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Bacterial lipopolysaccharide signaling pathways in mononuclear phagocytes involve protein and lipid kinases Herrara-Velit, Patricia 1997

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BACTERIAL LIPOPOLYSACCHARIDE SIGNALING PATHWAYS IN MONONUCLEAR PHAGOCYTES INVOLVE PROTEIN AND LIPID KINASES By Patricia Herrera-Velit B.Sc, Universidad Peruana Cayetano Heredia, Lima M.Sc, Universidad Peruana Cayetano Heredia, Lima  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standards  THE UNIVERSITY OF BRITISH COLUMBIA October 1997  In  presenting  degree  at  this  the  thesis  in  University  of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  publication of this  for  his thesis  or  scholarly her  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Pg-y^ftgjcL  4  I agree  may  representatives.  ffiy  ^HT)  that the  be  It  for financial gain shall not  ViicgjOV&tM ,<^Grf  requirements  the  I further agree  purposes  permission.  Department of  of  is  that  for  an  advanced  Library shall make it  permission for extensive  granted  by the  understood  head  that  be allowed without  of  my  copying  or  my written  I. ABSTRACT  Mononuclear phagocyte activation enhances the functional properties of these cells, and endotoxin (bacterial lipopolysaccharide) is one of the most potent agonists known to cause an activated state. This model was used to investigate signaling pathways that regulate macrophage activation. Exposure of mononuclear phagocytes to lipopolysaccharide resulted in rapid and dose-dependent increases In tyrosine phosphorylation of a set of proteins. This was due, at least in part, to increased tyrosine kinase activity. In particular, compared to untreated cells, lipopolysaccharide-treated monocytes showed increased activity of the Src-family tyrosine kinase pSJ/SS^".  In addition, both tyrosine kinase activity, as well as tyrosine phosphorylated  proteins without endogenous kinase activity, associated with the lipopolysaccharide receptor CD 14 in an inducible manner. Lipopolysaccharide treatment of monocytes also activated the lipid kinase, phosphatidylinositol 3-kinase in a dose andtimedependent manner. Activation of this enzyme resulted in increased levels of D3-phosphorylated phosphoinositides and this response was mediated through CD 14.  Activation of phosphatidylinositol 3-kinase also resulted in its  association with activated p5J/56 y without leading to a detectable change in the tyrosine f  n  phosphorylation state of the p85 regulatory subunitof the enzyme. Increased protein kinase activity is not restricted to tyrosine kinases, as lipopolysaccharide also activated serine/threonine kinases. Anion exchange chromatography of endotoxin-treated mononuclear phagocytes showed increased myelin basic protein-kinase activity eluting as two major peaks. Based on immunoreactivity and substrate preference, the earliest eluting peak, peak "one" was identified as p42/p44 mitogen-activated protein kinases.  Ill  Tyrosine phosphorylation of both p42 and p44 mitogen-activated protein kinases increased in response to lipopolysaccharide. The second peak of myelin basic protein-kinase activity, peak "two", was identified as protein kinase C-C,, an atypical isoform of protein kinase C. This conclusion is based on immunoreactivity, substrate preference, and cofactor independency of the enzyme. The cellular mechanisms regulating the activity of protein kinase C-C, were investigated. Protein kinase C-C, activation by lipopolysaccharide occurred downstream of phosphatidylinositol 3-kinase. This conclusion is based on two findings: (i) phosphatidylinositol 3-kinase inhibitors blocked activation of protein kinase C-C,, and (ii) transfection of cells with a dominant negative mutant of phosphatidylinositol 3-kinase impaired activation of protein kinase C-t; by lipopolysaccharide. Functional responses of mononuclear phagocytes dependent on signaling pathways involving phosphatidylinositol 3-kinase were examined. Phosphatidylinositol 3-kinase inhibitors abrogated lipopolysaccharide-induced adherence, but not adherence induced in response to phorbol 12-myristate 13-acetate in the monocytic cell line THP-1. In contrast, induction of transcription of cytokine genes by lipopolysaccharide was independent of phosphatidylinositol 3-kinase. In conclusion, examination of lipopolysaccharide signaling in mononuclear phagocytes showed that endotoxin activates both protein and lipid kinases including p53/56^ p42 and p44 isoforms of mitogen activated protein kinase and phosphatidylinositol 3-kinase through a CD-I 4 dependent mechanism. Activation of PKC-C, is phosphatidylinositol 3-kinase dependent and signaling through this lipid kinase regulates monocyte adherence, but not cytokine production induced by lipopolysaccharide. Given the requirement that monocytes must adhere  iv to endothelium, to other leukocytes and to extracellular matrix proteins in order to mediate functional responses, it may be possible to modify macrophage responses to lipopolysaccharide and perhaps other agonists by targeting phosphatidylinositol 3-kinase.  V  II. TABLE OF CONTENTS  I. ABSTRACT  ii  II. TABLE OF CONTENTS  v  III. LIST OF TABLES  ix  IV. LIST OF FIGURES  x  V. LIST OF ABBREVIATIONS  xii  VI. ACKNOWLEDGMENTS  xv  VII. INTRODUCTION •  A. MONONUCLEAR PHAGOCYTES  1  • B. LIPOPOLYSACCHARIDE •  C. LPS RECEPTORS AND LPS BINDING PROTEINS  1. Cell-associated LPS binding proteins 2. Soluble LPS binding proteins •  •  2  5 11  D. LPS INDUCED CELL SIGNALING  1. Protein Tyrosine kinases  16  2. Phosphatidylinositol 3-kinase  19  3. Protein kinase C  23  4. Ceramide activated protein kinase  26  5. Mitogen-activated protein kinases, p21 ^and other signaling proteins  27  E. THESIS RATIONALE AND OBJECTIVES  29  vi Vlll. MATERIALS AND METHODS •  •  A. MATERIALS  1. Antibodies  32  2. Reagents  33  B. METHODS  1. Isolation of monocytes and cell culture  .....34  2. Preparation of cell lysates a. High speed supernatants  35  b. Lysates for anion exchange chromatography  36  c. Lysates for analysis of phosphotyrosine content  36  3. Ras immunoprecipitation  36  4. Anion exchange chromatography  37  5. Immunoadsorption of kinase activity  38  6. Western blotting.  38  7. Immunoprecipitation  39  8. In-situ renaturation kinase assay  39  9. Immune complex kinase assay  40  10. PKC-; assays  40  11. Protein kinase assays...  41  12. In vitro PI 3-kinase assay  42  13. Cell labeling and extraction of inositol phospholipids  42  14. HPLC analysis of deacylated lipids  43  15. Transfection of U937 cells  44  16. RNase protection assay 17. LPS induced adherence of M<|>s 18. Protein determination  46 47 48  IX. RESULTS •  A. TYROSINE PHOSPHORYLATION AND TYROSINE KINASE ACTIVITIES INDUCED BY LPS  1. LPS-induced tyrosine phosphorylation of M<)> proteins 2. LPS-induced tyrosine kinase activity  49 49  3. Association of CD 14 with tyrosine kinases and phosphotyrosine proteins in response to LPS 4. LPS treatment activates pSJ/Sd^m M<t>s •  54 57  B. SERINE/THREONINE-KINASES ACTIVATED IN MOS IN RESPONSE TO LPS  1. LPS inducestime-and concentration-dependent increases in phosphotransferase activity towards myelin basic protein 2. Activation of p42/p44 mitogen activated protein kinase by LPS in PBM(j>s  .....61 64  3. Analysis of protein kinase activity using a MAP kinase-specific peptide substrate 4. LPS-induced activation of PKC-C, in monocytes  69  5. Immunoadsorption of PKC-t; activity  75  6. Biochemical characterization of PKC-C, •  65  78  C. PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION IN LPS-TREATED MoS.  1. Detection of ?td\nsZ, A ,SPj in vivom LPS-treated cells 2. HPLC analysis of PI 3-kinase metabolites in U937 cells  81 81  viii 3. Wortmannin inhibits the generation of Ptdlns3,4,5/j/>7 vivo. ,  4. LPS-induced activation of PI 3-kinase is CD 14-dependent 5. Kinetic analysis of PI 3-kinase activity in LPS-treated monocytes 6. LPS dose-response analysis for activation of monocyte PI 3-kinase  84 85 88 88  7. LPS treatment activates P53/564 and promotes its transient association 77  with PI 3-kinase  91  8. Tyrosine phosphorylation state of the M 85,000 subunit of PI 3-kinase r  9. Activation of PKC< is phosphatidylinositol 3-kinase-dependent. •  D.  E F F E C T S O F LPS  O N p21  •  E. PI 3 - K l N A S E A N D LPS-ELICITED F U N C T I O N A L R E S P O N S E S  r  a  s  AND THE V A V PROTOONCOCENE  96 98 106  1. Effects of PI 3-kinase inhibitors on LPS-induced enhancement of cytokine mRNA levels 2. PI 3-kinase and LPS induced adhesion of M<> js  108 108  X. DISCUSSION  116  XI. REFERENCES  135  ix III. LIST OF TABLES  Table 1. HL-14 template set Table 2. Effects of LPS on inositol phospholipids in U937 cells  47 84  X  IV. LIST OF FIGURES  Figure 1. Chemical structure of lipopolysaccharide Figure 2. Chemical structure of lipid A and the antagonistic lipid A partial structure, lipid IV A  Figure 3. Metabolism of phosphoinositides Figure 4. Structure of the atypical PKC isoforms  3  ...4 21 24  Figure 5. Cloning of the wild-type bovine p85a and dominant negative mutant Ap85  45  Figure 6. Tyrosine phosphorylation of M<> | proteins in response to LPS  51  Figure 7. Activation of protein tyrosine kinases in M<|>s in response to LPS  53  Figure 8. LPS induces the association of CD 14 with tyrosine kinases  55  Figure 9. Increased association of phosphotyrosine containing proteins with CD 14 is brought about by LPS Figure 10. Activation of 53/56>y in PBM(|>s treated with LPS n  56 58  Figure 11. LPS-dependent activation of piJ/itf^ in undifferentiated and D3differentiated U937 cells ...60 Figure 12. LPS treatment of M<|>s leads to increased myelin basic protein phosphorylating activity in atimeand dose dependent manner 62 Figure 13. Anion exchange chromatography of PBM<|>s show several peaks of MBP-phosphorylating activity stimulated by LPS  66  Figure 14. MBP and synthetic MAP kinase peptide substrate reveal distinct substrate specificities of peak 1 and peak 2 of MBP kinase activity  68  Figure 15. LPS induces activation PKC-t;  71  Figure 16. LPS induces activation of PKC-C activity in promonocytic cell lines  73  Figure 17. Immunoadsorption of the major peak of MBP kinase activity with anti-PKC-C antibodies  76  xi  Figure 18. Substrate preference and cofactor dependency of monocyte PKC-c^  79  Figure 19. Ptdlns3,4,5P levels in M<|>s after LPS treatment  82  3  Figure 20. HPLC analysis of deacylated inositol phospholipids from U937 cells  83  Figure 21. Effects of wortmannin on Ptdlns3,4,5P3 levels  86  Figure 22. CD 14-dependence of enhanced Ptdlns3,4,5P3 levels in LPS-treated human monocytes  87  Figure 23. Time course of PI 34dnase activity in LPS-treated cells  89  Figure 24. Dose dependence of PI 3-kinase activation in response to LPS  90  Figure 25. Association of pSJ/Sd^ with PI 3-kinase  '.  93  Figure 26. Inhibition of PI 3-kinase activity by herbimycin A  94  Figure 27. PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates  97  Figure 28; Abrogation of LPS-induced activation of PKC-t; by the PI 3-kinase inhibitors, wortmannin and LY294002 Figure 29. Expression of p85 in U937 cells Figure 30. PI 3-kinase activity in U937 cells transfected with either wild type, bovine p85a or dominant negative mutant, Ap85 Figure 31. Attenuation of LPS-induced activation of PKC-t; by a dominant negative mutant (Ap85) of the p85 subunitof PI 3-kinase Figure 32. Tyrosine phosphorylation of pP/^^in PBNtys  99 101 102 104 107  Figure 33. Effect of PI 3-kinase inhibitors on mRNA levels of different cytokines  110  Figure 34. LPS-induced adherence of THP1-WT cells in PI 3-kinase dependent  111  Figure 35. Adherence of THP1 -WT cells to fibronectin-coated wells  115  V. LIST OF ABBREVIATIONS  BSA  Bovine serum albumin  CAK  Ceramide-activated protein kinase  CPAE  Cow pulmonary artherial cell  ECL  Enhanced chemoluminiscence  EDTA  Ethylendiaminetetraacetic acid  EGTA  Ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetracetic acid  ELAM-1  Endothelial-leukocyte adhesion molecule-1  FCS  Fetal calf serum  FPLC  Fast performance liquid chromatography  G-CSF  Granulocyte-colony stimulating factor  GDP  Guanine 5'-diphosphate  GM-CSG  Granulocyte/macrophage-colony stimulating factor  GPI  Glycosylphosphatidylinositol  HBSS  Hank's balanced salt solution  HPLC  High pressure liquid chromatography  HRPO  Horse radish peroxidase  HUVEC  Human umbilical vein endothelial cells  ICAM-1  Intercelullar cell adhesion molecule-1  IL  Interleukin  KDO  2-keto-3-dexyoctonic acid  LAM  Lipoarabinomannan  LBP  LPS binding protein  LPS  Lipopolysaccharide, endotoxin  mAb  Monoclonal antibody  MAPK  Mitogen-activated protein kinase  mCD14  Membrane CD 14  MBP  Myelin basic protein  MOPS  3,-[N-morpholino]propanesulfonic acid  M(|>s  Mononuclear phagocytes  NHS  Normal human serum  PAF  Platelet activating factor  PAGE  Polyacrylamide gel electrophoresis  PBM(|>s  Peripheral blood mononuclear cells  PEI  Polyethylenimine cellulose  PIPES  Piperazine-N,N'-bis[2-ethanesulfonic acid]  PI 3-kinase  Phosphatidylinositol 3-kinase  PKB  Protein kinase B  PKC  Protein kinase C  PLC  Phospholipase C  PMA  Phorbol 12-myristate 13-acetate  PMN  Polymorphonuclear lymphocytes  PMSF  Phenylmethylsulfonyl fluoride  PNH  Paroxysmal nocturnal hemaglobinuria  PS  L-a-phosphatidyl-L-serine  Ptdlns  L-a-phosphatidylinositol  Ptdlns3P  L-a-phosphatidylinositol 3-phosphate  Ptdlns4P  L-a-phosphatidylinositol 4-phosphate  Ptdlns3,4P  L-a-phosphatidylinositol 3,4-bisphosphate  Ptdlns4,5P  L-a-phosphatidylinositol 4,5-bisphosphate  2  2  Ptdlns3,4,5P  L-a-phosphatidylinositol 3,4,5-trisphosphate  PTK  Protein tyrosine kinase  R-LPS  Rough LPS  RT  Room temperature  sCDI 4  Soluble CD 14  S-LPS  Smooth LPS  TGF-p  Transforming growth factor-p  TLC  Thin layer chromatography  TNF-a  Tumor necrosis factor-a  3  XV  VI. ACKNOWLEDGMENTS  I would like to give innumerable thanks to Dr. Neil Reiner for all the advice he has given me along this way and to the proficient use of red pen he has practiced in my writing. Special thanks to the people in the Biochemistry Laboratory from Universidad Peruana Cayetano Heredia, Lima and to my family -especially my mother- for their infinite patience, and to all the people in Neil's lab, in particular to Keith Knutson and Zakaria Hmama for their help. I would also like to acknowledge scholarship support from the Canadian International Development Agency.  1  VII. INTRODUCTION/LITERATURE REVIEW  MONONUCLEAR  PHAGOCYTES  Mononuclear phagocytes (M<|>s) are important components of both innate resistance and adaptive immunity. After extensive development in the bone marrow, relatively immature monocytes enter the circulation and migrate to tissue sites where they mature and differentiate into macrophages (1). Whether circulating or in thetissues,M<j)S are subject to a large variety of signals, both stimulatory and inhibitory in nature. This is especially true when homeostasis is altered, as occurs duringtissueinjury, inflammation and sepsis. Under these conditions, many of the innate properties of macrophages change, including their morphology and metabolism (2). These changes are associated with the induced expression of various gene products that enable the cells to perform functions otherwise not achieved by resting cells. This process of macrophage activation results in the appearance of an "immunocompetent" cell. Activated cells show enhanced responses to lymphokines and become highly secretory cells, with their secreted products exerting a wide variety of effects on many cells types (3). These products include lipid-derived inflammatory mediators such as platelet-activating factor (PAF) and prostaglandins, as well as chemokines that recruit other inflammatory cells to the site of injury, reactive oxygen species involved in microbial killing, cytokines such as tumor necrosis factor (TNF-a) with tumoricidal and microbicidal activities, and growth factors such as fibroblast growth factor and transforming growth factor-p (TGF-p) that promote repair of injured tissues. M<j)S  are essential not only for natural immunity, but they also have evolved to play critical roles  in acquired immunity. They are efficient in presenting foreign antigens to T cells, they bind  2 and phagocytose particles opsonized with complement, IgG, or other opsonins, and they respond to cytokines secreted by T lymphocytes and other cells (4-6). Activated Ntys play a central role, therefore, in the host response to external stimuli.  