitogen ( M A M ) (60). Donor T cells were utilized in this experimental model for several reasons. We could not find a suitable human T cell line that expressed the VB2 T C R determinant on the cell surface. Furthermore, the use of a mixed THP-1 and T cell co-culture system is the next logical step in a two-cell model because it replaces only one of the donor components. Since they are considered immature, cell lines may not have the level of co-stimulatory molecules required for the T cell-TSST-1- A P C model to release TNF-a into the culture supernatant. Further experiments may allow for the replacement of the donor T cells with human or animal V B  46  specific T cell lines which will further simplify the model and may be used to determine which of the two cell types actually produce TNF-a. Finally, this chapter also details experiments aimed at determining the level of THP-1 cell surface H L A - D R that is required for TSST-1 mediated TNF-a release. Previous workers have suggested that constitutive H L A - D R cell surface expression on THP-1 cells is quite low and may not bind (SAgs) effectively (60). Other workers have shown that IFN-y upregulates H L A - D R mRNA within cells and H L A - D R on the cell membrane of THP-1 cells (60,186,187). Specific Aims 1) To create a functional assay for TSST-1 pathogenesis in a THP-1 and monocyte-depleted donor T cell model. These conditions can be used to study TSST-1 mediated signal transduction in THP-1 cells prior to the recruitment of donor T cells. 2) To determine the conditions required for TNF-a release from the THP-1 and donor T cell coculture treated with TSST-1. 3) To compare the requirement for TSST-1-induced TNF-a release with LPS- induced T N F - a release when using THP-1 cell culture. 4) To determine if the constitutive level of H L A - D R on the THP-1 cell surface is high enough to bind to TSST-1, thereby allowing for TNF-a release, in the two-cell model.  3.2. Results. 3.2.1. RT-PCR analysis of IFN-y induced mRNA upregulation in THP-1 cells.  RT-PCR was used to determine the levels of H L A - D R and control p-actin mRNA expression in THP-1 cells following treatment with 0-400 U/ml IFN-y. Doses of 0 to 400 U/ml IFN-y did not induce noticeable increases in the expression of housekeeping P-actin mRNA  47  within 6 hours of treatment (Figure 5, Table III ). IFN-y induced 1.5-fold to 6-fold increases, respectively, in corrected H L A - D R mRNA expression over basal expression within 6 hours of treatment (Figure 5, Table III). Based on these results, the dose of 100 U/ml IFN-y was chosen for future studies. IFN-y (100 U/ml) did not induce a noticeable increase in a ~ 600 bp control p-actin mRNA housekeeping sequence between 12 and 36 hours after treatment (Figure 6, Table IV). IFN-y induced a 3.5-fold increase in H L A - D R mRNA expression, corrected for levels of P-actin mRNA, at 12 hours of treatment. The increase of H L A - D R mRNA at 12 hours after treatment with IFN-y (100 U/ml) is representative of two separate experiments (Figure 6, Table IV).  / 3.2.2. FACscan analysis of H L A - D R upregulation on THP-1 cells following treatment with IFN-y.  At each time point, IFN-y (100 U/ml) induced increases in relative anti-HLA-DR flourescence intensity on THP-1 cells when compared to time zero values. IFN-y induced a 22% increase, a 60% increase and a 75% increase over time zero anti-HLA-DR fluorescence intensity at 12, 24 and 36 hours, respectively (Figure 7). After 48 hours of IFN-y treatment, the levels of anti-HLA-DR flourescence staining on THP-1 cells decreased to a 22 % increase over time zero anti-HLA-DR fluorescence (Figure 7). These values were determined for 1 experiment using a population of 5000 THP-1 cells for both anti-HLA-DR and control staining of treatment groups.  -  48  u o =3  C  CU  u  s  D o o  11 g S z < —i a:  i  JH  CU  E  S-i  £  oB  9 « < vo  = -a  n  —J  S  43  <  CQ.  o  B,  y,  2o N  " tu  J  T3  >-i  0  tn <* tn Z  cn  >- S  tu  Z  P  m <  ~2  2  5  E  c ei  c 'cn cn CU  to Q Q  ca.  ^ S: < <o d  & cD  - *  cn  CD  .S  CU  P  cu <n'  2  I  ce da "•—<  E <2 IT) cd  49  Table III. The effect of different IFN-y doses on H L A - D R mRNA expression in THP-1 cells. IFN-y dose (U/ml) HLA-DR band density B-actin band density HLA-DR density /B- actin density Corrected HLA-DR density / basal H L A DR density  0  10  50  100  200  400  0.083  0.150  0.320  0.357  0.330  0.467  0.575  0.651  0.650  0.500  0.664  0.544  0.144  0.230  0.492  0.714  0.497  0.843  1  1.5  3.5  5  3.5  6  The densitometry readings for H L A - D R mRNA bands (from Figure 5) were divided by the readings for B-actin bands. The corrected H L A - D R mRNA band densities at each dose were then divided by the basal H L A - D R mRNA band density (0 U/ml IFN-y, 6 hours post-treatment). A dose of 100 U/ml IFN-y induced a 5-fold increase in corrected H L A - D R mRNA band density over basal (0 U/ml IFN-y) levels of expression.  50  CU  o ^ H i  CN  i  PH  ^  I  «  CU GO  cu  e  cu  o  £  cn cU  g  -rt —  O «  SH  O.  Q i  ed  <  C  A\ cu o  II  IE  E  u  T  c g  —i  ft  7Z  ^ i € § .2? 8 .Sao,  eg CQ.  E ^  1  u  2 < §> 2 w  Z «a  Q  cn  2  Z f- f" 1  .2  w  O D . c n 6 0  -3 <3  ^~  —  H U  C  c  X! <U o  is  P<  CU  PH  E  h i  5 >P o o  2 E 00  '£  51  H 2  43  Table IV. A time course for H L A - D R mRNA expression in THP-1 cells treated with a preoptimized dose of IFN-y (100 U/ml). 12 hours 0 hours 0.157 0.050 H L A - D R band density 0.296 0.316 B-actin band density 0.530 0.158 H L A - D R density /B-actin density 3.5 Corrected H L A - D R density/ 1 basal H L A - D R mRNA expression at time zero  52  — Anti-HLA-DR Control  0  10  20  30  40  50  60  Time (hours)  Figure 7. Relative flourescence intensity of THP-1 cells stained with anti-HLA-DR and anti-control antibodies following treatment with IFN-y (100 U/ml). The peak is seen at 36 hours after treatment. The values reported are the mean flourescence intensity and standard deviation for 5000 THP-1 cells per treatment point (n=l).  53  3.2.3. Determination of the sensitivity of the TNF-a enzyme-linked immunosorbance assay (ELISA). TNF-a expression in the THP-1 and donor T cell co-culture was determined by an enzyme-linked immunosorbance assay as described in Materials and Methods. A standard curve between 15 pg to 2000 pg/ml of TNF-a was used for each plate (n=6) and the optical density was determined for each standard as seen in Figure 8. The lowest detectable amount of T N F - a was determined to be 125 pg/ml using one-way analysis of variance (p < 0.0001) of the log transformed optical density readings at 490 nm. This sensitivity limit was determined as the TNF-a concentration with the highest O.D. 490 nm value that did not statistically differ from the O.D. 490nm values of all consecutive lower TNF-a concentrations. A Tukey-Kramer Multiple Comparisons test showed that a series of standards from 125 pg/ml to 15 pg/ml TNFa did not significantly differ from each other (P>0.05). A Bartlett analysis of the standard deviations (SDs) showed no significant difference among the standard deviations of the log transformed standards (p= 0.9999).  3.2.4. Release of TNF-a from THP-1 and T cell co-cultures treated with TSST-1 and IFN-y.  In preliminary experiments, a dose of 1 ng/ml TSST-1 ( M N 8) was optimized and shown to induce the release of TNF-a from the co-cultures at 12, 36, 60 and 84 hours after treatment. A l l THP-1 and monocyte-depleted T cell co-cultures treated with TSST-1 (1 ng/ml)  54  2.5  2.0H  1  1  1  1  1  1  1  1  0  250  500  750  1000  1250  1500  1750  i  i  2000  2250  Cone. TNF-a (pg/ml)  Figure 8. A standard curve for the determination of TNF-a from O.D. 490 nm readings. O.D. 490 nm readings were taken from 6 separate ELISA plates. The lowest sensitivity was determined by A N O V A to be 125 pg/ml TNF-a (PO.0001, n=6). Values are reported as means and S E M from 6 separate ELISA plates.  55  •  TSST-1  •  TSST-1 + IFN-y  O)  a  u c o o  Time (hours)  Figure 9. The release of TNF-a from THP-1 and T cell co-cultures treated with TSST-1 (1 ng/ml) in the presence or absence of IFN-y (100 U/ml). TNF-a release into the supernatant was measured by ELISA and was maximal 36 hours after treatment with TSST-1 in the presence or absence of IFN-y. The reported values are the means and the S E M of 3 independent experiments. The sensitivity of the ELISA was 125 pg/ml TNF-a.  56  released TNF-a at levels above the limits of detection of the ELISA (Figure 9, Figure 10) (n=3). The release of TNF-a from T cells and THP-1 cells was evident between 12 and 84 hours of TSST-1 treatment and peaked at 36 hours following treatment with TSST-1 (Figure 9, Figure 10). By observing the TNF-a release profile it appears that exogenous IFN-y was not required for TSST-1-induced TNF-a release (Figure 9) (n=3). THP-1: T cell co-cultures were unable to release measurable levels of T N F - a (<125 pg/ml) at 12, 26, 60 or 84 hours after treatment with RPMI 1640 alone without TSST-1 (data not shown, n=3). These co-cultures were also unable to release measurable levels of TNF-a into the culture supernatant at these time points following treatment with IFN-y (100 U/ml) alone (data not shown, n=3). Reported values are the means and SEMs of 3 independent experiments.  3.2.5. The effect of 1 ng/ml TSST-1 and 100 U/ml IFN-y on TNF-a release from donor T cells.  