LlPOPOL YSACCHARIDE (LPS)  Amongst the most potent stimuli known to lead to Md) activation are bacterial products. The classical and most thoroughly studied microbial product known to activate M(j>s is endotoxin or lipopolysaccharide (LPS), derived from cell walls of gram negative bacteria. Qualitatively, LPS is able to elicit many, but not all of the responses usually attributed to activated Ntys (7,8). As has already been mentioned, this may be necessary as part of a proper protective innate host response against gram negative infection. However, uncontrolled Nty activation can be deleterious to the host and many of the clinical manifestations of systemic bacterial infections such as fever, hypotension, and metabolic dearrangements can be attributed, at least in part, to the effects of LPS on Ntys (9). Numerous studies have been performed to delineate the structural components of LPS (10-13). The minimal structure of endotoxin consists of a lipid component termed lipid A and a core hydrophilic polysaccharide region formed minimally by at least one or two, 2-keto-3deoxyoctonic acid (KDO) residues or KDO derivatives (Figure 1). The composition of the hydrophilic region is usually considerably more complex, however, and is highly variable. In Enterobscteriacea, for example, it is formed by a heteropolysaccharide consisting of a core oligosaccharide and an O-specific chain (smooth- or S-LPS). The structure of the core region may be subdivided further into outer and inner components, the latter being formed by KDO and heptose residues (11), and the former consisting of the common hexoses D-glucose, D-  3  f  galactose and N-acetyl-D-glucosamine. Besides providing an attachment site for O-antigen, the function of the outer core is not known. The O-specific chain is a polymer of oligosaccharides, containing between two and eight sugar monomers. Further complexity is added by the fact that the composition of the repeating units in the O-side chain differs from strain to strain within a serotype and exhibits large structural variability. This region also functions as an important surface antigen and determines the serological specificity of the LPS variant and of the bacteria containing it The synthesis of the O-specific chain is determined by a cluster of genes termed rfb. A second type of LPS, the rough or R-type, lacks the O-specific chain, and was first identified in enterobacterial mutants with a defect in the rfb locus. Mutants are able to grow and multiply in vitro indicating that the 0-specific chain is not necessary for bacterial viability.  Polysaccharide  Phospholipid  1  1  O-Specific Chain (  Outer Core 1|  1|  Inner Core  1  Lipid A  ((  (  Figure 1. Chemical structure of lipopolysaccharide. From Hoist, O. etal. (13).  Lipid A is a phosphoglycolipid that, together with the KDO-containing inner core, is the most conserved structure of LPS. It contains a p( 1 -6) residue-linked disaccharide with one glycosidic and one non-glycosidic phosphoryl group and medium to long chained fatty acids  4 linked by amide and ester bonds (10-13). Despite being highly conserved, variations in its structure exist These derive from the type of hexoseamine residues present, the degree of phosphorylation, and the nature and length of the acyl groups (11). Most of the effects of endotoxins on animals or on cultured cells are attributable to lipid A, and certain chemical modifications of the constituents of lipid A result in a biologically inactive endotoxin. Lipid A expressing full endotoxic activity requires two hexosamine residues, two phosphoryl groups, and six fatty acids with defined lengths and distinct positions.  A  B  Figure 2. Chemical structure of lipid A (A) and the antagonistic lipid A partial structure, lipid IVA  (B). m= 14C-acyl group, I = 12C-acyl group. From Hoist, O. etal. (13).  Cells affected by LPS include not only those of the monocyte/macrophages lineage, but also endothelial cells, smooth muscle cells, granulocytes and megakaryocytes. Brief exposure to endotoxin or lipid A results in the production of bioactive lipids, reactive oxygen intermediates and cytokines including TNF-a, interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8)  5  and interleukin 10 (IL-10), in addition to a multitude of other mediators (3). These responses are observed in murine, rabbit and human Mfys of either alveolar, peritoneal or blood origin (8).  LPS RECEPTORS  AND  LPS BINDING  PROTEINS  Cell-associated LPS binding proteins  In the presence of serum, Ntys responses to LPS (or to lipid A) occur at concentrations in the picogram to nanogram/ml range. This sensitivity involves an LPS receptor at the cell surface, as well as a serum factor that facilitates LPS binding (14,15). CD 14, a myeloid differentiation protein present mainly on peripheral blood monocytes and macrophages, as well as on neutrophils, has been shown to function as an LPS receptor. CD 14 is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) tail (16) and is also found in serum in a soluble form. It appears that the GPI anchor may not be required for all the downstream events brought about by LPS binding. This conclusion is based on the finding that LPS is able to induce identical cellular responses in the 70Z/3 B lymphoma cell line, stably expressing CD 14, irrespective of whether it is expressed as an integral membrane protein or if it is anchored to the membrane by a GPI tail (17). Several experimental observations indicate the existence of other LPS cell surface receptors in M<> j s in addition to CD 14. For example, antibodies against CD 14 are not able to block completely responses to LPS when the latter is used in concentrations > 100 ng/ml. Similarly, promonocytic cell lines such as U937 and THP-1 express very low levels of CD14, but are still able to respond to LPS in concentrations > 100 ng/ml. Likewise, cell types that do not express CD 14 such as, 70Z/3, respond to concentrations of LPS in excess of 100 ng/ml  6  (18). Thesefindingsindicate that, both in CD 14 negative as well as in CD 14 positive cells, there appears to be other cell surface proteins that can bind LPS and transmit signals, albeit at concentrations higher than those required for signaling through CD 14. The function of CD 14 as an LPS receptor in M<> j s and polymorphonuclear phagocytes (PMNs) will be discussed in more detail below. Using a photoactivatable, radioiodinated LPS, Hailing etal. (19) described the presence of several proteins in human blood cells, including M<|>s, that are able to bind LPS in a specific manner under serum free conditions. A 73 kDa LPS-binding protein was noted to be present in the different cell types examined in this study. The question of whether this protein is at the cell surface or intracellular was not addressed in this work.  In the same study, when  lymphocytic cells were examined, several additional lower molecular weight LPS-binding proteins were detected, the most abundant having approximate subunit sizes of 50, 31 and 18 kDa. These secondary proteins were also present in the adherent monocytic preparations, although they were less prominent Surprisingly, even though several other LPS-binding proteins were reported, none had a size in the range of 55 kDa, potentially corresponding to CD 14. Lei and Morrison (20) described a similar LPS binding protein of 80-kDa in B and T lymphocytes and in murine macrophage membranes with specificity for the lipid A region of LPS. Approximately 5,000-10,000 molecules of this putative LPS receptor were detected on these cells indicating that it appeared to be less abundant than CD 14 [approximately 1x10  s  copies/monocyte (21)]. Chen and colleagues reported that addition of purified mAbs specific for the 80-kDa protein to bone marrow-derived macrophages from LPS-responsive C3H/HeN mice, led to activation of these cells for tumoricidal activity towards mastocytoma cells In vitro (22). In contrast, when these antibodies were used with M(|>s derived from LPS unresponsive  7 C3H/He] mice, no tumoricidal activity was observed, even though there was equivalent binding of mAb to cells from these two strains. Using affinity-purified rabbit antibodies with specificity for the-80 kDa protein, and a variety of techniques to detect this protein at the surface of thioglycollate-elicited peritoneal macrophages, Perera et ai. (23) were also unable to detect any difference in the surface expression of this putative 80-kDa LPS-receptor from cells derived from C3H/HeH and C3H/HeJ mice.  Taken together, these results indicate that LPS  hyporesponsiveness in macrophages from C3H/He] mice is not due to lack of this ~ 80-kDa LPS-binding protein, but to events distal to LPS binding. Although some of the data regarding this 70-80-kDa LPS binding protein suggest that it may be a functional receptor, recent findings have ailed this into question. Dziarski (24) reported in 1994, that a 70-kDa protein present on the surface of lymphocytes and macrophages, capable of binding LPS, was in reality, cell bound albumin. Species-specific antibovine, anti-human, and anti-mouse albumin antibodies recognized the 70-kDa protein on mouse and human cells according to the species of albumin that was present in the culture medium or in the serum in vivo, but not according to the species of the cells. Anti-albumin antibodies immunoprecipitated a radiolabeled 70-kDa protein from I-ASD-LPS cross-linked 125  THP-1 cells grown in the presence of FCS. Furthermore, soluble albumin perse was also found to bind radiolabeled-LPS in vitro. Lastly, this "LPS-binding protein' was shown to originate from the cell culture medium or from the serum in vivo, but was not produced by the cells. Thus, these results indicate that, this 70-80-kDa putative LPS receptor may be an artifact In addition to the proteins described above, some of which may function as specific receptors for LPS or lipid A, there are other macrophage receptors with well defined ligands that have also been shown to bind LPS. Among these, the scavenger receptor has been  8 reported to bind lipid A. For example, subsequent to binding to the scavenger receptor, lipid IV^, a bioactive precursor of lipid A, is metabolized to a less active form by the murine macrophage-like RAW 264.7 cells (25). Both binding and deactivation of lipid was shown to be mediated by the macrophage scavenger receptor. It seems unlikely, however, that this protein is involved as a functional LPS receptor in mononuclear phagocytes, since its natural ligand, acetylated LDL, is unable to block induction of TNF-a production by endotoxin. Similarly, Golenbock et al. (26) found that transfection of CHO cells with CD 14 confers responsiveness to LPS as assessed by arachidonate release. In contrast, transfection with the macrophage scavenger receptor failed to produce the same effect Thus, while not functioning as an LPS "signaling" receptor, the macrophage scavenger receptor may play an important role in clearance and detoxification of LPS in vivo. Members of the CD 18 or P2 complex of leukocyte integrins (CD11a-c/CD18), are known to participate in numerous cell-cell and cell-substrate interactions. In addition to functioning as adhesion molecules, P2 integrins on monocytes, macrophages and polymorphonuclear leukocytes (PMN) have also been shown to bind LPS (27).  Using  antibodies against this complex, Wright and coworkers (28) were able to block the binding of particulate LPS to M<> js when it was presented on the surface of bacteria or as LPS-coated erythrocytes. However, the importance of this complex in M<> | functional response to LPS is unclear for several reasons. First, antibodies to CD 18 do not prevent LPS-induced synthesis of TNF-a by whole human blood (29). Second, antibodies against CD1 la, CD1 lb or CD 18 do not block the LPS-induced secretion of IL-1 p and TNF-a by normal human monocytes (30). Finally, M<|>sfrompatients genetically deficient in CD 18 show in vitro responses to LPS that are indistinguishable from those observed using cellsfromnormal volunteers (31).  9  Recombinant human CD 14 expressed at the surface of Chinese hamster ovary (CHO) fibroblasts interacts rapidly with LPS in the presence of serum or LBP, being almost complete within 5 min and reaching maximal binding by 10 min (32). If LPS and LBP are preincubated before being presented to the cells, binding occurs in a similar manner either at 10 ° or 20 °C. This suggests that this process does not require extensive mobility of CD 14 within the cell membrane. The molar ratio of LPS bound to surface CD 14 varies from 8:1 to 20:1. One explanation for this high stoichiometry is that LPS is present, not as a monomer, but as an aggregate of 8 to 20-mers and binds to CD 14 as such. Another explanation is that CD 14 has multiple LPS binding sites. In this respect, it has been found that there are ten leucine-rich repeats within CD 14 that may be LPS binding motifs (33). If each leucine repeat has the capacity to bind one or two LPS molecules, this would allow for the observed stoichiometry. Several human, promonocytic cell lines have also been used to study the effects of LPS. THP-1, U937 and HL-60 are amongst the cell lines used most commonly. Although CD 14 can be detected in some of these cell lines (21,34) the level of expression of CD 14 is minimal, and these cells respond poorly to low concentrations of LPS in terms of IL-1 and TNF-a production. Treatment of immature THP-1 cells with vitamin D3 induces the expression of CD 14 in atimeand dose dependent manner. After 48-72 h of treatment with 100 nM of D3, maximal expression of CD 14 is observed (35-fold increase over baseline), whereas expression of other surface markers, such as CD 18 increase less substantially (35). As THP-1 cells differentiate in the presence of D3, they become increasingly responsive to LPS and LPSLBP complexes. This phenomenon is likely explained by the interaction of the LPS-LBP complex with CD 14 now present at the cell surface in greater numbers. Induction of competence for LPS cell activation is also seen in cells that are stably transfected with CD 14.  10 CD 14 transfected murine B-lymphoma cells respond to 1000-fold lower LPS concentrations than do parental CD 14-negative cells (18,36,37). Similarly, CHO fibroblasts, a CD 14 neptive, LPS-unresponsive cell line, also become responsive to LPS when transfected with CD 14 (26). CD 14 present at the surface of neutrophils appears to be indistinguishable from M<|>CD14. As is the case for monocytes, it is also anchored to the membrane of PMNs by a GPItail and is shed into the culture medium. M<t>s and PMNs derived from patients with paroxysmal-nocturnal hemoglobinuria (PHN), a disease characterized by a defect in the GPIanchoring mechanism of proteins, show decreased levels of mCD 14 (16,38,39). In vitro, cells from patients with PHN exhibit diminished functional responses to LPS, thus further substantiating the critical role of CD 14 in LPS signaling. The precise role of membrane CD 14 in LPS signaling is a subject of intense interest Some insight into this question was provided by Kuhns et al. (40) who reported the case of a 15-year old patient with recurrent bacterial infections who showed hyporesponsiveness to LPS, both in vivo and in vitro. The neutrophils and monocytes from this patient showed normal levels of various surface markers, including CD 14, and the binding of LPS to CD 14 was not differentfromthat observed using cellsfromnormal donors. In vitro incubation of M<|>s with LPS and IL-1 failed to induce the production of either TNF-a or granulocyte colony stimulating factor (G-CSF).  LPS priming of neutrophils for N-formyl-methionyl-leucyl-phenylalanine  (fMLP)-induced superoxide production was also deficient, while the cells responded to TNF-aand PAF-priming for fMLP-induced superoxide production. The defect in this patient's cells, appears to localize to an early upstream event in the signal-transduction pathway activated by  11  LPS and IL-1. Thus, although required for binding of LPS to Ntys, the presence of CD 14  perse  is not sufficient to confer functional responsiveness.  