Monocyte-depleted donor T cells were unable to release TNF-a into the supernatant at levels above the limit of detection (TNF-a < 125 pg/ml) in the presence of TSST-1 (1 ng/ml) at 12, 36, 60 and 84 hours (Figure 10) (n=3). Similarly, co-treatment of T cells alone with both TSST-1 (1 ng/ml) and IFN-y (100 U/ml) did not induce the release of measurable levels of TNF-a into the culture media (Figure 10). In addition, donor T cells did not induce the release of detectable levels of TNF-a when treated with either IFN-y alone (100 U/ml) or RPMI 1640 at 12, 36, 64 or 84 hours (n=3). Reported values are the means and SEMs of 3 independent experiments.  57  • T Cells alone I THP-1 alone  5000-.  ITHP-1/TCelfe 4000-  O)  3000-  a  o  2000-  c o o  J  i  _IZ£L_  12  60  36  84  Time (hours)  Figure 10. The release of TNF-a from cultures treated with TSST-1 (ng/ml). T N F - a release from T cells alone, THP-1 cells alone and co-cultures of THP-1/T cells treated with TSST-1 was measured by ELISA. The values reported are the means and SEM of 3 independent experiments. The sensitivity of the ELISA was 125 pg/ml TNF-a.  58  A. Time (hours) LPS LPS/PMB  12 1.1 ± 0.3 0.3 ± 0.1 2.110.0 0.5 ± 0 . 0  LPS/IFN-y LPS/IFN-y/ PMB (TNF-a levels as 10 pg/ml)  36 <125 <125 5.4 ± 2 . 6 0.2 ± 0 . 1  84 <125 <125 3.8 ± 2 . 7 0.3 ± 0.3  60 <125 <125 7.5 ± 5 . 7 0.7 ± 0.6  3  B. 15000i  LPS LPS/PMB  O) Q.  r  o i ooo-  LPS/IFN-y LPS/IFN-y/PMB  u c o o  5000  0  J3J  12  l  36  60  84  Time (hours) Figure 11. The release of TNF-a from THP-1 cells treated with LPS (1 ug/ml)in the presence or absence of PMB (10 ug/ml) and IFN-y (100 U/ml). A. and B. TNF-a release from THP-1 cells treated with LPS was measured by ELISA and occurred within 12 hours after treatment. The values reported are the means and SEM of 3 independent experiments. The sensitivity of the assay was determined to be 125 pg/ml TNF-a.  59  3.2.6. The effect of 1 ng/ml TSST-1 and 100 U/ml IFN-y on TNF-a release from THP-1 cells.  THP-1 cells treated alone with 1 ng/ml TSST-1 (MN8) did not release measurable levels of TNF-a (< 125 pg/ml) into the culture supernatant at 12, 36, 60 or 84 hours (Figure 10) (n=3). TNF- a release at these same time points was below the level of sensitivity of this test (<125 pg/ml TNF-a) when THP-1 cells were treated with 100 U/ml INF-y in the presence or absence of 1 ng/ml TSST-1. At 12, 36, 60 and 84 hours, THP-1 cells in RPMI 1640 did not release measurable levels of TNF-a into the culture supernatant. The results shown are the means and SEMs for 3 independent experiments.  3.2.7. TNF-a release from THP-1 cells treated with LPS and IFN-y. LPS (1 ug/ml) induced the transient release of T N F - a (1.1 ± 0.3) x 10 pg/ml from THP3  1 cells within 12 hours of treatment (Figure 11) (n=3). This LPS-induced TNF-a release was decreased, at 12 hours, in the presence of the LPS inactivating molecule P M B (10 ug/ml) to (2.5 ± 1.4) x 10 pg/ml of T N F - a (Figure 11). At later time points, T N F - a was not measurable (< 125 pg/ml TNF-a) in the cell culture supernatant of LPS-treated THP-1 cells. Upon co-treatment with LPS (1 ug/ml) and IFN-y (100 U/ml), THP-1 cells released sustained levels of TNF-a into the cell culture supernatant (Figure 11). At 36 hours following treatment with 1 ug/ml LPS and 100 U/ml IFN-y, the THP-1 cell culture supernatant contained (5.4 ± 2.6) x 10 pg/ml of TNF-a. This release of TNF-a was abrogated by co-treatment of cells 3  with P M B .  3.3 Discussion. This series of experiments shows that TSST-1-induced TNF-a release requires the presence of both THP-1 cells and donor T cells in co-culture (Figure 10, Figure 12). The A P C 60  TSST-l-T cell model contrasts to the LPS model of TNF-a release that only requires the presence of THP-1 cells (Figure 2, Figure 12). These new experimental results agree with previously published data which show that TSST-1 mediated TNF-a release required the presence of both donor monocytes and T cells. In contrast, multiple workers have shown that LPS induces TNF-a release from monocytic cells alone (36,67) and no other factors are required for the LPS-induced release of TNF-a from monocytic cells (36,67). The most important factor involved in TSST-1 mediated TNF-a release appears to be the interaction of the H L A - D R molecule with the VB2 TCR. Previous work in our laboratory indicates that in P B M C culture, mutations of either the TSST-1 M H C II-binding domain (213) or the TSST-1 TCR-binding domain (45) induced significantly lower levels of TNF-a release than wild type TSST-1. This series of experiments also indicated that THP-1 cells express enough constitutive H L A - D R to bind and present TSST-1 to T cells in a manner that induces TNF-a release (Figure 9). IFN-y will not be required to pre-activate THP-1 cells and increase plasma membrane levels of H L A - D R in future TSST-1 studies. The binding of TSST-1 to H L A - D R has been proposed to be the initial step in the bilateral engagement of TSST-1 to both APCs and T cells (58,214-217). However, there were initial concerns that THP-1 cells do not express enough constitutive H L A - D R molecules on the cell membrane to bind TSST-1 and present it to T cells (186). These concerns were also based on preliminary experiments by Al-Daccak indicated that H L A - D R levels on THP-1 cells were too low to bind to a M . arthritidis-derived SAg (MAM) (60). In the current study, preliminary experiments showed that THP-1 cells treated with IFN-y (100 U/ml) expressed increased H L A DR mRNA and cell surface protein levels within 12 hours (Figure 6, Figure 7). Furthermore, the peak levels of THP-1 H L A - D R expression occurred at 36 hours after treatment with IFN-y  61  LPS  TSST-  LPS + IFN-y  No TNF-a Release  4  TSST-1 +IFN-Y  TNF-a  THP-1 Cell  Figure 12. A summary of LPS, TSST-1 and IFN-y-induced events in THP-1 cells alone. TSST1 alone or in co-treatment with IFN-y cannot induce the release of TNF-a from THP-1 cells. In contrast, LPS is sufficient to induce the transient release of T N F - a from THP-1 cells. This LPS-induced T N F - a release can be sustained with exogenous IFN-y.  62  (Figure 7). These results agree with the published data which showed that IFN-y upregulates H L A - D R cell membrane protein levels on THP-1 cells within 12 hours of treatment (206). Our experimental results show that IFN-y co-treatment with TSST-1 was not required for TNF-a release from co-cultures (Figure 12). Exogenous IFN-y was not required, because THP-1 cells may express enough cell surface H L A - D R to bind to TSST-1. Another explanation could be that T cells in co-culture produce endogenous IFN-y that upregulates H L A - D R on THP-1 cells to levels that bind TSST-1 (218). By not using exogenous IFN-y in this model, we avoid potentially confounding variables in future signal transduction studies. For example, IFNy activates the J A K - S T A T pathway (219,220), the M A P kinase pathway (125) and late-acting P K C isoforms (187). In comparison to TSST-1-induced TNF-a release, IFN-y markedly enhances LPS-induced T N F - a release. Although TSST-1 mediated TNF-a release in a THP-1 and donor T cell model was detectable at 12 hours, these levels of cytokine decreased at later time points (i.e. 60 and 84 hours) (Figure 9, Figure 10). This TNF-a released in the culture supernatant could be degraded in the culture media or could become bound to soluble (221) and membrane-associated T N F receptors (TNF-R) (185,222) on either T cells or THP-1 cells (185,221). The scavenging of free TNF-a from cells that had previously released this cytokine was shown by Leeuwenberg and co-workers (221) and is probably involved in a TNF-a feedback inhibition (185,222). Multiple isoforms (30, 40, 44, 75 kDa) of soluble TNF-R are released within 48 hours by human P B M C s (221) and by THP-1 cells induced to release TNF-a (185). The binding of TNF-a to receptors and high occupancy of these receptors could prevent the activation of the transcriptional enhancer N F - K B in THP-1 cells and induce feedback transcription (83,183,208,222).  63  inhibition of cytokine mRNA  In comparison to the TSST-1 mediated results, the LPS-induced release of TNF-a from THP-1 cells does not require the presence of T cells or other T cell factors (Figure 11, Figure 12). However, this LPS-induced TNF-a release from THP-1 cells is transient and is not measurable at time points after 12 hours and could be the result of the LPS degradation or binding to TNF-R (185,221,222) (Figure 11). We are confident that this TNF-a release is due to LPS-binding to THP-1 cells because the co-treatment of THP-1 with LPS and an LPS inactivator, P M B markedly lowered levels of  TNF-a  release.  