Soluble LPS binding proteins  A major advance in the understanding of LPS signaling was the discovery of LPS binding protein (LBP), a plasma protein capable of binding LPS specifically and which is required for ligand binding to CD 14 (41,42). LBP is present in human serum at concentrations of < 5 ug/ml, and increases more than ten-fold after induction of an acute phase response, a phenomenon that develops rapidly after an infection where the concentration of a number of serum proteins increases dramatically. LBP has a predicted size of 50 kDa and is produced in the liver. Before secretion, LBP is glycosylated and circulates as a 60 kDa glycoprotein with a binding site for lipid A, the active component of LPS (43). The binding of LPS to LBP appears to be monomeric, with a dissociation constant of 10" M (43). The region of LBP between 9  amino acids 7 and 10 is necessary for signaling, but not for LPS binding, as constructs lacking this region, although capable of binding LPS, are unable to mediate LPS-dependent upregulation of IL-6 in U373 cells, integrin activation in neutrophils, or NF-KB activation in U937 cells (44). The N-terminal 197 residues of the protein are involved in LPS binding (45). In the presence of LBP, rabbit peritoneal exudate M<> js respond to LPS at < 1 ng/ml by secreting TNF-a. Under these conditions, cells also show more rapid induction of cytokine mRNA, higher steady-state mRNA levels and increased mRNA stability for  TNF-a  when  compared to serum-free conditions (46,47). Similar effects of LBP are observed in the responses of rabbit and human alveolar M<> js to LPS (48). The role of LBP is believed to be to bind endotoxin in blood and to transfer it to CD 14 at the cell surface. Thus, by making LPS  12 more readily available to the cell, LBP lowers the threshold concentration of LPS required to activate CD 14-positive cells (49). Wright and coworkers (50) described a distinct plasma constituent named septin, also able to bind to LPS. Septin activity is present at very high levels in plasma and is thought to be formed by at least two protein species requiring proteolytic degradation of one of the components for LPS opsonic activity. The biological activity of septin appears to be similar to that of LBP and a model has been proposed in which, like LBP, septin mediates binding of LPS to CD 14 in M<|>s and PMNs. According to this model, the concentration of LBP in serum under basal conditions -in the absence of an acute phase response- is too low to mediate efficient transfer of LPS to CD 14. This model proposes that under normal conditions, septin and not LBP is the principal means for opsonizing LPS for recognition by CD 14. As discussed above, over 90% of the total pool of CD 14 detectable in the circulation is accounted for by soluble CD 14 (sCD 14) with the remainder being associated with Ntys or PMNs. Soluble CD 14 is estimated to have a concentration in plasma of approximately 5 Lig/ml (51). There appears to be two sources for sCD 14: (i) mCD 14 is shed from the cell surface by protease digestion, and (ii) before export to the cell surface, some of the intracellular pool of CD 14 escapes the GPI-anchoring process and is secreted. The latter mechanism is supported by the finding that part of the sCD14 shed from cells spontaneously at 37 °C lacks ethanolamine present in the GPI-tail of normal mCD14 (16). Additional evidence that this form of sCDI 4, present in the culture supernatant of CD14 cells as well as in various body +  fluids, is not derived from mCD14, is its molecular size of 56 kDa. In contrast, sCD14 generated from proteolytic release of mCD14 has a molecular mass of 48 kDa. Proteins reserved for GPI anchoring have a C-terminal hydrophobic region of about 30 amino acids that  13 serve as a signal peptide for an enzyme responsible for GPI tailing. Normally, this peptide is replaced by the GPI anchor. Escape from this process, results in an unprocessed C-terminus and direct secretion. As demonstrated by Bufler ec al. (52), the larger (56 kDa) form of sCD14 is not truncated at the C-terminus implying that the protein escapes the GPI anchor attachment and is directly secreted. The surprising finding that some CD 14-negative cells are able to respond to LPS suggested the possibility that other LPS receptors besides CD 14 might be present in these cells, or that sCD14 present in blood might be involved in mediating these responses. In the presence, but not in the absence of normal human serum (NHS), CD 14-negative human umbilical vein endothelial cells (HUVEC) respond to LPS by producing increased levels of PAF (53), TGF-p and intercellular cell adhesion molecule I (ICAM-1) (54), and activation of the transcription factor NF-KB (55). Frey and coworkers (56) demonstrated that in the presence of NHS, HUVEC respond to LPS in the range of 1-10 ng/ml with increased production of endothelial-leukocyte adhesion molecule 1 (ELAM-1) after a 4 h treatment, and U373 cells produce IL-6 after 24 h of incubation with 0.1-100 ug/ml LPS. CD 14rnegative pulmonary arterial endothelial bovine cells (CPAE) also respond to LPS in the presence of NHS resulting in enhanced cell permeability and cell death. None of these effects of LPS in these two cell types are observed if serum is omitted or if it has been immunodepleted of sCDI 4 (56). If sCDI 4 acts in a manner similar to LBP, i.e. simply as a carrier for LPS, it would be expected that LBP could substitute for sCD14 in this system. However, the involvement of LBP in this system seems negligible as it is not able to support the responses brought about by the presence of sCD 14. In addition, Frey and coworkers found that IL-6 production by CD 14-negative U373 cells in the presence of NHS is also blocked by preincubation with anti-CD 14 antibodies, but  14 not by control anti-CD 18 antibodies. Haziot et al. (54) noted that LPS is able to bind to sCD14 directly, and that under serum-free conditions, 5-50 ng/ml of LPS elicits ICAM-1 expression in human endothelial cells if sCD 14 is present. However, working with lower concentrations of LPS (5 ng/ml), it was found that sCD 14-stimulated activation is enhanced in the presence of LBP. These results suggest that sCD 14 alone is sufficient for opsonization and presentation of LPS to CD 14-negative cells. As a corollary to this, for cells devoid of mCDI 4, the lack of surface anchor does not hinder the capacity of sCD 14 to bring LPS to the cell surface in a manner that elicits functional responses. This implies that sCD 14 may bind to additional proteins on the cell resulting in transduction of a signal. In this respect, Vita et al. reported specific and saturable binding of sCD 14 in a manner indicative of a receptor-ligand interaction, consistent with the presence of a cell surface binding structure for complexes of LPS andsCD14(57). The observed effects of sCD14 on CD 14-positive cells have been somewhat inconsistent Human M<|>s show increased synthesis of PAF when stimulated with LPS (1 ng/ml) in the presence of LBP. However, when sCDI 4 is added to LPS-LBP, a significant reduction of this response is observed (53). A similar effect is observed in peripheral blood M(|>s, where increased secretion of TNF-a in response to LPS in the presence of LBP is blocked by sCDI 4 in a dose dependent manner (58).  In the murine model of endotoxic shock, the use of  recombinant sCD 14 has proven beneficial as it is able to protect animals from LPS-induced mortality (58). These results indicate that the predominant effect of sCD 14 on CD 14 positive cells is inhibitory in respect to LPS action. By contrast,freshlyisolated PMNs show LPS-induced adherence even when sCDI 4 and LBP are present simultaneously (59). It has also been found (49) that the sensitivity of human M(|>s (IL-6 production) and neutrophils (adherence) to  15 complexes of LPS-LBP is the same as for complexes of LPS-sCD 14. Thus, depending upon the experimental conditions, the effects of sCDI 4 on CD 14+ cells have been observed to be either inhibitory or indifferent when LBP is also present, and in fact, sCDI 4 can substitute for LBP in some systems.  Several models can be proposed to explain how LPS might induce a signal through binding to CD 14. In the first model, when LPS or LPS-LBP bind to mCD14, the receptor directly initiates a signaling cascade leading to activation of the cells. The fact that anti-CD 14 antibodies mimic some LPS-elicited responses such as induction of homotypic adhesion (60), indicates that occupancy of CD 14 alone might be sufficient to elicit a response. CD 14, however, does not need to be the signal transducer perse. A protein analogous to gpl 30 in the IL-6 receptor complex could be active in the LPS system and be responsible for transducing the signal into the cell after LPS binds to CD 14. A second model may be envisioned in which CD 14 brings LPS to the membrane, acting like a shuttle, delivering it to a transmembrane coreceptor which is responsible for propagation of the signal. CD 14 may also function as a "pattern recognition" receptor with the capacity to bind structural components common to multiple microbial ligands, but without providing by itself ligand-specific responses. With respect to ligand binding, CD 14 appears not to discriminate between stimulatory molecules, and those that are antagonistic to the effects of LPS. For instance, CD 14 is capable of binding to lipid A, to soluble peptidoglycan from gram positive bacteria (61), or to lipoarabinomannan from Mycobacteria (62), as well as to antagonists, such as lipid IV (63), deacylated LPS (64,65), or LPS from Rhodobacter spheroides (66,67). A  Despite binding to CD 14, M<|) responses to these various ligands are disparate. These findings  16 point to the presence of an as yet unidentified element, distinct from CD 14 that is responsible for signal propagation in response to LPS which imparts functional specificity.  LPS-INDUCED CELL SIGNALING Protein Tyrosine Kinases  The functional consequences of M(|> activation by LPS have been studied extensively and are well described [reviewed in (8,9,13)]. However, the details of the intracellular events that take place after engagement of LPS at the cell surface are only now beginning to be elucidated. Ligand-induced protein tyrosine phosphorylation is a rapid and common event mediating subsequent intracellular responses through diverse receptors in many cell types (68). Increased protein tyrosine phosphorylation in response to LPS is also observed in murine and human M<f>s (69-71), and in B cells transfected with CD 14 (36,37). In the presence of sCD14, bovine and human endothelial cells also respond to LPS with transiently increased tyrosine phosphorylation (72). Tyrosine phosphorylation appears to be important for downstream events taking place after LPS stimulation. Treatment with the protein tyrosine kinase (PTK) inhibitor herbimycin prevents LPS-induced release of eicosanoid mediators from the murine M(|) cell line, RAW 264.7 (69). Herbimycin also blocks LPS-induced tumoricidal activity (73,74) and nitric oxide synthase activity of peritoneal Ntys (21). Protein tyrosine kinase inhibitors of the tyrphostin family have also been observed to protect mice against LPS-induced lethal toxicity (75). Inhibition of tyrosine phosphorylation by these various compounds appears to correlate with their capacity to block LPS-induced production of TNF-a and nitric oxide by M<)>s in vitro, as well as LPS-induced production of TNF-a in vivo.  17 The importance of tyrosine kinase activity for LPS-elicited responses of Ntys contrasts withfindingsin CHO cells transfected with CD14. CHO cells expressing CD 14 show M<b-like responsiveness to LPS as treatment with endotoxin results in the production of arachidonate in a dose and time dependent manner similar to that observed in M<|)S (26). However, in contrast to M<j>s, no increased tyrosine phosphorylation is observed in CD 14 positive CHO cells after LPS treatment Furthermore, in CD 14-transfected CHO cells, translocation of NF-KB by LPS, usually considered equivalent to its activation, is PTK-independent This conclusion is based upon thefindingthat LPS-induced translocation of NF-KB is not blocked by pretreatment with either herbimycin A or genistein (76). Thesefindingssuggest that tyrosine phosphorylation is not always an essential event in eliciting functional responses to LPS.  NF-KB  had been thought  to play a central role in the transcription of the IL-1 p gene. However, in contrast to the finding on translocation of NF-KB in CHO cells, LPS-induced expression of IL-1 p in M<|>s, is attenuated by PTK inhibitors. These apparently conflicting results, can be explained nevertheless, by the fact that even though treatment with tyrosine kinase inhibitors does not block  NF-KB  translocation, its ability to induce transcription is impaired under these conditions, indicating that translocation to the nucleus perse might not be sufficient to induce transcription (77). At least some of the tyrosine kinases that become activated in LPS-treated Ntys belong to the Src family of protein tyrosine kinases. In addition, treatment of macrophages with either LPS or LPS and interferon-gamma (IFN-y) has been shown to increase steady-state levels of piJ/itf -* (78,79). Stefanova etal. (80) demonstrated that in human Ntys,  ^58/6^and  7  77  LPS treatment results in the transient activation of pSJ/SS^,  ^58/64"^, and p5?fe . In this r  system, despite its lack of a transmembrane domain, CD 14 was shown to associate with v53/56fy in LPS-treated cells. In this regard, CD 14 is similar to other GPI-linked molecules n  18 such as decay accelerating factor, CD59, CD55, and CD48 which have also been reported to associate with Src tyrosine kinases in agonist-treated cells (81-83). Despite the unambiguous evidence that LPS brings about activation of Src kinases in M<t>s, it is not clear to what extent this is mechanistically important Thus, Meng and Lowell (84) recently showed that peritoneal and bone marrow-derived M<> js from hck-/-fgr-/-/yn-/-tr\p\e knock-out mice have no major defects in LPS-induced activation in terms of nitrite production and cytokine secretion. Similarly, it was found that the tumoricidal capacities of bone marrow-derived Mfys from these animals were appropriately enhanced after LPS stimulation in vivo. In the case of peritoneal M(j)S, induction of tumoricidal activity was only partially impaired. These results seem to indicate that despite the biochemical evidence that Hck, Fgr and Lyn are activated by LPS treatment of M<|)S, cells devoid of these proteins and expressing no other detectable Src-family kinases, appear to have normal functional responses to LPS. It is important to note, however, that the responses measured by Meng and Lowell were induced after prolonged exposure to IFN-y and LPS. Under similar conditions, Ziegler and coworkers (78) reported that exposure to LPS for 6-24 h resulted in increased expression of Hck in human macrophages and this was potentiated by priming of cells with IFNJ-y. Therefore, the possibility exists that incubation of macrophagesfromknockout mice for prolonged periods with LPS and IFN-y may induce the de novo expression of protein kinases -including Src-family kinases- that are not abundant under  basal conditions. These newly induced enzymes may be able to compensate for the absence of Lyn, Fgr, and Hck.  19  Phosphatidylinositol 3-kinase  Non-receptor tyrosine kinases have been shown to associate not only with cell surface receptors, but also with cytosolic proteins.  In this regard, the p85/p110 lipid kinase,  phosphatidylinositol 3-kinase (PI 3-kinase) has been found to associate with tyrosine kinases and with other tyrosine phosphorylated proteins in response to a variety of signals including those mediated by growth factors, cytokines and G proteins (85-87). Prior to the studies reported in this thesis, no relationship between LPS signaling and PI 3-kinase had been established. However, since PI 3-kinase is known to be activated in association with tyrosine kinases -some of which are known to be regulated by LPS-, we postulated that PI 3-kinase may be involved in LPS cell activation. PI 3-kinase phosphorylates the hydroxyl group at position 3 (D3) on the inositol ring of phosphoinositides (88).  