The relatively high  concentrations of LPS required for TNF-a release are probably due to the fact that THP-1 cells express very low levels of CD 14 (82) (Figure 2). In this undifferentiated THP-1 cell model, non-CD 14 low affinity LPS-receptors may bind LPS and induce TNF-a release through a wide variety of signal transdcution cascades (75,81,82) (Figure 2). In comparison to TSST-1-induced TNF-a release profiles, IFN-y significantly altered the LPS-induced TNF-a release profile (Figure 11, Figure 12). Co-treatment of THP-1 cells with LPS and exogenous IFN-y induces a sustained release of TNF-a that is measurable 84 hours after initial treatment (Figure 11, Figure 12). IFN-y may be increasing the level of secreted TNF-a by either sustaining the release of TNF-a or inhibiting the binding, uptake or degradation of TNF-a from the culture supernatant. Several workers have shown that THP-1 cells treated with LPS and IFN-y utilize signal transduction cascades that differ from cells treated with LPS alone. For example, LPS and IFN-y co-pre-treatment of THP-1 cells has a synergistic effect on 3'-0-(4-benzoyl) benzoyl (BZ) A T P activation of phospholipase D when compared to LPS and IFN-y pre-treatments alone (223). Although these pathways may not play roles in TNF-a release, co-treatments of THP-1 cells with LPS and IFN-y can apparently create signals within these cells which differ from signals produced solely by LPS or IFN-y. These  64  modified signal transduction cascades could then affect the level of proteins expressed by THP1 cells. Cramer and co-workers showed that treatment of THP-1 with both IFN-y and LPS increased the levels of mRNA of transporter associated proteins TAP-1 and TAP-2 when compared to levels in cells treated with LPS and IFN-y alone (212). The TSST-1-induced release of TNF-a from APC-T cell co-cultures requires the interaction of multiple factors other than the binding of TSST-1 to both H L A - D R and T C R (45,213). These factors include the interaction of cell adhesion molecules on both cells (53,224) and cytokine signalling between affected cells (61). The interaction of co-stimulatory molecules on THP-1 cells with those on human donor T cells probably plays an important role in TSST-1 mediated TNF-a release. Variations in the level of these molecules on THP-1 cells may affect the level of TNF-a released when compared to donor T cell cultures (36). Reduction of costimulatory molecule interactions with antibodies reduces the level of TNF-a released in cocultures treated with TSST-1 (36) and PBMCs treated with staphyloccocal enterotoxin B (SEB) (224). Krakauer and co-workers showed that antibodies against CD2, CD 11a, CD 18, ICAM-1 and CD28 decreased TNF-a and IFN-y release in human peripheral blood cells treated with SEB (224). In vivo CD28 double mutant (-/-) female C57BL/6 (H-2b) mice presensitized with D-galactodosidase were completely resistant to TSST-1 over an 80 hour period when compared to CD28 wild type (+/+) mice (53). This THP-1 and T cell model of TSST-1-induced TNF-a release has several weaknesses that should be addressed in future experiments. Unlike the LPS-induced TNF-a release from THP-1 cells, the cellular origins of TSST-1-induced TNF-a are unknown. Theoretically, T N F - a could be expressed by both THP-1 cells (67,185,225) and donor T cells (38). T N F - a is released by THP-1 cells treated with a variety of agents including 1-a, 25 dihydroxy vitamin D3  65  (l,25(OH2)D3) (225), LPS (67) and P M A (185). Recent work suggests that T cells are a minor source of both TNF-a and TNF-P in human peripheral blood mononuclear cells treated with TSST-1. Akatsuka and co-workers induced the upregulation of TNF-a and TNF-P mRNA in CD4+ and CD8+ lymphoblasts treated with TSST-1 and separated by flow-cytometry from H L A - D R transfected L cells (38). This increase in TNF-a and TNF-P mRNA levels correlated with an increased expression of low levels of TNF-a and TNF-P (1000 pg/ml) (38). In comparison, the flow-cytometry purified L cells did not show increases in TNF-a or TNF-P mRNA levels following treatment with TSST-1 or lymphoblasts (38). The low concentrations of released TNF-a from TSST-1-treated T cells indicate that T cells are secondary source of TNF-a in TSST-1 treated peripheral blood. The utilization of donor T cells also introduces other problems. The level of Vp2 molecules (226-228), co-stimulatory molecules and cytokine expression (33) may vary between samples of donor T cells. Density centrifugation-purified donor T cells may also contain small numbers of monocytes and B cells (229) that express HLA-isoforms. These contaminating APCs could complicate both cytokine and signal transduction studies by presenting TSST-1 to donor T cells. There was also some concern that the treatment of these residual-contaminating APCs with IFN-y could upregulate H L A - D R levels to levels that could cause TSST-1-induced TNF-a release. In an attempt to address these issues, monocyte-depleted T cells were treated with TSST-1 in the presence or absence of IFN-y (Figure 10). TSST-1 was unable to induce the release of measurable levels of TNF-a from monocyte-depleted T cells in the presence or absence of IFN-y (Figure 10). The lack of measurable levels of TNF-a release indicate that there were not enough H L A - D R expressing donor monocytes and B cells available to effectively present TSST-1 to donor T cells and induce TNF-a release (Figure 6). Furthermore,  66  IFN-y did not upregulate H L A - D R on APCs to levels that could induce TNF-a release by presenting TSST-1 to T cells (218). The utilization of human or animal T cell lines or donor T cells (230) in future experiments should help determine the cellular nature of TNF-a in this model. There has been an ongoing debate in the literature about the ability of TSST-1 to induce TNF-a release from monocytic cells. In the past, some researchers have suggested that TSST-1 can induce TNF-a expression in monocytic cells alone (231) while others have proposed that this expression requires the presence of APCs and T cells (36). Work by Espel and co-workers suggested that TSST-1 could induce increases in TNF-a mRNA in donor monocytes alone (231). In our study, THP-1 cells alone treated with TSST-1 were unable to release measurable levels of TNF-a within 12, 26, 60 or 84 hours of treatment (Figure 10). This new data agrees with previous results in our laboratory which have shown that donor monocytes treated with TSST-1 did not release measurable levels of TNF-a into the culture supernantant (36). The discrepancy between our data and the previously published work is most likely due to several factors including the purity of human monocytes, the source and purity of TSST-1 preparations. See and co-workers showed that commercially available TSST-1 contains impurities such as staphylococcal lipase which may induce T N F - a release from monocytic cells (169). It is also possible that increased levels of TSST-1-induced TNF-a mRNA were not translated into TNFa protein or that increased levels of translated TNF-a protein were not released from THP-1 cells. Finally, the purified donor monocytes used in earlier published work also contain low levels of T cells that may be just high enough to bind to SAg-APC complexes and induce TNFa release (36).  67  In a related debate, several workers have suggested that the T cell component of the APC-SAg-T cell model could be replaced with either conditioned T cell media or IFN-y (232). These workers implied that T cells are required to provide soluble factors that act in concert with TSST-1 or other SAgs to induce TNF-a release from monocytes. Our studies showed that THP-1 cells treated with TSST-1 and IFN-y were unable to release measurable levels of T N F - a into the supernatant (< 125 pg/ml) (Figure 10). This new data indicates that the T cell component of the APC-Sag-T cell model probably contributes factors that are more important than IFN-y in inducing TNF-a release from monocytes (232). These other factors could include SAg-TCR interactions with MHCII (45), accessory molecule interactions between T cells and APCs (43) and intercommunication by means of non-IFN-y cytokines (61). The treatment of THP-1 cells alone with TSST-1 controlled for the possibility of LPS contamination of either the cell cultures or toxin preparations. Previous workers have shown that the LPS-induced T N F - a release from THP-1 cells does not require the presence of T cells (67). Controlling for LPS contamination of TSST-1 treated THP-1 cells is important, because it is now known that TSST-1 can potentiate the in vivo effect of LPS in mice. For example, the co-injection of LPS (75 ug/mouse) and TSST-1 (15 ug/mouse) into B A L B / c mice produces levels of serum TNF-a and IFN-y that are higher than those induced by TSST-1 or LPS alone (233). This published work indicates that the presence of active LPS and TSST-1 in THP-1 cell cultures could lead to misleading results and interpretations. It was also important to determine the unregulated release of TNF-a from untreated THP-1 cells, monocyte depleted cells or co-cultures. Constitutive levels of TNF-a release from cell cultures can give one clues to the basal level of cytokine release as well as any pre-existing conditions that may be activating these cells (168). The biggest concern to us was that these cultures may contain low levels of the endotoxin that could synergistically act with TSST-1 to 68  induce cytokine release (97). The failure to measure free supernatant TNF-a in THP-1 cell culture, monocyte-depleted T cell culture and THP-l/T cell co-cultures treated with P M B indicated that there was no physiologically relevant LPS contamination as assessed by TNF-a release. This new data also indicated that these cells were not activated to induce T N F - a release by handling or manipulation prior to the addition of toxins. One major drawback of our study is that we did not look at the mRNA level of T N F - a in the T cell-THP-1 cell model treated with TSST-1 or the THP-1 cultures treated with LPS. During the planning of this research project, it was decided to address the release of cytokines instead of TNF-a mRNA expression for several reasons. The release of cytokines and their measurement with ELISA is more quantitative than the measurement of mRNA levels obtained by semi-quantatative RT-PCR assays (33). We were confident that TNF-a release reflected transcriptional upregulation and was not simply the result of increased processing and release of stored pro-TNF trimers or the translation of stored mRNA products.  Other TNF-a release  models show that LPS induces the upregulation of pro-TNF-a homotrimers in THP-1 cells within two hours of treatment with LPS (234). These 26 kDa pro-TNF-trimers are transported to the cell membrane of monocytic cells and have been shown to reduce TNF-a release from THP-1 cell following treatment with LPS (22). It is also unlikely that these upregulated levels of TNF-a proteins arise from stored mRNA, because TNF-a mRNA has a half-life of 25 minutes (231) and LPS treatment of monocytic cells activated TNF transcription factors (137). In conclusion, this series of experiments established a THP-1 and T cell model that could be used to study TSST-1 mediated signalling in THP-1 cells. This T cell-SAg-THP-1 cell model required the presence of both monocytic cells and T cells for TSST-1 mediated T N F - a release (Figure 10, Figure 12). The TSST-1-induced TNF-a release from co-cultures does not  69  require co-treatment with exogenous IFN-y to upregulate H L A - D R to levels that can present TSST-1 to T cells, thereby inducing TNF-a release. In comparison, LPS was sufficient to induce a transient release of TNF-a from THP-1 cells without the addition of either T cells or exogenous IFN-y. This LPS-induced TNF-a could be sustained by co-treating the THP-1 cells with both LPS and IFN-y. In the future, the study of the role that TSST-1 plays in cytokine release and cell signalling will be simplified by avoiding the addition of exogenous IFN-y. Hopefully, future work will also include a determination of the origin of TNF-a release in this system and the effect of TSST-1 on cytokine gene transcription and upregulation.  70  Chapter 4. The Effect of TSST-1 on Mitogen-Activated Protein Kinases in THP-1 Cells. 4.1. Introduction. This series of experiments attempted to determine the effect of TSST-1 on M A P kinase activity in THP-1 cells prior to the recruitment of T cells. The binding of TSST-1 to H L A - D R is the first step in TSST-1-mediated pathogenesis (214) and may induce signals that prepare, or prime, the THP-1 cell for its interaction with T cells (36). These priming activities may include the control of the THP-1 cell cycle (106), activation of ion channel activity (151,211) into the cytoplasm, the expression of co-stimulatory molecules on the cell membrane (235) and the preparation for release of co-stimulatory cytokines (61,236). With a better understanding of the TSST-1 induced priming signals in THP-1 cell culture, it will be easier to study signal transduction events in T cell-THP-1 co-cultures. There are a wide variety of intracellular signals that are activated by the binding of TSST-1 and related superantigens (Table II) to monocytic cells (28,173,182,211,236). Some of these signals such as P K C activation (168,173), tyrosine phosphorylation (196) and Ca i-fluxes 2+  (114,127,155) have been implicated in pathways upstream of ERK-1 and ERK-2. For example, TSST-1 induces the early activation of P K C in THP-1 cells (182) and this activation may control downstream E R K activity (152,153). Other researchers have shown that both SEB and SEE induce the activation of non-receptor protein-tyrosine kinases (PTKs) of the Src family in T cells (171). These Src-related kinases have also been implicated in M H C Il-mediated signalling (172) and influence the Ras-»ERK pathway (101,136). TSST-1 also activates pathways which have been shown to be downstream of ERK-1 and ERK-2. For example, TSST-1 treatment of THP-1 cells induces binding of AP-1 transcription enhancers N F - K B (175) 71  and the transcriptional promoter factor activator protein (AP-1) to D N A (183). A component of AP-1 in T cells, the nuclear factor of activated T cells (NF-AT),  has been shown to be a  downstream target of ERK-1/2 (156). G31Rmut TSST-1 was chosen as a comparison to TSST-1 induced ERK-1/2 signalling, because it binds to PBMCs with less affinity than TSST-1 (44). This lowered binding affinity is due to a single amino acid substitution in the H L A - D R binding domain of TSST-1 resulting in lower levels of TNF-a and T cell mitogenesis in P B M C culture (Figure 12) (44). It is possible that these differences in the physiological effects of G31Rmut TSST-1 and TSST-1 treatments correlate to differences in M A P kinase signalling in monocytic cells. LPS was used as a comparison to TSST-1 mediated signalling for two main reasons. First, we showed earlier (see Chapter 3) that LPS induces TNF-a release from THP-1 cells alone, while TSST-1-induced TNF-a release requires the presence of THP-1 cells and T cells (Figure 12). These differential requirements for TNF-a release probably involve differences in LPS and TSST-1-mediated signalling in THP-1 cells. Second, LPS induces signals through multiple upstream activators of ERK-1 and ERK-2 in human and animal monocytic cells (67,88,93). These upstream pathways include non-receptor protein-tyrosine kinases such as Src (165), Raf->ERK control pathways (101,136) and PI-3 kinases (165) which have been implicated as possible upstream activators of E R K kinases (115,149,150). Both LPS and TSST1 induce similar non-MAP kinase pathways that have been implicated in pathways upstream or downstream of ERK-1/2. Specifically, both LPS (154) and TSST-1 induce the activation of P K C isoforms (168,173), the Src family of protein-tyrosine kinases (165,171) and the downstream transcriptional enhancer N F - K B (137,175).  72  Specific Aims 1) To screen THP-1 cells for TSST-1 mediated early activation of M A P kinases in THP-1 cells prior to the recruitment of T cells. 2) To compare the effect of TSST-1, G31Rmut TSST-1 and LPS on early ERK-1 and ERK-2 activation and deactivation profiles in THP-1 cells. 4.2. Results. 4.2.1. The effect of P M A on M B P kinase activity in THP-1 cells.  The effect of P M A on THP-1 M A P kinase activity was analyzed by the M B P phosphotransferase assay of Bio-Scale Q eluent fractions. These results revealed the presence of three peaks of M B P kinase activity (Figure 13). The first peak between fractions 0 and 12 was referred to as Peak I (Figure 13). Peak II eluted in Bioscale-Q fractions 15 to 25 and expressed the highest levels of M B P kinase activity in fractions from both P M A and untreated cells (Figure 13). The basal level of Peak II M B P kinase activity in THP-1 cells was determined to be 7.54 ± 0.48 x 10 pmol-min'-ml" at two minutes. Within 5 minutes, P M A induced a 2-fold 5  1  increase, 1.56 ± 0.37 x 10 pmol-mhf'mr , in M B P kinase activity over basal activity (Figure 6  1  13). The third peak M B P kinase activity occurred between fractions 28 and 35 and was referred to as Peak III. M B P kinase activity results are reported as the means and standard errors of the means (SEM) from 3 independent experiments.  4.2.2. The effect of TSST-1 on M B P kinase activity in THP-1 cells.  TSST-1 (1 ng/ml) induced a 2-fold increase in Peak II M B P kinase activity, from basal levels of 7.54 ± 0.48 x 10 pmol-min'-ml" to 1.42 ± 0.12 x 10 pmol-mm'-ml" at 30 minutes 5  1  6  1  (Figure 14). Over a 30 minute period, TSST-1 induced a gradual increase in M B P kinase 73  activity. At 5 minutes following treatment with TSST-1, Peak II M B P kinase activity increased to 9.69 ± 1.85 x 10 pmol-min" mr (Figure 14). At 15 minutes following treatments, the level 5  1  1  of M B P kinase activity was determined to be 1.29 ± 0 . 4 1 x 10 pmole-min'-mr . There was 6  1  little noticeable change in the M B P kinase activities of Peaks I and Peaks III following TSST-1 treatment (Figure 14). The M B P kinase activities reported are means and S E M of 3 independent experiments.  4.2.3. The phosphorylation of ERK-1 and ERK-2 in M B P kinase Peaks I, II and III.  Fractions corresponding to Peaks I, II and III from cells treated with P M A (1 n M , 2 minutes), TSST-1 (1 ng/ml, 30 minutes) or RPMI-1640 medium alone were analyzed for the presence of both ERK-1 and ERK-2 by immunoblotting with anti-ERK-1/2 antibodies. Examination of Figure 15 shows that Peaks I, II and III from each treatment contained roughly the same levels of ERK-1/2  (Figure 15). The largest levels of ERK-1/2  specific  phosphotyrosine staining were seen in fractions corresponding to Peak I in all groups (Figure 16, Figure 17). ERK-1 and ERK-2 bands showed only weak or no tyrosine phosphorylation in Peak II and Peak III which was not increased following treatment with P M A or TSST-1 (Figure 16, Figure 17). Densitometry of ERK-1/2 specific phosphotyrosine staining (4G10) intensity corrected for ERK-1/2 protein concentration showed that both P M A (2 minutes) and TSST-1 (30 minutes) induced 2-fold increases over RPMI 1640 treated basal levels in Peak I (Figure 17). The densitometry results are expressed as the means and S E M of three separate experiments.  4.2.4. The effects of either P M A or TSST-1 on immunoprecipitated ERK-1/2 activity.  74  The effects of P M A or TSST-1 treatment on ERK-1/2 activity were examined for M B P phosphotransferase activity in fraction eluents following immunoprecipitation with anti-ERK1/2 antibody. It was observed that both P M A (2 minutes) and TSST-1 (30 minutes) induced a 40% increase in Peak I ERK-1/2 activity compared to RPMI 1640 treated basal levels (Figure 18) . In Peak II (Figure 18), there was a 23% increase in relative M A P K activity in THP-1 cells following treatment with 1 n M P M A (2 minutes) (Figure 18). In contrast, there was little detectable increase in Peak II M A P K activity from cells treated with TSST-1. For Peak III, both P M A and TSST-1 induced a 50% decrease in relative M A P kinase activity when compared to RPMI 1640 treated basal M A P kinase activity (Figure 18). The M B P kinase results were expressed as the mean and S E M of three separate experiments. 