The most thoroughly studied of the PI 3-kinase family is a  heterodimeric form comprised of a catalytic subunit (either pi 10a or pi 10p) and a regulatory subunit (p85a or p85p). The interactions of p85/p110 PI 3-kinase with other tyrosine phosphorylated signaling molecules appear to be mediated by SH2 domains in p85 and phosphotyrosine residues in the interacting proteins. A second mechanism for this association involves SH3 domains in p85 and proline-rich regions in the associated proteins (89,90), or vice versa (91). These interactions lead to the activation of the p 110 catalytic subunit of PI 3kinase resulting in increased levels of D-3-phosphorylated metabolites of phosphatidylinositol. Another PI 34dnase enzyme, pi lOy, present in neutrophils and U937 cells, is specifically activated by G protein py subunits (92). This PI 3-kinase does not interact with any of the p85 subunits described thus far, and possesses a pleckstrin homology domain near its amino terminus (93). A third PI 3-kinase family member has recently been described by Stephens  20 and coworkers. This Gpy-activated PI 3-kinase is a heterodimer comprised of a 120 kDa protein highly related to pi 10y, and a pi 01 subunit that is not substantially related to protein in current protein sequence databases (94). The tight association existing between these subunits seems to be the mechanism for activation of the enzyme, as it leads to an amplified effect of Gpy on the PI 3-kinase activity of pi 20. The roles played by PI 3-kinase metabolites in cell regulation are not completely clear. Levels of these metabolites, especially Ptdlns3,4P2 and Ptdlns3,4,5P3 (Figure 3), in resting cells are negligible. Cell activation by various agonists, including membrane lg cross-linking in B lymphocytes (95,96), IL-1 treatment offibroblasts(97), and fMLP activation of neutrophils (88), leads to transient increases in their concentration. This supports a role for these polyphosphoinositides as second messengers. In contrast to the activity displayed by the enzyme in vitro, where Ptdlns is the preferred substrate resulting in the formation of Ptdlns3P, levels of this metabolite change minimally in vivo in response to agonist treatment (85). The D-3 phosphorylated phosphoinositides are not substrates for phospholipases, thus they are not sources of either inositol phosphates, or diacylgiycerols. D3-phosphorylated phosphoinositides have been reported to bind to SH2 domains of Src and the p85 subunit of PI 3-kinase, to PH domains of Akt/PKB and to the non-receptor, Bruton's tyrosine kinase and to other proteins with novel phosphotyrosine binding domains [reviewed in (98)].  21  Ins1,4,5/>j  P'4K Ptdlns  r. Ai *oPI(4)P5K „ Ptdlns4/>  ?t<\\x\sZP  Figure 3.  J (  ,  /PLC  R N  Ptdlns^S/^^  Ptdlns3 4^ 7  Metabolism of phosphoinositides.  • Diacylglycerol  Ptdlns3 4 5/ /  /  ? J  Generation of different inositol-derived  metabolites and the enzymes involved. K= kinase; Pl/Ptdlns = phosphatidylinositol; PLC = phospholipase C; Ptase = phosphatase.  Several downstream signaling elements appear to be regulated by PI 3-kinase. This lipid kinase, for instance, appears to mediate activation of v70  ,  s6k  as treatment of cells with the PI  3-kinase inhibitor, wortmannin, prevents activation of \>70  s6l(  (99) and partially inhibits  activation of p42 and p44 MAP kinases by growth hormone (100), as well as platelet activating factor-induced MAPK activation in neutrophils (101). Similarly, activation p70*** through the platelet-derived growth factor (PDGF) receptor seems to be mediated by PI 3kinase, as activation of pJO  s6k  is reduced either in PDGF-treated cells expressing a receptor  unable to bind PI 3-kinase, or in normal cells after treatment with wortmannin (102). The protooncogene product, protein kinase B (Akt/PKB) is also activated by growth factors operating through PI 3-kinase. It has been shown that activation of PKB is inhibited by wortmannin and by coexpression of a dominant negative mutant of PI 3-kinase (103). In addition, Akt-1/PKB is able to bind to Ptdlns3,4,5P3 in vitro, but this binding does not affect  22 the enzymatic activity of the protein (104). Thesefindingssuggest that some other protein or factor may be required for activation of Akt/PKB in vivo, but that binding to phosphoinositides might recruit PKB to the membrane where it is activated in the context of other elements. Thus, PI 3-kinase, or its metabolites, may act as priming agents, in a manner analogous to the mechanism by which Ras is thought to prime c-Raf for activation. The involvement of PI 3kinase in the activation of Akt/PKB is reinforced byfindingsthat Akt can be directly regulated by phosphatidylinositol 3,4-bisphosphate both in vivo and in vitro (105). The PH domain present in the amino terminal region of PKB is known to bind to D3 inositol phospholipids in vitro (106), and PKB mutated at the PH domain is activated neither by PI 3-kinase in vivo nor  by Ptdlns3,4P2 in vitro (105). Other proteins with PH domains such as cytohesin and "general receptors for phosphoinositides" (CRPs), have also been shown to bind Ptdlns3,4,5P3 in vitro (107). A Ptdlns3,4P2-dependent protein kinase-1 (PDK1) is also directly activated by both Ptdlns3,4P2 and Ptdlns3,4,5P3, and interestingly, has been found to phosphorylate and activate Akt in vitro (108). Other potential targets of a PI 3-kinase cascade include some members of the protein kinase C family (PKC). Certain PKC isoforms have been reported to be activated by PI 3-kinase metabolites (109) and this aspect is discussed below. The functional alterations mediated by PI 3-kinase are only now being elucidated. PI 3kinase has been implicated in protein sorting, as the sequence of the catalytic subunit pi 10, shows homology with another lipid kinase Vps34p, a yeast enzyme implicated in sorting of proteins to vacuoles (86,110). In neutrophils, functional responses to chemotactic peptides have been linked to activation of PI 3-kinase. Thus, the PI 3-kinase inhibitors wortmannin and LY294002 have been shown to block B cell proliferative responses (111), fMLP-induced superoxide production (112,113), and neutrophils treated with wortmannin develop  (  -  23 oscillatory changes in F-actin content (114). In contrast to observations in neutrophils where PI 3-kinase does not appear to be involved in cytoskeletal rearrangements (113,114), treatment of T cells with wortmannin leads to collapse of the actin architecture (115).  Protein kinase Cand LPS cell signaling  An important, ubiquitous family of protein kinases also known to be involved in diverse signaling pathways in mammalian cells is protein kinase C (PKC). PKC participates in cellular differentiation, maturation and proliferation. This family of enzymes includes multiple isoforms with different biochemical characteristics and distinct patterns oftissuedistribution. Eleven isoforms of PKC have been identified in mammalian tissues thus far, and they have been divided into three subfamilies (116): classical or conventional PKCs (cPKC), new or novel PKCs (nPKC) and atypical PKCs (aPKC). cPKC isoforms (a, pi, pll, y) are activated by diacylgiycerol (DAG) and C a . Tumor-promoting phorbol esters act as analogues of the physiological +2  activator DAG, and activate some PKC isoforms in vitro. The nPKC isoforms (8, e, rj, 9) do not require C a  2+  and exhibit enzymatic activity in the presence of phosphatidylserine (PS) and  DAG or phorbol esters, whereas the activities of the members of the third PKC subfamily, aPKC (t;, X/i) are not affected by either DAG, C a  +2  or phorbol esters.  Regulation of atypical PKC subfamily members is noteworthy for marked differences from that of other PKC isoforms. Phorbol esters have been shown to have profound effects on several members of the PKC family. Brief exposure of cells to phorbol 12-myristate, 13-acetate (PMA), for instance, results in translocation of some PKC isoforms to the membrane resulting in enhanced enzymatic activity. As well, prolonged exposure of cells to this tumor promoting agent results in reduced expression and downregulation of certain PKC isoforms [reviewed in  24 (117)]. In contrast, phorbol ester treatment of cells does not translocate PKC< to the membrane (118), does not bind to it (119), and fails to alter its expression (120). The primary structure of aPKC family members explains, at least in part, their atypical biochemical characteristics (Figure 3). For example, atypical PKQ have only the first of two cysteine-rich zinc-finger motifs, common to all other isoforms of PKC. These cysteine-rich motifs have been reported to function in the phospholipid-dependent binding of tumor-promoting phorbol esters or DAG, thus explaining the insensitivity of the aPKC isoforms to PMA. Likewise, the C2 domain present in cPKC members confers calcium sensitivity upon conventional isoforms of the enzyme. Both nPKCs and aPKC lack a C2 domain and hence are insensitive to C a  2+  (Figure  4) (121,122).  Regulatory Domain  VI Zeta Lambda/ Iota  Cl  Catalytic domain  C2  V3  C3 V4  C4  V5  X A  Cys-Motif  ATP-binding site  Figure 4. Structure of the atypical PKC isoforms. From Zhou G. etal. (122). The regulation of aPKC family members in vivo, especially of PKC-t;, is not yet fully understood, but the substrate preferences of these enzymes and the effects of different cofactors that modulate their activities in vitro have been examined (123). As stated above, in  25 contrast to other PKC isoforms, PKC-i; expression is not downregulated by chronic exposure to phorbol esters. Depending on the assay conditions, the cell type, or whether it is a recombinant protein, wide variations in the biochemical characteristics of PKC< have been reported. Several investigators have shown the activation of PKG< by PI 3-kinase metabolites. In particular, Ptdlns3,4P and Ptdlns3,4,5P3 have been shown to stimulate the activities of PKC-e; and PKC-i 2  (109,124). The role of PKC in LPS signaling has been somewhat controversial. Findings supporting the participation of PKC in LPS signaling include experiments in which pretreatment of J774 cells with the PKC inhibitor H-7, abrogates LPS-stimulated tumoricidal activity (125). Similarly, it has also been shown that LPS transiently activates a PKC in human monocytes and treatment of these cells with H-7 completely blocks TNF-a and IL-1 p secretion in response to LPS (126). Intracellular levels of the endogenous PKC activator DAG, were not, however, significantly modified by endotoxin in this study, indicating activation of a DAG-independent PKC isoform. The identity of the specific isoform(s) involved was not determined. Translocation of PKC has frequently been associated with its activation, and this phenomenon has been reported for different effectors, especially for phorbol esters. Thioglycolate-elicited murine peritoneal M<|>s show LPS-induced translocation of PKC-p to the cytoskeleton. This effect is observed in LPS-responsive C3H/HeN mice, but not in LPShyporesponsive C3H/He] mice.  Nevertheless, cells from the latter animals show PKC  translocation in response to PMA (127). Results with murine macrophages, however, have not been consistent in respect to PKC activation. For instance, Hamilton and coworkers were unable to show LPS stimulation of Ca /DAG/PS-dependent PKC in murine peritoneal M<|>s +2  (128).  26 Numerous PKC-isoforms have been found to be expressed in primary M<> js and in M<> j cell lines. For instance, thioglycolate-elicited peritoneal M<|>s from C3H/HeN (LPS-sensitive), and C3H/He] (LPS-hyporesponsive) mice express the conventional PKC-p isoform, as well as two members of the nPKC family, PKC-5 and PKC-s. PKC<, as well as PKCp-ll and PKC-s were observed to be expressed in the murine M<> | cell line ]774 (129), and PKC-p, and PKC-a have been detected in human monocytes by Western blot analysis (130).  The human  promonocytic cell line U937 expresses, both PKC-pi and PKC-pll, as well as PKC-£ (131). The participation of PKC-t; in downstream events such as activation of NF-KB has been demonstrated in several different systems. For example, activation of NF-KB is observed in Xenopus laevis oocytes after insulin treatment or following microinjection of PC-phospholipase C (PC-PLC). This effect is abrogated when cells are microinjected with peptide Z, a specific PKC-d; inhibitor based on the pseudosubstrate region of the enzyme (132). Likewise, overexpression of PKC-t; in NIH-3T3 fibroblasts is sufficient to stimulate a persistent translocation of functionally active  NF-KB.  Transfection of a kinase-defective, dominant  negative mutant of PKC-c; dramatically inhibits tcB-dependent transactivation of a reporter gene (133) as well as activation of NF-KB by sphingomyelinase in NIH-3T3 cells (134). As discussed previously, LPS brings about the activation of NF-KB in M<|>s and this suggests the possibility that PKC-c; may play a role in the induction of functional responses elicited by LPS.  Ceramide-activated protein kinase  Ceramide-activated protein kinase (CAK) is a 97 kDa membrane-associated Ser/Thr protein kinase. The enzyme appears to be identical to kinase suppressor of Ras previously detected in Caenorhabditis elegans and Drosophila (135). CAK is activated in vitro by  27 ceramide, but not by other lipids such as diacylglycerol or arachidonic acid, and is able to phosphorylate Raf-1 very efficiently in vitro (136). LPS has also been shown to activate CAK in HL-60 cells and in human neutrophils in a CD 14-dependent manner. Activation of this enzyme by LPS, in contrast to its activation by IL-1 or TNF, appears to occur independent of the hydrolysis o f sphingomyelin and the generation o f ceramide (34). Rather, LPS appears to act by mimicking the second messenger function o f ceramide in these cells. Not surprisingly then, the LPS and ceramide signaling pathways appear to be regulated by some common elements. Macrophages from C3H/Ou] mice not only fail to respond to LPS, but also do not respond to ceramide analogues that enhance expression o f LPS-inducible genes in LPS sensitive mice (137). These findings suggests the possibility that CAK may be involved in LPS stimulusresponse coupling.  Mitogen-activated protein kinases, p2 l  ras  and other signaling proteins  An additional signaling pathway potentially activated by LPS is one involving activation o f mitogen activated protein kinases (MAPK). Indeed, human and murine M(()S exposed to LPS, show increased tyrosine phosphorylation o f a number o f proteins, including the 42 and 44 kDa isoforms o f MAP kinase (MAPK2 and MAPK-1, respectively, also known as ERK2 and ERK1) (70). Dong et al., demonstrated that cells from the LPS-responsive strain C3H/HeN show increased tyrosine phosphorylation o f numerous proteins including three with subunit sizes o f 35, 41 and 45 kDa. Based upon immune complex kinase assays, the 41 and 45 kDa proteins appear to be respectively, MAPK2 and MAPK1 (138). In contrast, activation o f MAPK was not observed in cells from LPS-unresponsive C3H/He] mice. In human neutrophils, in addition to the activation o f MAPK1 and MAPK2, exposure to LPS results in tyrosine  28  phosphorylation and activation of p38 MAPK. This occurs in a dose-dependent manner through CD 14 (139). Cloning and characterization of p38 has confirmed it to be member of the MAPK family (140). Increased tyrosine phosphorylation of p38 is also observed in response to LPS in 70Z/3 cells transfected with human CD 14, and in peritoneal exudate M<> js from LPS-responsive mice (C3HeB/FeJ). On the other hand, cells derivedfromLPSunresponsive C3H/HeJ mice do not respond to LPS with increased phosphorylation of p38. On the other hand, exposure of these cells to hyperosmolarity does activate p38. Because M<|>s from hyporesponsive mice express normal levels of CD 14, these results indicates that the defect in cellsfromthese mice is localized to a functional element located distal to the LPS receptor and proximal to p38/p42/p44 MAPKs and CAK (140). Activation of MAP kinases by growth factors and mitogens has been shown to involve a series of phosphorylating enzymes. One of the earliest events observed after engagement of certain cell surface receptors is the activation of p21 and with it, activation of a protein ras  kinase cascade consisting of the Raf-1 kinase, the MAP kinase activator, MAP/ERK kinase (MEK), and ultimately MAP-kinases [reviewed in (141,142)]. The involvement of p21 in ras  the activation of MAPK by LPS in M<)>s is as yet not clear. The murine macrophage cell line BAC-1.2F5, when stimulated with LPS shows increased MAP kinase activity in a Raf-dependent manner. This is also independent of increased p21 * activity (143), whereas in the same /3  system, activation of MAP kinase via colony stimulating factor 1 (CSF-1) involves activation of both Raf-1 kinase and p21 . The different signaling pathways recruited by these two agonists ras  may underlie the contrasting responses elicited by these compounds in M(|>s. Thus, CSF-1 stimulates proliferation of M(|>s, while LPS inhibits cell growth and stimulates differentiation and activation. In contrast to these findings, Geng et al. (144) reported that LPS stimulation of  29 human M<|>s results in activation of $53/56 y l  n  and this is associated with tyrosine  phosphorylation of Vav, and activation of p21 . The product of the protooncogene vav is a ras  guanine nucleotide exchange factor for Ras, specifically expressed in cells of lymphoid lineage that becomes tyrosine phosphorylated upon cell activation. Nevertheless, these findings remain to be confirmed since in other studies, LPS did not activate Ras or Vav in human monocytes (Herrera-Velit P. and N. Reiner, unpublished, see below).  