4.2.5. Anti-phosphotyrosine blotting of THP-1 whole cell lysates treated with TSST-1, G31Rmut TSST-1, LPS and RPMI-1640.  Anti-phosphotyrosine staining of proteins from total cell lysates showed that LPS (1 ug/ml) (Figure 19) was unable to induce any noticeable change in protein tyrosine phosphorylation within the 40 kDa to 44 kDa range in THP-1 cells when compared to RPMI 1640 treated control cells (n=5) (Figure 19). When compared to RPMI 1640 treatments, LPS did not induce any other protein tyrosine phosphorylation patterns in THP-1 cells (n=5) (Figure 19) . Treatment of THP-1 cells with TSST-1 and G31Rmut TSST-1 (Figure 20) also did not induce any change in early tyrosine phosphorylation within the 40 kDA to 44 kDa range when compared to RPMI 1640 treatments (Figure 20). TSST-1 and G31Rmut TSST-1 did not induce any noticeable change in protein tyrosine phosphorylation, within 30 minutes, of other proteins when compared to RPMI 1640 treatments (n=5) (Figure 20).  75  Fraction #  Figure 13. Activation of M B P kinases in P M A treated cells. M B P kinase activity in Peak II increased 2-fold over basal activity in THP-1 after 5 minutes of treatment. There was little noticeable increase in Peak I or Peak III activities at 2 or 5 minutes. The reported values are the means and S E M of 3 independent experiments.  76  2.0  Basal - A - 5 minutes 15 minutes -•-30 minutes  1.5  1.0  0.5  10  20  30  40  Fraction #  Figure 14. Activation of MBP kinases by TSST-1. Wild type TSST-1 induced a 2-fold increase over basal levels in peak II after 30 minutes of treatment. There was little increase in Peak I and a slight increase in Peak III within this time period. The values reported are the means and SEM of 3 independent experiments.  77  CN  CM  •  n  ON  H 60  H  < OH  — i c rn O  CN i—i  CN  c  o  H  - - H  c  u  E  €  u H  u, c  u  CD  cd  u w H  c  03  | ^  t-7-  oc  78  —i  CN  i  H OQ  2 Pi £ 6  OQ  •a cu  y  PH CD  2 S C+H  i—i  O  H  •-> ra 2  ra  so  ° H  f—'  ^ H  hH  H ^ ^ H  PC O  IS (?  i—i  P H,  d w so —  ,  §  , "r! -3 S  c 3  M  H  cu ^5  ^ P H  cu  PH  ra « M  3 —i d  ra  ° 8"  CO  O Tf  ^  2 ©  1  c  CO cu  C  o ra ra hJ  PH  cu  cu  m o CN  cn —•  CN  T-H  oo  79  3  2 * ra  ra Q  5 ^ ra  i  £  p a so ra  PH  cu  CD  d P _H ra T > — ' H b w  £  2  <! cu  J  .s s0H  .SP €  LU  d .2  SO  CO  *"f  o ^ ra o ^ cu wO  CU  CU  _5  CN  °d ^ § Q  CU  H  d  CO  V  OH  3 ^ <  oo § c .5  CU •*-> cu  IH  ^ a  O  cn  CU  PH  CU  0)  i—i  2  rn r- c cu M CD  SO  *-  'O  I&i  PH  *£>  •£ S & x  I—1 & ^ OH  PH  a  £3  7 s  cu J9 <  O  . 05  cu  u  2  S3 P H  GO  ~  ^  Tf  1  OS  w  o t  cu  "aj  H as cu d ra _  cu  d  '3 ra PH  ra cu  — 1ft  '"g  • PMA •TSST-1  o Ti-  to  a. a. i_  >» iCN  A UJ  Peak  Figure 17. ERK-1/2 specific phosphotyrosine staining in Peak I, II and III eluent fractions following treatment of THP-1 cells with TSST-1 and P M A . Densities of phosphotyrosine staining were corrected for ERK-1/2 staining intensity and expressed as a ratio over basal activity following RPMI 1640 treatment. Peak I shows a 2-fold increase in ERK-1/2 specific phosphotyrosine staining intensity after treatment with either P M A or TSST-1. Both Peaks II and III show little increase in ERK-1/2 specific phosphotyrosine staining after treatment with TSST-1 or P M A . The values reported are the means and S E M of 3 independent experiments.  80  2i  PMA treatment for 2 min.  •>  o  TSST-1 treatment for 30 min.  CO o  CD  o. 0. > o CO CN  or UJ  Peaks  Figure 18. Relative ERK-1/2 activity in THP-1 cells after treatment with TSST-1 and P M A . ERK-1/2 were co-precipitated from Peaks I, II and III from eluents of TSST-1, P M A and RPMI 1640 basal treatments. ERK-1/2 activity was measured by M B P kinase assays of the antiERK-1/2 reactive immunoprecipitates and expressed as a ratio over basal activity. The largest increase in levels of TSST-1 and PMA-induced ERK-1/2 activity were measured in Peak I, with little change in Peaks II and markedly decreased ERK-1/2 activity in Peak III compared to RPMI 1640-treated basal levels. The reported values are the means and S E M of 3 separate experiments. 81  ON CD  NO  co  l-H  CD  21 3  |  T3  CN +H C  d  o «*>  o  3  o  cd C/3  11  PH HJ  o c NO  Q  PH  !-)  o •* NO  43  O "-  CN  CH Cfl  13&  1  Cd  ^  CD  u «c  2  |  d Cd oo . | M 3 CD B CM CD  J  13 CN ON  — cu  &  o oo  d  I i  O r-m  CO  S  §  2  co  o  J  1/1  la •—  CD  1  <+H  O CD _>  cd  52 g %  00  — ' CO  ^  i—1  OH fi  CO w  cd  .to  CD  a «  m so  CD  3  r-  'cu  J  3 §J 1 ,2? o ^ 'S 2  a C3 B P CD  JD  "o  CM MJ  1 CN c  O  co •  §  o  OH  . O  CD  CD  T5  '55  £  C cd o C  CD  CD  DO  CN rn  9  NO  CD  c cd  w  m  ^H  NO  o o •* CD  ^  CD  9 2 OH  co  -3 —  <  co  B § H  ON  K.  S  £ £  1  £ ^  1—1  NC  -1-  oc  m  CN  CD  CO  "CT  NJ  «  CD  W  '—'  d  1  HJ  cd  PH  CD * 5  tu 82  C  — CN CQ O  3£  u u c c i—5  cd —J  r—  s£  09 co  H  3  3  O  OO  cn 0 9 O H -a  r—H  | £ K  CN  c  co  .  co cu  CU  ^  •§ S 3  ^ -2 g s h B 'C .2 x g S 3 u a - O 3 cS m c2 S  .-3  U  Ib  r-H  Cd  b^  o  ^  ,!(*)&  CO  tu  "3 s cu 53 .2  f 1 ii  o o™ cu ' J 8 J  -i . 'f  /-N  3 g co c,u .g• i g o H g «n  CL,  0 0 CO ^ 3 CO ~ H ^  a c  •s bHi £ I - oico S  C&  cu  g . H '55 i—l o ^ CU  c u S  a cu C H  co  O  -3  H  W  cl  Tt  O  CU  M  co CU  3 3 3 - 3  „ ^  S3  C  co  © m  «  +H  *J  •=  CN  2  g  3  CU  sa  H cx —  3 CO —i CU  •-2 83  8 3  ^  2  111  • g  NO  ^ H  g  cd  T3  < £  tu tu  00  •O  cCO u^ a ^ .2  CO C £  g  4.2.6. Immunoprecipitated ERK-2 activity from ultracentrifuged cell lysates of THP-1 cells treated with either TSST-1, G31Rmut TSST-1, LPS or control medium.  ERK-2 specific kinase activities from control and treated cells were examined by immunoprecipitation of ultracentrifuged lysates followed by M B P kinase assays. TSST-1 induced both activation and deactivation of ERK-2 within 30 minutes when compared to baseline activity in RPMI-1640 control media (n=6) (Figure 21 A). Figure 21 A is a representative sample of the TSST-1 induced ERK-2 activation and deactivation seen in THP-1 cells in 6 independent experiments. The mean and S E M area under the TSST-1- induced E R K 2 activation curves (Figure 21 A , shaded areas) were calculated to be 829 ± 324 min»%ERK-2 activity (n=6). The mean and S E M area under the TSST-1- induced deactivation curves (Figure 21 A , unshaded areas) were calculated to be 226 ± 66 min*%ERK-2 activity (n=6). Furthermore, the median of the areas under the TSST-1-induced activation curve did not differ from the median of the areas under the TSST-1-induced deactivation curves. A Wilcoxin signed rank test comparing paired areas under the TSST-1-induced deactivation and activation curves gave a P value of 0.0938 (two-tailed, n=6). G31Rmut TSST-1 induced both ERK-2 activation (Figure 21 B, shaded areas) and deactivation (Figure 2IB, unshaded areas) when individual values from each experiment were analyzed (Figure 21 B). Figure 21 B is representative of 6 independent experiments. The mean and S E M area under G31Rmut TSST-1-induced activation curves (n=6) were calculated to be 888 ± 250 min* %ERK-2 activity (Figure 22). In comparison, the mean and S E M area under the G31Rmut TSST-1-induced deactivation curves (n=6) were calculated to be 107 ± 44 min*% ERK-2 activity (Figure 22). The median of the areas under the G31Rmut TSST-1-induced activation curves were significantly greater than the median of the areas under the G3 IRmut  84  I-  CD > CD  to  8 8>  I }  CD  PH  +-» cd  o S CO  2 cci  cd  pajoojjoo %  c3 £  cn E  o  2-*a3 pejoajjoo % cd  O CN  AljAIJOB pe»08JJOQ  4 .5 > >  %  X  C  CD >tn  85  CD J-H  1500  **  I TSST-1 I G31Rmut TSST-1 •LPS  1000  u CO CM  HI  CO CD  500-  0  -500  Activation  Deactivation ERK-2 activity  Figure 22. Areas beneath gross ERK-2 activation and ERK-2 deactivation curves. The areas under the curves were calculated as decribed in Materials and Methods. A Wilcoxin signed rank test found a significant difference between medians of areas under G3 IRmut TSST-1 induced ERK-2 activation and deactivation curves (P=0.0313, n=6). Differences between activation and deactivation areas under the curve following TSST-1 or LPS treatments were not statistically significant The values reported are the means and S E M from 6 independent experiments. 