Thesis Rationale and Objectives  General Objective Activation of M<)>s in response to extracellular signals is essential to induce immunological competence and maximal effector functions. While there is an extensive body of knowledge about the functional changes in M<|>s after activation, less known is about the signaling pathways used to bring about these changes. The activation process is known to involve a series of intracellular events including activation of protein kinases, gene transcription and new protein synthesis. To understand cell regulation leading to Nty activation, it is important to define the signaling elements involved in the process. As LPS is one of the most potent agonists of M<|>s, the studies described below examined signal transduction events taking place in response to LPS and their potential relationship to M<> j activation.  Hypotheses and Specific Objectives - Hypothesis 1.  LPS-mediated cell signaling in human Ntys involves activation of tyrosine  kinases signaling through the LPS receptor, CD 14.  30 Rationale: Increased protein tyrosine phosphorylation and activation of tyrosine kinases is a common phenomenon resulting from engagement of cell surface receptors with different agonists. LPS is a potent effector of M<> | responses and tyrosine phosphorylation of different cellular proteins in response to LPS has been reported for M<> j s of different origins. This signal may originate at the Nty membrane through CD 14, a monocyte differentiation, cell surface molecule described as an endotoxin receptor.  Hypothesis 2. LPS brings about the activation of PI 3-kinase leading to increased levels of D3phosphorylated phosphoinositides. Rationale: Both receptor and non-receptor tyrosine kinases have been shown to associate with the lipid kinase, PI 3-kinase, leading to its activation. PI 3-kinase phosphorylates position D3 of inositol phospholipids resulting in a dramatic and transient increases in the intracellular levels of these novel second messengers. This lipid kinase associates with different proteins, including tyrosine kinases, and activation of PI 3-kinase results from this association. LPS has been found to activate several protein tyrosine kinases in myeloid cells, thus indicating the possible stimulation of PI 3-kinase through a pathway involving activated tyrosine kinases.  Hypothesis 3. LPS treatment of M<|>s involves the activation of p21 and the Ras guanine m  nucleotide exchanger Vav. Rationale: Activation of different enzymes, including MAPK, PKC and PI 3-kinase has been reported to be linked to activation of the protooncogene product p21 . Activation of Ras ras  may occur through Vav, a guanine nucleotide exchanger for Ras present in several lymphocytic cells. Activation of M<|>s and neutrophils by different agonists, including LPS, has  31 been shown to result In activation of MAPK and some isoforms of PKC. This indicates that some LPS responses may be Ras-dependent  Hypothesis 4. LPS activation of PI 3-kinase, leading to increased levels of D3-phosphorylated inositol phospholipids, results in activation of PKC-c;, an atypical isoform of PKC. Rationale: The activation of PI 3-kinase by different agonists results in increased levels of D3 phosphoinosiddes. These metabolites have been shown to activate atypical isoforms of PKC-c; in vitro. LPS-treatment of M<|>s results in activation of a PKC isoform independent of calcium  and DAG for its activity. These features are characteristic of members of the aPKC subfamily, including PKC-^.  Hypothesis 5. LPS activation of Nty-PI 3-kinase regulates monocyte adherence and cytokine induction. Rationale: LPS treatment of M<|)S results in increased production of different cytokines, and enhanced cellular adhesion. LPS activates PI 3-kinase in M<|)S, and in T cells and neutrophils this lipid kinase has been linked to changes in F-actin content and to cytoskeletal rearrangements.  These findings suggest the possibility that PI 3-kinase may regulate  monocyte adherence and cytokine production in response to LPS.  32  Vlll. MATERIALS AND METHODS  A. MATERIALS  /. Antibodies:  Horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG was from BioRad laboratories (Mississauga, Ontario, Canada). Upstate Biotechnologies (Lake Placid, NY) were the suppliers of: mouse monoclonals anti-phosphotyrosine (4G10, #05-321), and anti- PI 3kinase, N-SH2, antibodies (UB93-3), and rabbit polyclonal antibodies against-MAP kinase (anti-ERK-1-CT), pan PKC, human lyn kinase ($51/56^"), PI 3-kinase, and PKC-C. A second polyclonal anti-PKC-C (C-20), anti-p70 S6 kinase (C-18), mouse monoclonal anti-PKC (MC5), and goat polyclonal anti-PI 3-kinase pi 10a (C-17) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). GIBCO (Burlington, Ontario, Canada) was supplier of a third rabbit polyclonal anti-PKC-C. Goat anti mouse HRPO IgG was purchased from Cedarlane Laboratories Ltd. (Hornby, Ontario, Canada). Mouse monoclonal anti-MAP kinase (#036600 recognizing both ERK1 and ERK2) was from Zymed Laboratories Inc. (San Francisco, CA). Anti-CD 14 monoclonal antibody 3C10 was a generous gift from Dr. Wesley C. Van Voorhis (University of Washington, Seattle, WA), rat monoclonal anti-p21 antibody Y13ras  259 was a gift from Dr. Vince Duronio (University of British Columbia, Vancouver, B.C.), and rabbit polyclonal antibody against p97 was a gift from Dr. Amnon Altman (Division of Cell KJ|,  Biology, La Jolla Institute of Allergy and Immunology, La jolla, CA).  33 2. Reagents  The following reagents were from Sigma Chemical Company (St. Louis, MO): agarose, ammonium acetate, aprotinin, benzamidine, boric acid, bovine serum albumin (BSA), bromophenol blue, dimethyldichlorosiline, cAMP-dependent protein kinase peptide inhibitor (TTYADFIASGRTGRRNAIHD), ethylendiaminetetraacetic  acid, disodium salt, dihydrate  (EDTA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) FicollPaque, p-giycerophosphate, herbimycin A, histone lll-S, isoamyl alcohol, lauryl sulfate, sodium salt (SDS), leupeptin, mineral oil, 3-[N-Morpholino]propanesulfonic acid (MOPS), myelin basic proteinfrombovine brain (MBP), pepstatin A, phenylmethylsulfonylfluoride(PMSF), La-phosphatidylinositol (Ptdlns), L-a-phosphatidylinositol 4,5-bisphosphate (Ptdlns4,5,/2)» L-a7  phosphatidyl-L-serine (PS), piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES), phorbol 12myristate 13-acetate (PMA), protamine sulfate, protein A Sepharose, RNase A, sodium chloride, soybean trypsin inhibitor, Trizma Base, Trizma hydrochloride, Tris-saturated phenol, xylene cyanol, and wortmannin. Reagents for polyacrylamide gel electrophoresis, protein Aagarose and Dc protein assay kit were from BioRad Laboratories. Peptide E and MAPK peptide substrate (APRTPGGRR) were from Upstate Biotechnology.  Rainbow molecular weight  markers, reagents and film for enhanced chemiluminiscence (ECL), P04 ", 5'[a- P]UTP, 32  3  32  triethylammonium salt (3000 Ci/mmol), [H]Ptdlns4/>and [H]Ptdlns4,5/^> were purchased 3  3  from Amersham International (Oakville, Ontario, Canada). ATP, CTP, GTP, and UTP (all 10 mM), and 100 mM dithiothreitol (DTT), nuclease-free water, 5X transcription buffer, bacteriophage T7 RNA polymerase, recombinant RNasin RQ1, RNase-free DNase, and proteinase K werefromPromega Corporation (Madison, WI). Yeast tRNA, formamide, RNase TI, TRIzol, LipofectAMlNE werefromGibco BRL. Human AB+ serum was provided by the  34 Canadian Red Cross (Vancouver, BC). RPMI 1640, phosphate-free RPMI 1640, Iscove's methylcellulose, penicillin/streptomycin and Hank's Balance Salt Solution (HBSS) were from Stem Cell Technologies (Vancouver, British Columbia, Canada). Lipopolysaccharide (£. coli. 0127:B8) wasfromDifco Laboratories (Detroit, Ml). Microcystin-LR, LY294002, and a,25dihydroxy-vitamin D3 were from Calbiochem (San Diego, CA). [y- P]ATP (sp. act. 3000 32  Ci/mmol) was obtainedfromDupont (Wilmington, DE). Mono Q HR5/5 columns and protein G-Sepharose werefromPharmacia Biotech Inc. (Piscataway, NJ). Unless specified otherwise, all reagents were of the highest quality available The human monocytic cell lines U937 and THP-1 were from the American Type Culture Collection (Rockville, MD). THP-1 cells stably transfected with CD 14 (THP1-WT) were provided by Dr. R. ]. Ulevitch (Scripps Research Institute, La jolla, CA).  B. METHODS  1. Isolation of monocytes and cell culture.  White cell enriched fractions of peripheral blood were obtained from normal human volunteers by the Cell Separator Unit of the Vancouver Hospital and Health Sciences Center (Vancouver, British Columbia) and fractionated by centrifugation (800 x ^for 15 min) over Ficoll Hypaque as previously described (145). After three washes in HBSS, the cells were resuspended in RPMI 1640 with 10% human AB + serum at 10 viable cells per ml, dispensed 7  into 150 cm cell cultureflasksat a density of 1.5 x 10 cells perflask,and incubated at 37 2  8  °C in a humidified atmosphere (5% CO2, 95% air [vol/vol]) for 45 min. Non-adherent cells were removed by vigorous washing with divalent cation-free HBSS (37 °C) and theflaskswere replenished with RPMI (without serum). The monolayers were allowed to equilibrate for 1 h  35 before further treatment Adherent monolayers prepared in this manner were 89% _+ 5% monocytes by morphologic and phagocytic criteria as determined by microscopic examination of preparations stained with Diff-Quik (CanLab, Vancouver, British Columbia). Cells were then either left untreated (controls) or incubated with different concentrations of LPS solubilized in RPMI + 10% human serum (final serum concentration 0.1%). Incubations were terminated by rinsingflaskswith ice-cold phosphate buffered saline (PBS: 1.0 mM NaH2P04, 8.1 mM NaHP04, 154 mM NaCI, pH 7.4) and monolayers were immediately frozen under liquid nitrogen and stored at -70 °C before further analysis. Cell lines were cultured in RPM I supplemented with 10% heat inactivated fetal calf serum (FCS) and incubated in a humidified atmosphere (5% CO2, 95% air [vol/vol]). THP1 -WT cells were maintained in a similar manner except that the medium was supplemented with G418 (350 ug/ml) for selection of positive transfectants. Unless otherwise stated, 12 to 15 h prior to incubation with LPS, cells were rendered quiescent in RPMI without FCS at a concentration of 5 x 10 cells/ml. 5  Following stimulation with LPS, cells were lysed  immediately and the detergent soluble material was frozen at -70 °C until further analysis.  2. Preparation of cell lysates  a. High speed supernatants Monolayers were rapidly thawed by the addition of ice-cold extraction buffer (pH 7.5), containing 20 mM Hepes, 12.5 mM p-glycerophosphate, 5 mM EDTA, 2 mM ECTA, 1 mM Na V04, 0.2 mM PMSF, 2.5 mM benzamidine, 5 mM 2-mercaptoethanol, 100 nM 3  microcystin, 2 ug/ml aprotinin, 2 ug/ml pepstatin, and 2 ng/ml leupeptin. Cells were dislodged and transferred to prechilled, Dounce glass homogenizers and disrupted by 20 strokes  36 on ice. After disruption, extracts were centrifuged at 100,000 x g for 60 min at 4 °C and high speed supernatants were assayed directly for protein kinase activity. b. Lysates for anion exchange chromatography Extracts were prepared by lysing cells on ice (20 min) in fast performance liquid chromatography (FPLC) extraction buffer (1 % Nonidet P-40, 12.5 mM MOPS, pH 7.5, 12.5 mM p-glycerophosphate, 2 mM EGTA, 1.0 mM Na V04, 1 mM PMSF, 100 nM 3  microcystin, 10 ug/ml aprotinin, 10 ug/ml leupeptin, and 10 ug/ml pepstatin). Lysates were centrifuged at 16,000 x g to remove insoluble material and were passed through a 0.2 um filter prior to chromatography. c. Lysates for analysis of phosphotyrosine content Cells lysates for phosphotyrosine analysis, and for anti-Lyn, anti-Vav, anti-CD 14, and anti-PI 3 kinase immunoprecipitates were prepared by addition of cold phosphotyrosine (PY) lysis buffer for 15-30 min at 4 °C (1% Triton X-100, 20 mM Tris-HCI, pH 8, 137 mM NaCI, 10% glycerol, 2 mM EDTA, 1 mM Na V0 , 5 mM NaF, 100 nM microcystin, 1 mM 3  4  PMSF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 10 ug/ml aprotinin; 1 ml per flask, for approximately 4 x 10 cells in suspension). Detergent-insoluble material was removed by 6  centrifugation for 15 min at 16,000 x g'm the cold.  3. Ras immunopredpitation  Adherent monocytes were incubated in phosphate-free RPMI for 1 h and labeled with [P]orthophosphate for 2 h (2 mG/150 cm flask). Following stimulation, medium was  32  2  removed and cells were rinsed in cold PBS. To increase labeling of cells, adherent monocytes  37 were removed from tissue cultureflasksby incubation with 5 ml of cold HBSS (divalent cationfree) and gentle scraping. Cells were resuspended in Pi-free R.PMI, and cell viability was assessed by trypan blue dye exclusion (~90% viable cells). After 1 h, cells were centrifuged and resuspended in RPMI + 0.5 mG/ml P-orthophosphate and incubated for 3 additional h. 32  Cells were then solubilized in ice cold buffer consisting of 50 mM Hepes (pH 7.4), 1% Triton X-100, 100 mM NaCI, 5 mM MgCl2, 2 ug/ml leupeptin, 1 uM pepstatin, 10 ug/ml soybean trypsin inhibitor and 40 ug/ml PMSF and centrifuged in a microfuge for 1 min. Supernatants were transferred to new tubes and were immediately made up to 500 mM NaCI, 0.5% sodium deoxycholate, and 0.05% SDS. Protein A-Sepharose beads were precoated with 0.1% BSA, rabbit anti-rat IgG antiserum (1 h) and anti^K^ antibody (Y13-259; 1-2 h) and added to the lysates. After incubation for 45-60 min, the beads were washed 4timeswith wash buffer (50 mM Hepes, pH 7.4, 0.1% Triton X-100, 500 mM NaCI, 0.005 % SDS, 5 mM MgCI) 2  and resuspended in elution buffer (2 mM EDTA, 2 mM DTT, 0.5 mM GTP and 0.5 mM GDP). Samples were incubated at 67 °C for 20 min after which they were centrifuged and 10 ul of supernatant were loaded into polyethylenimine (PEI) cellulose plates with fluorescent indicator. Samples were developed in 1.0 M  KH2PO4,  pH 3.4 and radioactive spots detected  on X-ray film were compared with unlabeled standards (GDP and GTP) detected using a UV lamp.  4. Anion-exchange chromatography  Detergent extracts were loaded onto a Mono Q FPLC column pre-equilibrated in buffer A (12.5 mM MOPS, pH 7.5, 12.5 mM p-giycerophosphate, 2 mM EGTA, and 0.5 mM Na3V04). Proteins were resolved with a 20 ml linear gradient of 0-0.8 M NaCI in buffer A  38 at a flow rate of 0.5 ml/min. Fractions of 0.25 ml were collected and aliquots were assayed for protein kinase activity or inimunoreactivity as described below. No significant MBP phosphorylating activity could be detected either in the column flow-through or wash fractions prior to initiation of the salt gradient  5. Immunoadsorption of kinase activity  Cell lysates (1 mg) prepared in FPLC-extraction buffer from LPS-treated U937 cells, were incubated with either 7.5 ug of two commercially available rabbit anti-protein kinase C-C. antibodies (Santa Cruz Biotechnology and Gibco) or with 7.5 ug of monoclonal anti-PKC antibody (recognizing a, p and y isoforms) for 2-3 h at 4 °C. Immune complexes were then incubated for 1 h with either protein A-agarose or protein G-Sepharose. Solid phase complexes were washed 2timeswith FPLC extraction buffer by centrifugation at 14,000 x g for 1 min and the supernatant fraction was subjected to a second immunoprecipitation with the same antibodies. Immunoadsorbed supernatants were fractionated by Mono Q chromatography as described above and fractions were analyzed by immunoblotting and protein kinase assays.  