86  TSST-1-induced deactivation curves. A Wilcoxin signed rank test comparing paired values under the G31Rmut TSST-1-induced activation and deactivation curves gave a P value of 0.0313 (two-tailed, n=6). LPS-induced levels of mean ERK-2 activity can also be divided into distinct phases of activation (Figure 21 C, shaded areas) and deactivation (Figure 21 C, unshaded areas). The LPS-induced changes in ERK-2 activity seen in Figure 21 C are representative of 6 independent experiments (Figure 21 C). The mean and S E M area under the LPS-induced activation curves (n=6) were calculated to be 363 ± 94 min«%ERK-2 activity (Figure 22). In comparison, the mean and S E M area under the LPS-induced deactivation curves (n=6) were calculated to be 234 ± 98 min»%ERK-2 activity (Figure 22). The median of the areas under the LPS-induced ERK-2 activation curves did not differ from the median of the areas under the LPS-deactivation curves. A Wilcoxin signed rank test comparing paired values under the LPS-induced activation and deactivation curves gave a P value of 0.6875 (two-tailed, n=6). It was also decided to compare the effect of TSST-1, G31Rmut TSST-1 and LPS on the medians of areas under ERK-2 activation curves (Figure 21 A , B, C, shaded areas, Figure 22). There was no measurable difference between the medians of the areas under the ERK-2 activation curves for TSST-1, G31Rmut TSST-1 and LPS treatments. A Wilcoxin signed rank test between the medians of areas under TSST-1 and G31Rmut TSST-1-induced ERK-2 activation curves gave a P value of 0.8438 (two-tailed, n=6). The medians under the TSST-1 and LPS-induced ERK-2 activation curves were not significantly different and had a Wilcoxin signed rank P value of 0.5625 (two-tailed, n=6). Furthermore, a Wilcoxin signed rank test between the medians of areas under the G31Rmut TSST-1 and LPS-induced ERK-2 activation curves gave a P value of 0.0938 (two-tailed, n=6).  87  We also wanted to compare the effects of TSST-1, G31Rmut TSST-1 and LPS on the medians under the ERK-2 deactivation curves (Figure 21 A , B, C, unshaded areas, Figure 22). There was no significant difference between the medians of the areas under the ERK-2 deactivation curves for TSST-1, G31Rmut TSST-1 and LPS treatments (Figure 22). A Wilcoxin signed rank test between the medians of areas under TSST-1 and G31Rmut TSST-1induced ERK-2 deactivation curves gave a P value of 0.0938 (two-tailed, n=6). The medians under TSST-1 and LPS-induced ERK-2 deactivation curves were not significantly different and had a Wilcoxin signed rank P value of 1.000 (two-tailed, n=6). Finally, a Wilcoxin signed rank test between the medians of areas under the G31Rmut TSST-1 and LPS-induced ERK-2 deactivation curves gave a P value of 0.3125 (two-tailed, n=6).  4.2.7. Immunoprecipitated ERK-1 activity from ultracentifuged lysates of THP-1 cells treated with TSST-1, G31Rmut TSST-1, LPS and control media.  TSST-1 induced a pattern of  ERK-1 activation (Figure 23 A , shaded areas) and  deactivation phases (Figure 23 A , unshaded areas) when compared to RPMI 1640 control medium activity (Figure 23 A). The values in Figure 23 A are representative of 6 independent experiments. The mean and S E M area under the TSST-1-induced ERK-1 activation curves (Figure23 A , shaded areas) (n=6) were determined to be 288 ± 90 min»%ERK-l activity (Figure 24). In comparison, the mean and S E M area under the TSST-1-induced ERK-1 deactivation curves (Figure 23 A , unshaded areas) (n=6) were determined to be 170 ± 90 min»%ERK-l activity (Figure 24). There was no significant difference between the medians of paired areas under TSST-1-induced activation and deactivation curves. A Wilcoxin signed rank test between the medians of areas under paired TSST-1-induced activation and deactivation areas gave a P value of 0.6875 (two-tailed, n=6).  88  li  3 £  o  t/3  >  «  S  B ~  3  s PH Bi  Q. Q-  tn ai o  C  s  pa»oaiioo % c  I 0>  E  332 A4|A!J0B  PH  89  H  750-  •TSSI-1 •G31W1SST-1 •LPS  ">  VP  o ro •  250  UJ  r  X  (0 CD  -250-  ERK-1 activity  Figure 24. Areas beneath gross ERK-1 activation and ERK-1 deactivation curves. The areas under the ERK-1 curve were calculated as per the Materials and Methods. The areas are reported as the means and S E M of 6 independent experiments.  90  G31Rmut TSST-1 also induced ERK-1 activity profiles with distinct activation (Figure 23 B , shaded areas) and deactivation (Figure 23 B, unshaded areas) phases when compared to RPMI 1640 control medium activity (Figure 23 B). Figure 23 B shows G31Rmut TSST-1induced ERK-1 activation and deactivation profiles that are representative of 6 independent experiments. The mean and S E M area under the G31Rmut TSST-1-induced activation curves (n=6) were calculated as 424 ± 1 4 2 min«%ERK-l activity (Figure 24). In comparison, the mean and S E M area under the G31Rmut TSST-1-induced deactivation curves (n=6) were determined to be 129 ± 58 min»%ERK-l activity (Figure 24). There was no statistical difference between the medians of the areas under the paired G31Rmut TSST-1 activation and deactivation curves. A Wilcoxin signed rank test between paired G31Rmut TSST-1-induced activation and deactivation areas gave a P value of 0.2188 (two-tailed, n=6). LPS induced both the early activation (Figure 23 C, shaded areas) and deactivation (Figure 23 C, unshaded areas) of ERK-1 in THP-1 cells. The LPS-induced ERK-1 activation and deactivation profiles in Figure 23 C are representative of 6 independent experiments. The mean and S E M area under the LPS-induced gross activation curves (n=6) were calculated to be 298 ± 1 1 7 min«%ERK-l activity (Figure 24). In comparison, the mean and S E M area under the LPS-induced deactivation curves (n=6) were calculated to be 339 ± 128 min«%ERK-l activity (Figure 24). There was no significant difference between medians of paired areas under LPSinduced activation and deactivation curves because a Wilcoxin signed rank test between these values gave a P value of 0.6875 (two-tailed, n=6). It was also decided to compare the effect of TSST-1, G31Rmut TSST-1 and LPS on the medians of areas under ERK-1 activation curves (Figure 23 A , B, C shaded areas, Figure 24). There was no significant difference between the medians of the areas under the ERK-1 activation curves for TSST-1, G31Rmut TSST-1 and LPS treatments. A Wilcoxin signed rank 91  test between the medians of areas under TSST-1 and G31Rmut TSST-1-induced ERK-1 activation curves gave a P value of 0.4374 (two-tailed, n=6). The medians under the TSST-1 and LPS-induced ERK-1 activation curves were not significantly different and had a Wilcoxin signed rank P value of 1.000 (two-tailed, n=6). A Wilcoxin signed rank test between the medians of areas under the G31Rmut TSST-1 and LPS-induced ERK-1 activation curves gave a P value of 0.4375 (two-tailed, n=6). We also wanted to compare the effects of TSST-1, G31Rmut TSST-1 and LPS on the medians under the ERK-1 deactivation curves (Figure 23 A , B , C unshaded areas, Figure 24). There was no significant difference between the medians of the areas under the ERK-1 deactivation curves for TSST-1, G31Rmut TSST-1 and LPS treatments. A Wilcoxin signed rank test between the medians of areas under TSST-1 and G31Rmut TSST-1-induced ERK-1 deactivation curves gave a P value of 1.000 (two-tailed, n=6). The medians under TSST-1 and LPS-induced ERK-1 deactivation curves were not significantly different and had a Wilcoxin signed rank P value of 0.1563 (two-tailed, n=6). Finally, a Wilcoxin signed rank test between the medians of areas under the G31Rmut TSST-1 and LPS-induced ERK-1 deactivation curves gave a P value of 0.1536 (two-tailed, n=6).  4.3. Discussion.  The results of these signalling studies indicated that TSST-1, G31Rmut TSST-1 and LPS  induce the early activation of ERK-1 and ERK-2 in THP-1 cells. The mutant toxin  G31Rmut TSST-1 induces an ERK-2 activity profile that differs from both TSST-1 and LPSinduced profiles. Specifically, G31Rmut TSST-1 favors the ERK-2 activation phase over the ERK-2 deactivation phase (Figure 21 B, Figure 22). In contrast, both LPS and TSST-1 induce 92  ERK-2 activity profiles that favor neither activation nor deactivation phases (Figure 21 C, Figure 22). This pattern of differential activation was not seen in TSST-1, LPS or G31Rmut TSST-1-induced ERK-1 signalling (Figure 23, Figure 24). It is likely that this G31Rmut TSST1 effect on ERK-2 is related to the fact that the G31Rmut TSST-1 binds to the H L A - D R molecule with lower affinity than TSST-1 and induces lower levels of TNF-a release and T cell mitogenesis in PBMCs (44). It is also likely that both TSST-1 and LPS induce similar patterns of ERK-1 and ERK-2 activity, because they activate common non-MAP kinase pathways that may modulate M A P kinase activity in THP-1 cells (67,88,93,137,165,171,175). The early activation of both ERK-1 and ERK-2 by LPS reported here agrees with early activation profiles described in previously published data (Figure 21 C, Figure 23 C, Figure 22, Figure 24). For example, LPS induces the early activation of M A P kinases in murine macrophages (88) and in H U V E C cells (237). The transient nature of this early ERK-1 and ERK-2 activation is probably due to rapid dual dephosphorylation of ERK-1 and ERK-2 tyrosine and threonine residues (181) by one or more of at least eight dual specificity phosphatases (112) including MKP-1 and PAC-1 (113). The G31Rmut TSST-1 mutant induces an ERK-2 activity profile that favors activation over deactivation (Figure 21 B, Figure 22, Figure 25). It is possible that this difference between wild type and mutant toxin-induced signalling could be due to the lowered binding affinity of G31Rmut TSST-1 to H L A - D R (44). These differences  between toxin-induced ERK-1  signalling profiles may also be reflected in the low levels of TNF-a released by PBMCs treated with G31Rmut TSST-1 in comparison to wild type toxin treatments (44). The preference for G31Rmut TSST-1-induced ERK-2 activation, versus deactivation, may be due to sustained activation of upstream M E K (121), the inhibition or decrease of M K P activity (113,114) or both (Figure 25). G31Rmut TSST-1 could also induce MAPK-activating pathways that differ 93  from TSST-1 and LPS in a wide variety of upstream kinases including Ras—>Raf (101,104,128,129), Ras->MEKKs (138,139) and Ras-independent events (148). In contrast, G31Rmut TSST-1 and TSST-1 both equally induce medians of areas under ERK-1 activation and deactivation curves (Figure 23, Figure 24). In this case, both the wild type and mutant toxins may be signalling through common upstream ERK-1-activation and deactivation pathways (153,168,170,238). This similarity between TSST-1 and G31Rmut TSST-1-induced ERK-1 signalling differs from the effect that these reagents have on ERK-2 and indicate that G31Rmut signal differently through ERK-1 and ERK-2. Differential activation of ERK-1/2 by the same reagent has been described by previous workers and varies between the reagents and cell cultures used (104,122,123). For example, Sutherland showed that anti-CD40 antibodies induced differences between ERK-1 and ERK-2 activity profiles in human WEHI231 B cells (122). Schematically, G31Rmut TSST-1 could be inducing the early activation of upstream M A P K K s and MKPs in a manner that has different effects on ERK-1 and ERK-2 (Figure 25, Figure 26). Both TSST-1 and LPS induced similar ERK-1 and ERK-2 activity profiles (Figure 21) and induce equivalent areas under ERK-2 activation and deactivation curves (Figure 22). These similarities indicate that LPS and TSST-1 may be activating ERK-1 and ERK-2 through common upstream pathways. Currently, both LPS and TSST-1 have been shown to activate some common pathways that have been implicated in E R K control. These common pathways could be upstream protein-tyrosine kinases (PTKs) (33,125,164,170,171,238) and P K C (152,153,168). It is also possible that LPS and TSST-1 signal similarly through ERKs because they activate similar down-stream events such as AP-1 transcription control mechanisms (156,175) (Figure 4).  94  These patterns of TSST-1, G31Rmut TSST-1 and LPS-induced ERK-1 and ERK-2 activation were not noticeable when assayed for by anti-phosphotyrosine blotting (Figure 19, Figure 20). The failure of LPS to induce phosphotyrosine staining in the 38 kDa to 47 kDa ERK-1/2 range (Figure 19) contrasts with previously published data. Previous workers used LPS to upregulate ERK-1 and ERK-2 tyrosine phosphorylation and activity in CD14-positive monocytic cells (86-88,93). In comparison, we used undifferentiated THP-1 cells that have been shown to express very low levels of C D 14 (82). LPS still induced the release of T N F - a (82) and the activation of signal transduction pathways in these immature THP-1 cells (137), likely, in a CD14-independent fashion (75,81,95). These CD14-independent mechanisms could still phosphorylate and activate both ERK-1 and ERK-2. However, it is possible that the levels of physiologically relevant ERK-1 and ERK-2 tyrosine phosphorylation were not measurable given the conditions of this assay (239). It is also possible that LPS, TSST-1 and G31Rmut TSST-1-induced tyrosine phosphorylation could also occur through other related pathways that could not be assayed well with anti-phosphotyrosine staining. For example, SAP kinase activation is tyrosine phosphorylation-dependent but this tyrosine phosphorylation is not detectable with available anti-phosphotyrosine antibodies (88). Starvation and differentiation of cell culture prior to treatment may also affect the levels of tyrosine phosphorylation visualized following treatment with bacterial toxins. Serumstarved, or quiescent, eukaryotic cells often respond to environmental stimuli in a different manner than unstarved cells and are often used in LPS-induced signalling studies (85). For example, unstarved human monocytes treated with LPS produced higher levels of superoxide ((LV) when compared to quiescent monocytes (240). These different physiological responses may be due to activation of different signal transduction pathways within serum-starved cells. Weiglein and co-workers showed that quiescent HeLa cells had lower levels of ERK-1/2 95  tyrosine phosphorylation but showed higher levels of P M A induced ERK-1/2 bandshifting compared to unstarved cells (239). TSST-1 also induces the early activation of non-MAPK M B P kinases that co-elute with ERK-1/2 during anion exchange chromatography (Figure 14) but did not react with anti-ERK1/2 antibodies during immunoprecipitations (Figure 18) and immunoblotting (Figure 15, Figure 16). It is possible that these kinases have been mistaken for M A P kinases in earlier and more cursory studies (118,123,140,241). At the beginning of this series of experiments, we used anion exchange column chromatography as a method for separating THP-1 proteins that could be screened for TSST-1 and P M A induced ERK-1/2 activity (88). This method uses a NaCl gradient to separate proteins on the basis of their pi values. Very negatively charged proteins retain strong ionic bonds with the -NH4 ions on the column and must be eluted with high +  concentrations of NaCl (i.e. close to 0.8M NaCl) in the elution buffer. Proteins with fewer negatively charged residues do not create strong ionic bonds with the - N H } ions on the column +  and will elute with lower concentrations of salt in the elution buffer.  The Peak II fractions  obtained by the treatment with control media, P M A or TSST-1 (Figure 13, Figure 14) correlated to a NaCl concentration range, between 0.3 M to 0.5 M NaCl, where M A P kinases are expected to elute (88,109,242-244). P M A was initially used as a positive control for M A P kinase activation because of its reported activation of M A P kinases in monocytic cells (86,116), other eukarytoic cells (126,243) and pathways downstream of ERK-1/2 (110). In these experiments, P M A (Figure 13) induced a rapid activation of Peak II M B P kinase activity that was maximal at an earlier time point than TSST-1-induced M B P kinase activity (Figure 14). This early PMA-induced M A P K like activity agreed with the published data where P M A induced an early and transient M A P kinase activity (70,85,88). Unfortunately, this series of experiments did not determine i f the 96  PMA-induced M B P kinase activity was transient or sustained. In comparison, the TSST-1 induced late and sustained M B P kinase activity is similar to the late ERK-1/2 activities described in the literature (100,103,121,123). We originally thought that these late activities could be due to M A P K s , because late sustained M A P kinase responses are involved in a wide variety of physiological events including cell migration (108), upregulation of structural proteins (121) and cell maturation events (103,123). We now feel that a large portion of these increases in Peak II M B P kinase activity (Figure 13, Figure 14, Figure 18) are due to non-MAP kinases. Previous workers showed that this central peak can contain M B P kinases other than ERK-1 and ERK-2, including the PKC-c^ isoform (85) and P K B . The presence of P K C in a central elution profile may be relevant in this study because both TSST-1 (182) and P M A (70) activate P K C in eukaryotic cells. There is a growing list of kinases which can phosphorylate M B P in vitro and may be mistaken for E R K 1/2 in eukaryotic cells. These M B P kinases include T A K (195), the cytosolic active component of P K C known as protein kinase M (PKM) (245), the novel protein kinase N (PKN) (246) and protein kinase Fa/glycogen synthase kinase-3 (Fa/GSK-3) (247). Upon  anti-phosphotyrosine  phosphorylation  blotting  and  anti-ERK-1/2  immunoprecipitation followed by M B P kinase assays, it was concluded that the Peak II region of eluents from P M A and TSST-1-treated THP-1 cells contain ERK-1/2 isoforms as well as other M B P kinases (Figure 16). The levels of P M A and TSST-1-induced ERK-1/2 activity (Figure 18) and ERK-1/2 tyrosine phosphorylation, a marker of enzyme activation (244), (Figure 17) were negligible in Peak II fractions from selected time points. These time points were chosen from an examination of M B P kinase activities in eluents from P M A and TSST-1 treated cells (Figure 13, Figure 14). For example, the 30 minute time point for TSST-1 treatment was chosen because it contained the maximal M B P kinase activity present in the 97  experimental group (Figure 13, Figure 14). In comparison, the 2 minute P M A treatment time point was chosen because it was close to the maximal M B P kinase activity levels (5 minutes) and peak fractions for P M A (2 minutes) showed ERK-1/2 band-shifting in preliminary experiments. Surprisingly, we found that these peaks contain a large number of non-ERK-1/2 proteins (Figure 16, Figure 17) which react with anti-phosphotyrosine antibodies (Figure 15) (85,195). It is possible that these other protein include non-ERK-1/2 M B P kinases (237) which may mask any changes in TSST-1 and PMA-induced ERK-1/2 activity profiles. If the nonERK-1/2 M B P kinase masking effect is taken into account, then it is possible that P M A and TSST-1-induced changes in ERK-1/2 tyrosine phosphorylation activity are occurring at other time points. For example, Tao showed that both ERK-1 and ERK-2 can become dephosphorylated and deactivated following earlier periods of activation (96). The largest changes in P M A (2 minutes) and TSST-1 (30 minutes) induced ERK-1/2 activity (Figure 18) and tyrosine phosphorylation (Figure Figure 15, Figure 17) occurred in elution peaks not previously associated with M A P kinases (88,242). In support of the phosphotyrosine blot data (Figure 16, Figure 17), Peak I showed the largest increases in E R K 1/2 activity expressed as a ratio over basal activity for both P M A (2 minutes) and TSST-1 (30 minutes) treatments (Figure 18). In comparison, both P M A (2 minutes) and TSST-1 (30 minutes) induced decreases in Peak III ERK-1/2 activity (Figure 18). The presence of Peak I increases and Peak III decreases in ERK-1/2 kinase activity (Figure 18) indicate that TSST-1 (30 minutes) and P M A (2 minutes) both induce transient ERK-1/2 kinase activation and deactivation in THP-1 cells. It is possible that these changes in P M A and TSST-1-induced Peak I and Peak III ERK-1/2 activity are either not physiologically relevant or they may represent the true ERK-1/2 activity in the selected fractions following the removal of non-ERK-1/2 M B P kinases. 