6. Western blotting  Samples were subjected to SDS-PAGE and separated proteins were transferred to nitrocellulose membranes. Membranes were blocked overnight at 4 °C, with 3% BSA dissolved in TBT-T (20 mM Tris, pH 7.4, 137 mM NaCI, 0.1% Tween 20) and immunoblotting was carried out by incubating the membranes for 2 h at room temperature (RT) with different monoclonal antibodies or with polyclonal rabbit antibodies raised against proteins of interest as described in figure legends. Immunoreactive bands were then detected  39 by incubation (1 h at RT) with horseradish peroxidase (HRPO) conjugates of either goat-antimouse or goat-anti-rabbit (1/5,000 dilution) antibodies and enhanced chemiluminescence (ECL) reagents (Amersham). When appropriate, after developing, antibodies were removed from membranes by incubating in stripping buffer containing 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol and 2% SDS at 50 °C for 30 min. Membranes were then blocked and reprobed with antibodies of interest as just described.  7. Immunoprecipitation  Cell lysates (-500 ug protein) were incubated with either a monoclonal antibody specific for PI 3-kinase, rabbit anti-Lyn antiserum or irrelevant control antibodies (normal mouse serum and normal rabbit serum, respectively) overnight at 4 °C. Immune complexes were collected by incubation with protein A-agarose for 1 h and washed 5timeswith lysis buffer. Samples were resuspended in Laemmli sample buffer (125 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 2% 2-mercaptoethanol plus bromophenol blue) and proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting.  8. In-situ renaturation kinase assay  Whole cell lysates prepared in PY lysis buffer were used for detection of protein tyrosine kinases in an in situ renaturation kinase assay with a polydispersed tyrosine kinase substrate, [poly(Glu,Tyr) 30 to 94 kDa)] as described by Durocher etal. (146). Briefly, samples were mixed with substrate and separated by PAGE. After electrophoresis, SDS was removed from gels, proteins were denatured in guanidine hydrochloride, and renatured by incubating in buffer containing 0.04% Tween and 10% sucrose (four washes for a total of 19 h). Gels were  40  equilibrated in 10 mM Hepes pH 7.4 plus 10 mM 2-mercaptoethanol, 10 mM MgCl2, 5 mM MnCl2 and 0.1 mM Na3VC»4 and kinase assays were performed in the same buffer with the addition of 100 uCi [y - P]ATP. Gels were soaked in 1 M KOH for 1 h at 56 °C to 32  eliminate signals from non-tyrosine kinase enzymes and after staining and drying, phosphorylated bands were detected by autoradiography.  9. Immune complex kinase assay  Immune complexes precipitated with anti-Lyn antiserum, monoclonal anti-CD 14 antibodies or appropriate controls plus protein A-agarose, were washed threetimeswith lysis buffer and four times with kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2). Washed immune complexes were suspended in 40 ul kinase assay buffer (20 mM Hepes, pH 7.4, 5 mM MnCI , 5 mM MgCI , 10 uM ATP, 10 uCi [y- P]ATP) and incubated at RT for 15 2  2  32  min. Reactions were stopped by adding 40 ul of 2X sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 2% 2-mercaptoethanol with bromophenol blue). Samples were boiled for 10 min and subjected to SDS-10% PAGE. Gels were dried and exposed to X-ray film at -70 °C.  10. PKC-C assays  Quiescent THP-1 cells were incubated with 1 ug/ml of LPS for different times or left untreated. When the effects of PI 3-kinase inhibitors were analyzed, cells were incubated with either 32 uM LY294002 or 100 nM wortmannin prior to LPS treatment for 10 min unless otherwise stated. Cells were immediately lysed for 30 min at 4 °C in lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCI with protease and phosphatase inhibitors at  41  the same concentrations as described for PY lysis buffer). Lysates were precleared with protein A-Sepharose and PKC< was immunoprecipitated with rabbit polyclonal anti-PKC-i; (5 ug per sample, Upstate Biotechnology). Kinase activity was measured in immunoprecipitates using MBP as substrate as previously described (115). Quantitation of kinase activity was done by excision of the band corresponding to MBP arid scintillation counting.  Incorporation of  radioactivity into MBP in the absence of lysate was used as background and was subtracted from radioactivity present in the immunoprecipitates.  / /. Protein kinase assays  Aliquots of either high speed supernatants or fractions from Mono Q chromatography (5 pi) were assayed for MBP phosphotransferase activity as described (147). When substrate preference of FPLC fractions was analyzed, substrates were used as indicated in the corresponding figures. Substrate phosphorylation was carried out in a final volume of 25 pi of kinase assay buffer (pH 7.5) containing 12.5 mM MOPS (pH 7.5), 12.5 mM pglycerophosphate, 2 mM EGTA, 0.5 mM Na V04, 2 mM DTT, 10 mM MgCI , MBP (0.5 3  2  mg/ml), 75 ng/ml cAMP dependent protein kinase inhibitor peptide and [y- P]ATP (50 32  uM). Reactions were allowed to continue for 10 min at 30 °C, unless otherwise stated, and were terminated by spotting 22 pi of the assay mixture onto Whatman P-81 phosphocellulose filter squares (1.5 x 1.5 cm ). Filters were washed sixtimeswith ice-cold 175 mM phosphoric 2  acid over a period of 60 min and incorporation of radioactivity was determined by liquid scintillation counting.  42 12. In vitro PI 3-kinase assay  Aliquots of cell lysates normalized for protein content (300-500 ug protein) were incubated overnight at 4 °C with either antibody (mAb) to PI 3-kinase, anti-Lyn antiserum or with appropriate control antibodies. Immune complexes were adsorbed onto protein A-agarose for 1 h. When 4G10 was used, it was first incubated with protein A-agarose for 1 h and cell lysates were then added for 2-4 h. Immunoprecipitates were washed twice with lysis buffer and threetimeswith 10 mM Tris-HCl, pH 7.4. PI 3-kinase activity was measured as described (148). Briefly, immunoprecipitates were incubated with 10 ug of sonicated Ptdlns and 10 uCi of [y- P]ATP in the presence of adenosine in afinalvolume of 40 ul. Reactions were carried 32  out for 15 min at room temperature and stopped by the addition of 0.1 ml of 1 N HQ and 0.2 ml of chloroform:methanol (1:1, v/v). Lipids were separated on oxalate-treated silica TLC plates using a solvent system of chloroform: methanol: water: 28% ammonia (45:35:7.5:2.5, v/v/v/v). Plates were exposed to X-rayfilmat -70 °C. Incorporation of radioactivity into lipids was quantitated by excising the corresponding portions of the TLC plate followed by liquid scintillation counting.  13. Cell labeling and extraction of inositol phospholipids  After adherence of monocytes to cultureflasks,cells were incubated for 30 min at 4 °C with cold HBSS, without Ca , or M g +2  +2  and removed gently with a cell scraper.  Monocytes were then pelleted by centrifugation and resuspended in phosphate-free RPMI in sterile polypropylene tubes. Cells were incubated at a density of 2-4 x 10 /ml for 1 h at 37 6  °C and labeled with 0.5 mG/ml PC>4 " for an additional 2 h. Samples containing equal 32  3  numbers of cells were treated or not with LPS for the indicated periods oftime,and incubations  43 were terminated by the addition of 3.75 volumes of methanohchloroform (2:1, v/v). Extraction of lipids was performed as described (95). Phases were separated by the addition of 1.25 volumes each of 2.4 N HQ and chloroform, and the lower organic phase was recovered after centrifugation. The upper phase was reextracted with 1 volume of chloroforrmmethanol (2:1, v/v). The combined lower phases were washed twice with 1 volume of methanohO.I M EDTA in water (1:0.9, v/v) and then dried under liquid nitrogen, resuspended in chloroform and loaded into oxalate-treated TLC plates for separation using a solvent system of npropanol:2M acetic acid (13.7:7, v/v). Plates were exposed to X-ray film at -70 °C and spots were excised and counted as described above.  14. HPLCanalysis of deacylated lipids  U937 cells cultured in RPMI plus 10% FCS were incubated in Pj-free RPMI (without serum) and labeled for 3 h with 0.5 mG/ml P04 " as done for normal human Ntys. Cells 32  3  were recovered by centrifugation and resuspended at 4 x 10 /ml before treatment with LPS 7  (100 ng/ml) for either 5 or 10 min. Lipids were extracted as described above. Dried lipids were deacylated by adding 1.8 ml methylamine solution (methanol/25% methylamine/nbutanol, 45.7:42.8,11.4, v/v/v) for 50 min at 53 °C (148). Samples were dried in vacuo, resuspended in water, and extracted three times with equal volumes of n-butanol:light petroleum ethenethyl formate (20:4:1, v/v/v).  The aqueous phase was dried again,  resuspended in water and stored at -70 °C until analyzed by HPLC on a Partisil SAX ion exchange column. Column runs were calibrated by coinjecting ADP and ATP as internal standards. Samples were separated with a 60 min linear gradient of 0 to 0.25 M ammonium phosphate, pH 3.8 followed by a 50 min gradient of 0.25 to 1 M ammonium phosphate, pH  44 3.8. Fractions (0.5 ml) were collected, mixed with scintillation fluid (Ecolite+, ICN) and quantitated by liquid scintillation counting.  The elutiontimesof deacylated Ptdlns3/',  Ptdlns3,4P2 and Ptdlns3,4,5P3 were determined by preparing these metabolites in an in vitro kinase assay with anti-PI 3-kinase immunoprecipitates using Ptdlns, Ptdlns4P and Ptdlns4,5P2 as substrates. The elutiontimesof deacylated Ptdlns4/'and Ptdlns4,5/^ were determined using [H]Ptdlns4P and [ H]Ptdlns4,5P . 3  3  2  15. Transfection of U937 cells  pSRa-based mammalian expression plasmids, containing the entire coding regions of either wild-type bovine  p85a  or mutant bovine  p 8 5 a (Ap85a)  were kindly provided by Masato  Kasuga (Kobe University School of Medicine, Kobe, Japan) (Figure 5). The mutant has a deletion of 35 amino acids from residues 479-513 of bovine p 8 5 a and the insertion of two other amino acids (Ser-Arg) in the deleted position. This alteration prevents the association of mutant p 8 5 a with the pi 10 catalytic subunit. However, mutant p 8 5 a is able to compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant (149).  45 A.  SR.M (EcoRI)/wild type p 8 5 = S R a / W p 8 5 Pstl  SRM(EcoRI)vector:  Hindi Accl Sail  EcoRI  —(—  BamHI  Smal  polyA  —i—  -+-  EcoRI cut  S m a l cut  T  SRM(EcoRI)vector/ wild-type p85  Kpnl  T  ^  polyA  polyA  p85 wild EcoRI-StuI fragment from original cDNA of p85a in Bluescript  B. S R a ( X b a l ) / A p 8 5 = S R a / A p 8 5 EcoRI SRa(Xbal) vector  Xbal  — i —  EcoRI  Kpnl polyA  1—  —I—  EcoRI cut  Kpnlcut  T  T  polyA Xbal site  T SRa(Xbal)/Ap85  Ap85 EcoRI-Xhol fragment from p85acDNA  A  EcoRI  SH3  polyA  +  Xhol-Kpnl fragment by PCR  Xhol  Xba site  TT  TT  SH2  1  SH2  k  Kpnl  Figure 5. Cloning of wild-type bovine p85a (A) and dominant negative mutant Ap85 (B).  46 U937 cells were grown to a density of 4-8 x 10 cells/ml in RPMI-1640 media 5  supplemented with 10% heat-inactivated FCS. Cells were washed and resuspended in 800 ul of antibiotic-free RPMI (without FCS). Transfection was done using LipofectAMiNE according to the protocol supplied by the manufacturer. pSRa plasmids containing either wild-type or mutant p85a were cotransfected along with pMCI neo-polyA, a plasmid encoding resistance to the antibiotic, G418 sulfate. DNA/liposome complexes were added to the cells followed by a 5 h incubation after which the cultures were supplemented with RPMI containing 10% FCS, penicillin, and gentamicin. Expression of foreign DNA was allowed to proceed for 2 days followed by the addition of 350 ug/ml of C418. After 4 days in G418, the cells were suspended in Iscove's methylcellulose supplemented with 2-mercaptoethanol, FCS, BSA, G418 and glutamine. The cells were incubated at 37 °C for ten days and colonies were picked and resuspended in 400 ul of RPMI + 10 % FCS supplemented with G418. Thereafter, cells were maintained in medium containing 350 ug/ml G418.  16. RNase Protection Assay  Cells were incubated with LPS (0.1 or 1 ug/ml) for 2 h and RNA was extracted from cells using Trizol according to the manufacturer's instruction. Equal amounts of RNA (5-10 pg) were subjected to an RNase protection assay as described by Hobbs et al. (150). One cytokine-specific probe template set, HL-14, was assembled from EcoRI -linearized and purified subclones. The template set synthesized a group of P-labeled riboprobes specific for several 32  cytokines (Table I). Radiolabeled anti-sense RNA probes were prepared and hybridized in excess with test RNA dissolved in hybridization buffer (80% formamide, 1 mM EDTA, 400 mM NaCI, 40 mM PIPES, pH 6.7) for 12-1 5 h at 56 °C. Single stranded unhybridized RNA  47 was digested with RNase A and RNase Tl. After treatment with a mixture of proteinase K (0.5 mg/ml) and yeast tRNA (100 mg/ml), RNA duplexes were isolated , dissolved in 80% formamide and dyes, and resolved in 5% acrylamide/8 M urea sequencing gels. Dried gels were placed on X-ray film and developed at -70 °C.  Table 1. HL-14 Template Set Cytokine  Protected mRNA  IL-6  320 bases  IL-10  294  IL-1a  269  TNF-p  220  GM-CSF  208  TGF-p  170  IL-1 p  149  TNF-a  124  L32  76  GeneBank Accession Number for nucleotide sequence for protected RNA is described in reference (150).  17. LPS induced adherence of M<j)S  Control protein (BSA) or fibronectin (20 mg/ml) dissolved in 50 mM bicarbonate buffer (pH 9.6) were added to 96 well cell culture dishes and incubated for 1 h at room temperature or overnight at 4 °C. Wells were blocked with 1% BSA in RPMI for 1 h at RT  48 and washed twice with RPMI. Cells were then added and incubated with the PI 34dnase inhibitors, wortmannin or LY294002 or with anti-CD 14 antibodies (10 ug/ml). Cells were stimulated with 1 ug/ml LPS overnight at 37 °C, washed and fixed with methanol/acetone (1:1 v/v) for 10 min at 4 °C. Fixative was removed and cells were stained for 10 min with a 0.05% solution of crystal violet dissolved in 20% ethanol. After washing and drying, remaining material was solubilized in methanol and adherence was measured as absorbance at 570 nm. Alternatively, cells were added directly to uncoated wells, incubated with inhibitors or with anti-CD 14 antibodies and then treated overnight with LPS as described above. After removing non-adherent cells, wells were observed at 200X magnification under an inverted microscope and the number or cells per field was taken as an estimate of adherence.  18. Protein determination  Protein concentrations of high speed supernatants were determined by Bradford method using Bio-Rad protein reagent and BSA as standard (151). Protein concentrations in detergent solubilized samples were determined by a modified Lowry method using Dc protein assay kit and BSA as standard (152).  49 IX. RESULTS  A. TYROSINE PHOSPHORYLATION AND TYROSINE KINASE ACTIVITIES INDUCED BY LPS IN M<DS /. LPS-induced tyrosine phosphorylation ofM<(> proteins  Treatment of peripheral blood M<t>s (PBM<|>s) with 100 ng/ml of LPS led to a rapid increase in tyrosine phosphorylation of several proteins as observed by phosphotyrosine immunoblotting with 4G10 antibodies (Figure 6A). Responses were observed within 2 min of treatment and returned almost to basal levels by 30-40 min.  The major tyrosine  phosphorylated bands showed approximate subunit sizes of 35-38 kDa, 42 kDa, 77-79 kDa and  117-120 kDa.  Enrichment of  phosphotyrosine  containing proteins by  immunoprecipitation with 4G10 allowed the detection of a similar pattern of tyrosine phosphorylation (e.g. 117-120 kDa band, bottom arrow head in Figure 6B is also observed in Figure 6A), and also of additional bands, including a protein of approximately 1 55 kDa (Figure 6B, top arrow head;). For comparison, parallel cell cultures were treated with 100 nM PMA. Although some proteins were phosphorylated in response to both agonists, the presence of phosphotyrosine-containing proteins unique to each agonist was detected (i.e. a 105 kDa band observed after PMA treatment, Figure 6B).  