98  Other MBP Kinases  LPS THP-  Equivalent ERK-2 Activation/Deactivation  G31Rmut TSST-1 THP-1  TSSTTHP-1  Equivalent ERK-1 Activation /Deactivation  Preferred ERK-2 Activation  Figure 25. The effect of TSST-1, G31Rmut TSST-1 and LPS on ERK-1/2 activity in THP-1 cells.  99  In conclusion, TSST-1, G31Rmut TSST-1 and LPS induce the early activation of E R K 1 and ERK-2 in THP-1 cells (Figure 25). When activity is measured as an area under the activation and deactivation curves, all 3 toxins induce equivalent levels of ERK-2 activation and deactivation (Figure 22). Using the same measurement techniques, it is also evident that all 3 toxins induce the same levels of ERK-1 activation and deactivation within 30 minutes (Figure 24) . However, the mutant G31Rmut TSST-1 induces different patterns of ERK-2 activity than that of both TSST-1 and LPS (Figure 24, Figure 25). More specifically, G31Rmut TSST-1 induces an ERK-2 activity profile that favors activation over deactivation (Figure 22, Figure 25) . In comparison, both LPS and TSST-1 induce ERK-2 activity profiles that favor neither activation nor deactivation (Figure 22, Figure 25). A s a contrast to ERK-2 signalling profiles, all three toxins induced ERK-1 activity profiles that do not favor activation or deactivation (Figure 24). This reported difference between TSST-1 and G31Rmut mediated ERK-2 signalling may be a result of the lowered affinity that G31Rmut TSST-1 has to H L A - D R and may be reflected by lowered levels of T N F - a release and T cell mitogenesis in PBMCs (106). Surprisingly, LPS-induced ERK-1/2 activity did not differ from TSST-1-induced ERK-1/2 activity. These similarities in signalling may be a reflection of the common pathways that both toxins have been shown to activate. Finally, this series of experiments indicates that ERK-1 and ERK-2 are only one group of M B P kinase that can be activated by TSST-1 treatment of THP-1 cells (Figure 25). Postimmunoprecipitation M B P kinase assays and immunoblots indicate that there are a wide variety of proteins between 32 kDa->302 kDa that may be activated by TSST-1 (Figure 15). It may be more fruitful in the future to focus on these non-ERK-1/2 kinases because they may play a more physiologically important role in the response of monocytic cells to TSST-1.  100  Chapter 5. Conclusions and Future Research Directions. 5.1. Models of TSST-1 and LPS mediated TNF-a release. The main objective of the cytokine experiments was to determine conditions where TSST-1 and LPS induced TNF-a release in THP-1 cell culture. These conditions could then be used to study TSST-1 and LPS-induced signalling in THP-1 cells prior to the recruitment of T cells. Previous workers in our laboratory have shown that TSST-1 induced T N F - a release in human P B M C culture and that this TNF-a release requires the presence of both APCs and T cells (36). We also knew that LPS could induce TNF-a release in differentiated THP-1 cell culture without the input of other stimuli (67). Even though it was understood that TSST-1 induced TNF-a release required APCs and T cells, we wanted to replace human monocytes with THP-1 cells. We decided that the THP-1 cell line provided a level of reproducibility that could not be found in primary human monocytes. TSST-1-induced TNF-a release requires the presence of both THP-1 cells and T cells. This TNF-a release was measurable within 12 hours and was maximal at 36 hours following treatment with TSST-1. We also found that the level of H L A - D R on THP-1 cells was sufficient to bind TSST-1 and present this Sag to T cells in a manner that could induce TNF-a release. Specifically, this TNF-a release did not require exogenous IFN-y for the upregulation of H L A DR on THP-1 cells. From our results, it was also apparent that exogenous IFN-y could not replace T cells in the APC-Sag-T cell model as have been suggested by other (232). In comparison to the two cell model of TSST-1 mediated TNF-a release, LPS could induce the release of TNF-a from THP-1 cells alone. This release occurred within 12 hours and decreased to unmeasurable levels in THP-1 cells treated with LPS without exogenous IFN-y. THP-1 cells co-treated with both LPS and IFN-a released measurable levels of TNF-a after 12  101  and 84 hours of treatment. The mechanics of this LPS mediated signalling are in question, because it is not known i f LPS utilizes CD14-dependent (76,81) or CD14-independent mechanism of binding to undifferentiated THP-1 cells.  It is also unknown which CD 14-  independent mechanism would be utilized in this system and it is possible that LPS utilizes multiple mechanisms to bind and signal in THP-1 cells (75,94). The differences between TSST-1 and LPS mediated TNF-a release indicate that these reagents signal differently within THP-1 cells. It is also clear that TSST-1 can mediate signals within THP-1 cells in the absence of T cells (182,183). However, these signals are not sufficient to induce pathological conditions such as TNF-a release and T cell mitogenesis (44). In comparison, LPS induces intracellular signals that can induce the release of TNF-a from THP-1 cells alone.  5.2. TSST-1 mediated activation of ERK-1 and ERK-2: A comparison with G31Rmut TSST-1 and LPS mediated signalling. TSST-1 induced activation of M A P kinase in THP-1 cells within 30 minutes of treatment. It is possible that the earlier inability to measure this activation was due to masking of ERK-1 and ERK-2 activity by other M B P kinases (195,246,247). Upon immunoprecipitation of ERK-1 and ERK-2, it was evident that TSST-1 induces activation of both M A P kinase isoforms within 30 minutes. Although the amplitudes of TSST-1 mediated ERK-1 and ERK-2 activation were quite low (88), this mean activation followed an activation-deactivation profile common in other models of M A P kinase mediated signalling (237). Similarly, LPS induces  102  LPS  TSST-1  G31Rmut TSST-1  THP-1  THP-1  PBMCs  TNF-a  No TNF-a  No TNF-a  v  Equivalent ERK-2 Activation/ Deactivation  Equivalent ERK-1 Activation/ Deactivation  Preferred ERK-2 Activation  4  4  MAPKK/MAPKP Cycle?  MAPKK Sustained? Deactivate MAPKPs?  Figure 26. The roles of TSST-1, G31Rmut TSST-1 and LPS in pathophysiology.  103  ERK-1 and ERK-2 activity profiles in THP- 1 cells that do not favor either activation or deactivation. This similarity between TSST-1 and LPS ERK-1/2 signalling may be due to the activation of common pathways in THP-1 cells. G31Rmut TSST-1 induced different ERK-2 activity profiles when compared to TSST-1 mediated signalling. This mutant toxin induced an activity profile that favored activation over deactivation. The differences between TSST-1 and G31Rmut TSST-1 signalling could be due to the mutations present in the M H C II binding domain of the G31Rmut TSST-1 mutant molecule (44). In comparison, G31Rmut TSST-1 induced an ERK-1 activity profile that included activation and deactivation phases but did not favor one phase over another. This mutation which results in a decreased affinity of G31 Rmut TSST-1 for M H C II may also induce different levels of activity of both M A P K phosphatases or different pathways upstream of M A P K s .  5.3. Future directions of research. This series of experiments have set the groundwork and conditions for the study of M A P kinase activation by TSST-1 in co-cultures of THP-1 cells and T cells. The isolation of M A P kinase in either THP-1 cells or T cells could be accomplished by the poisoning of either THP-1 cells or T cells with PD 098059 which is a selective inhibitor of MEK-1 and M E K - 2 (118). Questions about the origin of TNF-a and other cytokines in the 2-cell model could be addressed by either human T cells clones or non-human T cell lines (81,232). The utilization and analysis of the role of T cell components in TSST-1 mediated M A P kinase activation would also be addressed (232). The study of the role of TSST-1 signalling in other related and unrelated kinases could be studied in THP-1 cells alone or in the presence of T cells. Both SAP kinase and p38 HOG-1 (122) have been implicated in pro-inflammatory cytokine signalling (117), LPS-induced  104  signalling (88,248) and LPS mediated TNF-a transcriptional control (159) in eukaryotic cells. Pathways upstream of SAP kinase and HOG-1 have also been implicated in apoptosis which is currently being studied in our laboratory (249). These cousins of ERK-1 and ERK-2 may play a role in TSST-1 mediated signal transduction as well. Other unrelated or peripherally related pathways such as P K C pathways may also be fruitful targets for TSST-1 signalling studies (182). It may also be profitable to study M A P kinase phosphatases (112,181,250) or M A P K kinases (119,251) in the pathogenesis of TSS.  105  Bibliography 1. Chesney, P. J. 1983. 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