2. LPS induced tyrosine kinase activity  The observed tyrosine phosphorylation of the proteins in whole cell lysates could be a consequence of autophosphorylation of tyrosine kinases occurring after cell activation. The proteins undergoing tyrosine phosphorylation could also be substrates of tyrosine kinases that become activated in response to LPS. To examine directly for the presence of activated  50  tyrosine kinases, a renaturation kinase assay was performed using whole cell lysates from control and LPS stimulated PBM<j>s. This assay takes advantage of the fact that some protein kinases regain their enzymatic activity after PAGE and renaturation. By renaturing the enzyme in the polyacrylamide gel an approximate estimate of the molecular mass of the protein can be obtained. As seen in Figure 7, PBM<|>s show at least three bands with renaturable tyrosine kinase activities with apparent subunit sizes of 47-49, 53-55 and 60-65 kDa (arrow heads). The activities of these proteins are increased in LPS treated cells as early as 2 min and return to near basal levels after 30 min. Prolonged exposure of gels showed two additional bands of approximately 75 and 79 kDa (n=2).  + 15 Mw 200-  97-  69-  45 -  30  I  + 2  + 5  + 10  + 15  LPS  30  +  -  PMA  15  15  min  52  Mw 200-  C  2  5  10  15  30  PMA  Figure 6. Tyrosine phosphorylation of M<> | proteins in response to LPS. Adherent M<> js were incubated for the indicated times with LPS (100 ng/ml dissolved in RPMI + 10% HS, final HS concentration = 0.1%), 100 nM PMA (15 min), or medium alone. Reactions were stopped by rinsing theflaskswith cold PBS, cells were snap frozen, and kept at -70 °C until processing. Lysates were prepared as described in Materials and Methods and (A) 50 ug protein/lane were loaded onto a 10% polyacrylamide gel. Alternatively, (B) 250 pg of lysatesfromcontrol or LPS treated cells were immunoprecipitated with anti-phosphotyrosine antibodies and immune complexes were electrophoresed.  After electrophoresis proteins were transferred to  nitrocellulose membranes and blocked overnight at 4 °C with 3% BSA in TBST. Blots were incubated with 4G10 (10 ng/ml) for 2 h, followed by goat anti-mouse HRPO-conjugated secondary antibody and developed by ECL according to the manufacturer's instructions. The migration of molecular mass marker proteins are shown in kDa.  53  .J C  2  5  10  15  30  min  Figure 7. Activation of protein tyrosine kinases in M<|>s in response to LPS. Peripheral blood M<|>s were left untreated for 10 min (C) or were stimulated with 100 ng/ml LPS and reactions were stopped after the indicatedtimes.Lysates were boiled together with Poly(Glu,Tyr) and electrophoresed in a 10% polyacrylamide gel. Proteins were renatured in the gel and enzyme activity was detected by incorporation of [y- P]ATP as described in Materials and Methods. 32  Gels were stained and radioactive bands were detected by exposure to X-ray film at -70 °C (n = 3). The migration of molecular mass marker proteins are shown in kDa.  54 J. Association of CD 14 with tyrosine kinases and phosphotyrosine proteins in response to LPS  As many receptors are known to associate with tyrosine kinases after binding of their ligands, the association of CD 14 with tyrosine kinases was investigated. In vitro kinase assays using CD 14 immune complexes and performed in the absence of exogenous substrate showed the presence of several protein kinases in the range of 55-66 kDa. Enzymatic activities were increased in lysates from LPS stimulated cells, and although thetimecourse varied from donor to donor, increased activities were observed as early as 2 min, peaked by 5-10 min and decreased by 30-40 min of LPS treatment (Figure 8). CD14 associated not only with tyrosine kinases, but also with other tyrosine phosphorylated proteins lacking kinase activity. For example, 4G10 immunoblotting of CD 14 immunoprecipitates showed a tyrosine phosphorylated band of 170-180 kDa (Figure 9) in lysates from LPS-treated cells, whereas no immunoreactivity was observed in immunoprecipitates prepared using a control antibody. Tyrosine phosphorylation of this protein was increased in response to LPS in atimedependent manner, was maximal at 2-5 min and returned nearly to basal levels after 20 min of treatment  55  Mw  C  5  10  20  40  min  Figure 8. LPS induces the association of CD 14 with tyrosine kinases. Peripheral blood M<> js were left untreated for 10 min (C) or treated with 100 ng/ml LPS for the indicated times and lysed as described above.  Equal amounts of lysates (750-1000 ng/ml) were  immunoprecipitated with either anti-CD 14 monoclonal antibodies or an isotype matched control overnight at 4 °C. After washing with lysis buffer, immunoprecipitated material was subjected to an in  vitro  kinase assay without exogenous substrate in the presence of [y- P]ATP 32  as described in Materials and Methods. After 15 min, reactions were stopped by boiling for 10 min with Laemmli buffer, and samples were electrophoresed in a 7.5% polyacrylamide gel. Gels were stained, dried and exposed to X-ray film for detection of radioactive proteins. Decreased intensity of bands in the lane corresponding to 10 min are a result of underloading of the lane and not to decreased activity, as other experiments showed kinase activities comparable to or higher than 5 min of LPS treatment  Shown is one of four similar  experiments with cellsfromdifferent donors. Migration of molecular mass marker proteins are shown in kDa.  56  C  2  5 IgG2  20  C  b  2  5  20  min  a-CD14  Figure 9. LPS induces association of phosphotyrosine containing proteins with CD 14. After stimulation of PBNtys with medium alone for 5 min (C) or with LPS for the indicated times, cells were lysed and equal amounts of protein were immunoprecipitated with anti-CD 14 as described for Figure 8.  Immunoprecipitated material was electrophoresed in a 7.5%  polyacrylamide gel and proteins were transferred to nitrocellulose membranes for detection of tyrosine phosphorylated bands with 4G10 as described for Figure 6 (n = 3). Migration of the molecular mass marker proteins are shown in kDa.  57 4. LPStreatment activates p53/S6 y in M<j>s f  n  Treatment of PBM<j>s with LPS has been shown to bring about the activation of several Src-family tyrosine kinases including pSJ/SS^", pJ^and p5^ * (78-80). These responses c  are CD 14-dependenL In the case of p53/56 y , this involves physical association of the kinase ,  n  with CD 14 (80). When \>53/56^ immunoprecipitates were examined for kinase activity in an immune complex kinase assay, the enzyme was found to be more active in LPS treated samples when compared to control PBM(|>s (Figure 10A, B). This effect of LPS on $53/56^" activity was also examined in U937 cells. Whether left undifferentiated (Figure 11 A) or differentiated with vitamin D3 for 72 h (Figure 11B) this cell line showed increased activity of pS3/S6 y after 10 min treatment with 0.1 or 1 ug/ml /  n  LPS. As expected, pS3/S6 y activity in immunocomplexes was markedly reduced if cells were f  n  preincubated for 4 h with the tyrosine kinase inhibitor, herbimycin A (Herb) (5 ug/ml).  5  C  10  20  40  3  C  5 10 20 40 time (min)  min  59  Figure 10. Activation of pSJ/Sd^" in PBNtys treated with LPS. (A) Cells were left untreated ("C") or treated with 100 ng/ml LPS for the indicatedtimes(min). Cell extracts were prepared and equal amounts of protein (-500 mg) were used for immunoprecipitation overnight with anti-lyn antibodies (or normal rabbit serum as control, not shown) as described under Materials and Methods. Immune complex assays were performed in the absence of exogenous substrate and proteins separated by SDS-PAGE as described for Figure 8. (B) After exposure of gels to X-ray film, radioactive bands were excised from the gel and quantified by liquid scintillation counting. Activity is expressed as a ratio with respect to control untreated cells (n = 3, except for t= 40 min where only one determination was done).  60  mm 0  0.1  1.0  DMSO  0 0.1  1.0  p.g/ml LPS  1.0  ixg/ml LPS  Herb  B  • 0  0.1  1.0  DMSO  0 0.1 Herb  Figure 11. LPS-dependent activation of pSJ/SS^y" in undifferentiated and D3-differentiated U937 cells. U937 cells were either left undifferentiated (A) in normal culture medium as described in Materials and Methods, or were treated with 100 nM 1,25-dihydroxy vitamin D (D ) for 72 h (B). Twelve to 15 h before treatment with LPS, cells were left in RPMI (no 3  FCS) and incubated with 5 ng/ml herbimycin A or vehicle alone (DMSO) for 4 h. Cells were then stimulated for 10 min with the indicated amount of LPS or left untreated. Immunoprecipitates were prepared with anti-Lyn antibodies and immunocomplex kinase assays were performed as for Figure 10.  61  B.  SERINE/THREONINE KINASES ACTIVATED IN  MO>S  IN RESPONSE TO LPS  /. LPS inducestime-and concentration-dependent increases in phosphotransferase activi towards myelin basic protein  Cytosolic preparations from cells stimulated with LPS for 30 min showed a dosedependent increase of MBP-phosphotransferase activity. The stimulatory effects of LPS were first apparent with concentrations as low as 10 pg/ml, and maximal with 10-100 ng/ml (Figure 12A). LPS-elicited increases of MBP kinase activity were dependent on the incubation time. Increased enzymatic activity was first detected at 10 min of LPS treatment (100 ng/ml) and peaked by 20-30 min of stimulation (Figure 12B). By 40-60 min of LPS treatment, MBP kinase activity returned to a level similar to untreated, control cells.  In four different  experiments, activation of 2-5 fold over basal levels was observed with 100 ng/ml LPS.  0  103 10-210 1 100 101 102 LPS (ng/ml)  B 6  C  5 10 20 30 40 time (min)  63  Figure 12. LPS treatment of Ntys leads to increased myelin basic protein phosphorylating activity in a dose and time dependent manner. (A) PBNtys were stimulated with different concentrations of LPS for 30 min, or (B) with 100 ng/ml LPS for the indicatedtimes.High speed supernatants were prepared and protein kinase assays were performed using MBP as substrate as described in Materials and Methods. After 10 min the reactions were stopped by spotting the samples on phosphocellulose filter paper. Filters were washed fivetimesin 0.85% (v/v) o-phosphoric acid and incorporated radioactivity was measured by scintillation counting. Activity is expressed as a ratio with respect to control untreated cells; shown is one of three experiments with similar results.  64 2. Activation ofp42/p44 mitogen activated protein kinase by LPS in PBM<f>s  Following exposure of cells to LPS (10 or 100 ng/ml) for 30 min two distinct peaks of LPS-activated MBP phosphotransferase activity were resolved by chromatography of cell lysates on Mono Q (Figure 13A). In a series of experiments with cells from different donors, both peaks of activity were consistently observed. A third peak of MBP phosphotransferase activity eluting later in the gradient was also present, but this was not consistently observed in preparations from different donors. Fractions from Mono Q peaks one and two were subjected to SDS/PAGE on 10% gels and transferred to nitrocellulose for immunoblotting. Using rabbit polyclonal antibodies that recognized both ERK-1 and ERK-2 members of the MAP kinase family, two proteins with approximate subunit sizes of M 42,000 and M 44,000 were detected (denoted by the two r  r  arrowheads in Figure 13B, D, E) in fractions under peak one (the earliest eluting peak) from both control (Figure 13B) and LPS-treated cells (Figure 13D). In contrast, no immunoreactive MAP kinase protein was detected infractionscomprising either Mono Q peak two or three (data not shown). The anomalous appearance of p44 MAP kinase in fraction 33 (the distorted bands in Figures 13B and 13E) may have been due to the presence of a large amount of nonspecific protein of similar size (detected by staining with amido black, data not shown) that eluted in thisfractionand caused increased migration of p44 MAP kinase. This band could also be accounted for by the 38 kDa protein kinase Hog that is known to become tyrosine phosphorylated in neutrophils in response to LPS and is weakly recognized by erkl-CT antibodies (139). Membranes that had been immunoblotted with anti-MAP kinase antibodies were stripped and re-probed with the anti-phosphotyrosine monoclonal antibody 4G10. Two  65  proteins also of subunit sizes M 42,000 and 44,000, that coincided precisely with the p42 r  and p44 isoforms of MAP kinase were shown to become tyrosine phosphorylated within activated peak one fractionsfromLPS-treated cells (denoted by the two arrowheads in Figure I3E,fractions30 and 31). No evidence for tyrosine phosphorylation of these proteins was found in the corresponding Mono Qfractionspreparedfromextracts of untreated monocytes (Figure 13C). Other protein species showed increased tyrosine phosphorylation in fractions from extracts of LPS-treated cells, but not in extractsfromcontrol cells (data not shown). Although the identities of the latter proteins have not been determined, their presence is consistent with the observation that treatment of monocytes with LPS was observed to provoke increased tyrosine phosphorylation of multiple proteins as detected in whole cell immunoblots (Figure 6).  J. Analysis of protein kinase activity using a MAP kinase-specific peptide substrate  To characterize further the nature of the kinase activities present in Mono Q fractions from LPS-treated cells, in vitro kinase assays were carried out in parallel using either MBP or a MAP kinase-specific peptide substrate  APRTPGGRK  (147,153). As shown in Figure 14A,  LPS-activated phosphotransferase activity towards MBP was detected in Mono Q fractions from both, peaks one and two. In contrast, only fractions from Mono Q peak one demonstrated stimulated activity towards the MAP kinase-specific peptide (Figure 14B).  B  «- 46  Untreated + erkl-CT  < 46  c Untreated + PY Ab LPS ° + erkl-CT LPS + PY Ab  * < •  25  "~  ^—  28  30  —  31  ^  33  Fraction number  _  35  4 6  67  Figure 13.  Anion exchange chromatography of PBNtys shows several peaks of MBP-  phosphorylating activity stimulated by LPS. After 30 min of incubation with LPS (10 or 100 ng/ml) or vehicle (RPMI + 10% HS), detergent lysates were prepared and fractionated by anion exchange chromatography on a Mono Q column. Proteins were separated by a 20 ml linear gradient of 0-800 mM NaCI in buffer A (12.5 mM p-glycerophosphate, 12.5 mM MOPS, 2 mM EGTA, 0.5 mM ortho-vanadate). Fractions of 0.25 ml were collected and (A) aliquots were analyzed for MBP-kinase activity as described under Materials and Methods using MBP as substrate. Aliquots (75-100 ul) of fractions from control (B, C) or LPS treated samples (D, E) were analyzed for the presence of p42/p44 mitogen activated protein kinases by immunoblotting with erkl-CT (B, D), an antibody recognizing both isoforms of the enzyme. After stripping the blots of erkl-CT antibodies, the membranes were reprobed with 4G10 (C, E) for the detection of tyrosine phosphorylated proteins as for Figure 6. Arrowheads shown in B, D and E correspond to positions of p44 and p42 MAP kinases.  68  A  Traction number Figure 14.  MBP and synthetic MAP kinase peptide substrate reveal distinct substrate  specificities of peak one and peak two of MBP kinase activity.  After lysis and FPLC  fractionation, samplesfromcontrol (untreated) or LPS (100 ng/ml, 30 min) treated PBM(j>s were assayed for kinase activity using (A) myelin basic protein as described for Figure 12, or (B) the synthetic peptide (APRTPGGRR) as phosphate acceptors. Mono Q peak one was comprised of fractions 27-34 and peak two of fractions 35-42. For ease of reference between A and B, fractions 31 (Peak one) and 36 (Peak two) are labeled in both panels (arrows) (n=2).  69 4. Lipopolysaccharide-induced activation of protein kinase C-£ in monocytes  FractionsfromMono Q peaks one and two were also subjected to immunoblotting with anti-pan PKC antibodies. A prominent immunoreactive band with an approximate size of M  r  81,000 was detected in peak twofractionsfromMono Q that contained LPS-stimulated MBP phosphotransferase activity (data not shown). A corresponding protein, with an identical elution profile of subunit size M 80,000 was also detected in peak two fractions prepared from r  extracts of untreated cells. In contrast no immunoreactive PKC was detected in Mono Q peak onefractionsobtainedfromLPS-treated cells. Based upon size, this protein was believed likely to represent a PKC and indicated the possibility that the LPS-activated MBP kinase activity detected in peak two may be due to PKC. Compared to lysates preparedfromuntreated cells, the mean increase in activity of the major MBP-phosphotransferase activity peak was 2.5 ± 0.7 (mean ± S.E.M., n = 6) fold. Since this activity (Figure 15A) was detected in the absence of calcium, phospholipids, or diacylglycerol, this indicated that this activity was a lipid-independent isoform of PKC. The possible presence of PKC-C. was analyzed by immunoblotting fractions with an isoform-specific antibody. Immunoreactivity for PKC-C was observed in fractions corresponding to the peak of kinase activity (Figure 1 5B). The antibody recognized a protein of - M r 80,000-85,000 which was sometimes resolved into a doublet or triplet of closely migrating proteins. The specificity of antibody reactivity with this M r -80,000 protein complex was confirmed by peptide competition. Thus, as shown in Figure 15B, an excess of the PKC-C, peptide used as immunogen to raise the antibody specifically abrogated recognition of these bands, whereas non-specific reactivity with other proteins was unaffected.  70  The effects of LPS on PKG< were also examined in two human, promonocytic cell lines, THP-1 and U937. After Mono Q chromatography the MBP kinase activities exhibited similar elution profiles than that observed in peripheral blood monocytes, and immunoreactivity for PKC-£ corresponded with peak 2 of activity (Figure 16). Immunoprecipitation of PKC-C, from THP-1 cells showed increased activity towards MBP in the lysates from LPS treated samples (1 ug/ml) as compared to untreated cells (Figure 16C). LPS treatment resulted in a transient increase in kinase activity which was maximal by 10 min.  71  A 800  3  koo  o-l—»  P-T""9  20  y  9  30  i~7—i—|—i—i—  40  50  60  fraction #  B antibody  antibody & peptide  PKC-C-^ *  »§^  37  38 40  37  Fraction #  38 40  z n  72  Figure 15. LPS induces activation of PKC-C. (A) Human monocytes were incubated with LPS (100 ng/ml) or medium alone (final serum concentration, 0.1 %) for 1 5 min. Cells were then and fractionated by Mono Q anion exchange chromatography and fractions were assayed for MBP kinase activity as described in Materials and Methods and in Figure 13. The data shown arefromone experiment and are representative of results obtained in > 7 independent experiments. (B) Detection of PKC-C immunoreactivity infractionscorresponding to the major peak elutingfromthe column at 390-420 mM NaCI. Aliquots of the fractions (125 u.1) were analyzed for the presence of PKC-C by immunoblotting with anti-PKC-^ antibody. Specific recognition of the Mr 80,000-85,000 protein by anti-PKC-C, antibody was determined by immunoblotting in the absence or presence of peptide used to raise the antibody. Bands that were eliminated by preadsorbing the antibody with the competing peptide were deemed specific. The results shown are from one of three similar experiments.  (|iu/u|iu/|ouid)  74  c  time (min) Figure 16. LPS induces activation of PKO< activity in promonocyte cell lines. THP-1 or U937 cells were rendered quiescent in serum-free RPMI for 12 to 15 h and subsequently stimulated (15 min) with 1 ng/ml of LPS or medium as described in Materials and Methods. (A) Cells were lysed and subjected to Mono Q chromatography. MBP kinase activity in aliquots (5 ul) of each fraction from each cell line was determined as described in the legend to Figure 10. (B) Detection of PKC-C, immunoreactivity in fractions corresponding to the major peaks was done as described in the legend to Figure 14 (n = 3). (C) THP-1 cells were treated with LPS for the indicatedtimesand PKC-C, was immunoprecipitated from cell lysates with polyclonal anti-PKC< antibodies (Upstate Biotechnology). immunoprecipitates were measured using MBP as substrate.  In vitro kinase activities in the  75  5. Immunoadsorption of PKC-C activity  Immunodepletion was used to examine further whether the peak of MBP kinase activity was due to PKC-<. Prior to fractionation on Mono Q, lysates preparedfromLPS-treated U937 cells were either untreated or immunoadsorbed with either of two anti-PKC-£ antibodies. Anti-PKC antibodies able to recognize a, p, and y PKC isoforms were used as a control for specificity. As shown in Figure 17A, immunoadsorption of lysates with anti-PKC-i; antibodies resulted in a significant reduction in peak activity when compared to fractionsfromeither unadsorbed lysates or to lysates immunoadsorbed with control anti-PKC antibodies. In fact, peak MBP kinase activity was reduced by treatment with anti-PKC-^ to a level essentially equivalent to that observed in fractions from control (non-LPS-treated) cells. Immunoadsorption of MBP kinase activity also resulted in removal of PKC-C immunoreactivity from Mono Q fractions (Figure 1 7B) and this was observed with anti-PKC-C antibodies from two different sources. When either anti-cPKC (left panel, Figure 17B) or anti PKC-C antibodies were used for immunodepletion, a non-specific high molecular weight band was immunoprecipitated.  76  lysate pretreatrnent:  anti-PKC-C  immunoadsorbed  no antibody  PKC<  30  34  36  37  38  40  30 34  Fraction #  36  37  38  40  77  Figure 17.  Immunoadsorption of the major peak of MBP kinase activity with anti-PKC-C  antibodies. U937 cells were either stimulated with 1 ug/ml of LPS or medium alone (final serum  concentration,  0.1%)  and  detergent-soluble  lysates were  prepared for  immunoadsorption as described in Material and Methods. Lysates from medium control and LPS-stimulated cells were either untreated or subjected to immunoadsorption with either rabbit anti-PKC-C antibodies (Cl and C2, Gibco or Santa Cruz) or anti-cPKC antibodies (specificity control). (A) One mg of lysate from each sample was then fractionated by Mono Q chromatography and MBP kinase activities were measured as described in Materials and Methods. The data shown are from one of three independent experiments that gave similar results. (B) Aliquots of fractions from LPS-treated cells analyzed by immunoblotting with antiPKC-C to confirm depletion of PKC-C.  78 6. Biochemical characterization of PKC-C  Since PKC-£ is known to behave differently from other PKC family members, experiments were done to biochemically characterize the putative PKC< in Mono Q peak two. Aliquots of Mono Q fractions containing PKC< were analyzed for activity using multiple substrates as shown in Figure 18A.  Of the different substrates tested, the kinase  phosphorylated MBP (0.2 mg/ml), peptide e (84 uM), and S6 peptide (0.1 mM) with similar efficiencies. In comparison, activities towards kemptide (0.2 mg/ml), histone (0.2 mg/ml), and protamine sulfate (0.2 mg/ml) were lower. This profile of substrate preferences is consistent with previous reports for PKC-t; (109,118). Cofactor requirements for PKC-C, have also been found to differ from those of other PKC isoforms (109,154). Figure 18B shows the activity of the kinase toward MBP in the presence or absence of various PKC activators and cofactors. Ptdlns3,4,5Pj, an activator of PKC-C, enhanced the activity of the kinase in Mono Q fractions prepared from control cells (Figure 18B), but not in those preparedfromLPS-treated cells. Unlike Ptdlns3,4,5/j, neither ,  arachidonic acid (alone or with diacylglycerol) nor phosphatidylserine significantly enhanced the activity of the kinase when compared to the activity detected in the absence of added lipids (Figure 18B). PMA, a known activator of several PKC isoforms other than PKC< was also tested in the presence of PS and Ca . The addition of PS, C a , and PMA together, 2+  2+  resulted in an approximate 35% increase in activity when compared to activity in the absence of cofactors (Figure 18C). In contrast, fractions corresponding to cPKC (i.e. 15-16) showed a robust activation in response to the combination of PMA, PS, and C a . 2+  79  80  Figure 18. Substrate preference and cofactor dependency of monocyte PKC-£. Detergent lysates of human monocytes were prepared and fractionated by Mono Q chromatography. (A) Kinase activities were determined in peak 2 fractions (5 ul) using various substrates in the absence of cofactors. MBP, protamine sulfate, histone, and kemptide were used at 0.2 mg/ml. Peptide e was used at 84 uM and S6 peptide at 100 uM. Activity is referred to MBP (100 5). (B) Kinase activity was measured in the absence (Cont) or presence of various cofactors (Ptdlns3,4,5P, 0.1 uM; AA, 33 uM; PS, 12.5 uM) using MBP as substrate. (C) Peak kinase 3  activity was determined in the fractions corresponding to both PKC< and cPKC (fractions 1516) in the presence of 50 nM PMA, 100 uM free Ca , and 25 uM PS or absence of 2+  cofactors (Control). Results are expressed as percent of activity measured in the absence of any factors which was taken as 100%.  The values shown are the mean + s.e.m. of 3-4  independent experiments. *p=0.04 (control vs. PtdlnsP); **p=0.04 (control vs. PMA), 3  both by Student's t test  81 c. PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION IN LPS-TREATED M<DS  /. Detection ofPtdlns3,4,5Pj in vivo in LPS-treated cells  Activation of PI 3-kinase results in rapid and transient increases in the production of D3phosphorylated phosphoinositides. The effects of LPS treatment on the levels of these metabolites were examined in peripheral blood M(j>s as well as in the monocytic cell line U937. Cells were labeled with P-orthophosphate and were then incubated in the presence or absence of LPS. 32  Phospholipids were extracted and analyzed by thin layer chromatography. As shown in Figure 19, when compared to control cells, the incorporation of P into Ptdlns3,4,5P3 was increased in LPS 32  treated monocytes. This effect was relatively rapid and transient with maximal accumulation of Ptdlns3,4,5P3 observed between 10 min and 15 min. Using cells from different donors, the magnitude of this increase ranged between 2- to 11 -fold relative to control cells.  2. HPLC analysis of PI 3-kinase metabolites in U937cells  When normal human M(|>s were labeled with P-orthophosphate and lipids were extracted 32  for HPLC analysis it was found that the incorporation of radioactive material into the least abundant inositol phospholipids was not high enough to detect in this system (data not shown). This appeared to be due to relatively low specific activity of the monocyte ATP pool. In contrast, the ATP pool in the human promonocytic cell line U937 labeled to higher specific activity and was used, therefore, to study the effects of LPS on PI 3-kinase metabolites in vivo. As shown in Figure 20 and Table 2, treatment of cells with 100 ng/ml LPS brought about increased levels of the PI 3-kinase metabolites Ptdlns3,4P, and Ptdlns3,4,5P3. LPS treatment also resulted in 2  increased levels of Ptdlns4P, and Ptdlns4,5P2- These effects were observed as early as 5 min after addition of LPS and were persistent through 10 min (data not shown) of treatment  82  |  |  <" PIP3  time (min)  Figure 19. Ptdlns3,4,5P3 levels in M$s after LPS treatment (a) Human Ntys were labeled with [ Pj] for 2 h before stimulation with LPS (100 ng/ml) for the indicated periods. Lipids 32  were extracted and separated by TLC using a solvent system of n-propanol:2 M acetic acid (13.7:7, v/v) as described in Materials and Methods.  (b) Spots corresponding to  Ptdlns3,4,5P3 were excised and analyzed by liquid scintillation counting. The data shown are from one of three independent experiments.  ( £  . 0 i x ) -ui-d-3  84  Table 2.  Effects of LPS on Inositol phospholipids in U937 cells.  [ P]-labeled 32  phosphoinositides were extracted from control cells and cells treated with LPS for 5 min (100 ng/ml) as described under Materials and Methods and Figure 20. The data shown are the ratios of values observed in LPS-treated versus control, untreated cells (mean ± S.E.M., n = 3).  LPS 5 min Ptdlns3P *  1.24 ± 0.07  Ptdlns4P  1.16±0.1  Ptdlns3,4P  1.73+0.32  Ptdlns4,5P  1.39 ± 0.16  2  2  Ptdlns3,4,5P **  2.02 ±0.19  3  *p = 0.018 vs. control; **p = 0.014 vs. control  J. Wortmannin inhibits the generation ofPtdlnsJ, 4,5 Pj in vivo  Increased accumulation of Ptdlns3,4,5P in LPS-treated cells was likely related to 3  increased specific activity of PI 3-kinase in vivo and this was supported by experiments (described below) in which PI 3-kinase activity was examined directly in vitro in cell lysates. Nevertheless, LPS-induced inhibition of a phosphatase that acts on Ptdlns3,4,5P could also 3  have contributed to increased detection of this metabolite. To examine this possibility, Pj32  labeled cells were pretreated for 10 min with 100 nM wortmannin, a concentration considered  85 to have relative specificity for PI 3-kinase (114). Cells were then either left untreated or incubated with LPS. As shown in Figure 21, pretreatment of monocytes with wortmannin completely abrogated the LPS-induced increase in Ptdlns3,4,5P3. Taken together, these findings provide direct evidence for activation of PI 3-kinase in vivo by LPS.  4. LPS-induced activation of PI 3-kinase is CD 14-dependent  Many functional responses elicited by LPS in M<|)S are mediated through the cell surface receptor CD 14 (29) and antibodies to CD 14 are able to block a variety of LPS-induced responses (80). Incubation of Pj labeled cells with 3C10 (10 ug/ml for 30 min prior to LPS 32  treatment), a monoclonal antibody that blocks CD 14, completely abrogated the LPS-induced increase in Ptdlns3,4,5P3 (Figure 22, lane 4). This effect was specific to CD 14, since the expected increase in Ptdlns3,4,5P3 in LPS-treated cells was not prevented by pretreatment with an irrelevant isotype (lgG2b) matched monoclonal antibody, OKT4 (Figure 22, lane 6).  86  <-PlP  3  <- Ori C  2  5  10  20  40  C 10 <-+Wt-»  St  +>  > o  10  20  10  time (min)  Figure 21. Effects of wortmannin on Ptdlns3,4,5P3 levels. M<|>s were labeled with [ Pj] for 2 32  h as described and treated with 100 nM wortmannin or vehicle (MeSO) for 10 min. Cells 2  were then treated with 100 ng/ml LPS for the indicatedtimes,(a) Lipids were extracted and analyzed by TLC as indicated in Figure 18. The position of Ptdlns3,4,5P3 prepared in  vitro  and separated under the same conditions is shown (St). The signal in this lane migrating between the origin and PIP3 corresponds to [y- P]ATP from the in 32  vitro  reaction, (b) Spots  corresponding to Ptdlns3,4,5P3 were excised and quantified by scintillation counting. Activity is expressed as a ratio compared to control samples. Data shown are from one of three independent experiments.  87  1  Figure 22.  2  3  4  5  6  CD 14-dependence of enhanced Ptdlns3,4,5P3 levels in LPS-treated human  monocytes. M<|>s were labeled with [ Pj] for 2 h and left untreated (lanes I and 2), or were 32  incubated with 10 ng/ml 3C10 (lanes 3 and 4) or an irrelevant isotype matched control antibody (OKT4, lgG2b) Canes 5 and 6) for 30 min before treatment with 100 ng/ml LPS (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) for 10 min. Lipids were extracted and analyzed as described above (n = 3).  88  5. Kinetic analysis of PI 3-kinase activity in LPS-treated monocytes  Treatment of monocytes with LPS brought about a substantial increase in PI 3-kinase activity assayed in vitro (Figure 23). Cell lysates were immunoprecipitated with a monoclonal antibody specific for PI 3-kinase and enzyme activity was measured in an in vitro kinase assay using PI as substrate. Increased PI 3-kinase activity in samples from LPS treated cells was detected as early as 2 min and was consistently maximal after 10 min of treatment (range, 2.55.7 fold greater than control samples using cells fromfivedifferent donors). PI 3-kinase activity in LPS-treated cells returned to near basal levels by 40 min.  6. LPS dose-response analysis for activation of monocyte PI 3-kinase  Activation of monocyte PI 3-kinase was dose dependent and was detected with LPS concentrations as low as 10 pg/ml. Maximal activation was achieved at 1-10 ng/ml of LPS (Figure 24A).  Immunoprecipitation of PI 3-kinase from cell lysates yielded comparable  amounts of the M 85,000 regulatory subunitfromboth control and LPS-treated cells as r  detected by immunoblotting with a polyclonal antibody against p85 (Figure 24B). This result indicates that increased enzyme activity detected in samples from LPS-treated cells was not an artifact related to preferential immunoprecipitation of PI 3-kinase protein from these samples. Moreover, thisfindingprovides direct evidence that LPS treatment brings about an increase in the specific activity of monocyte PI 3-kinase.  89  PIP  •  •  •  •  •  §  C  2  5  10  20  40  < Ori min  b 5  o < 1  0  10  20  30  40  time (min)  Figure 23. Time course of PI 3-kinase activity in LPS-treated cells. Monocytes were incubated for the indicatedtimeswith LPS (100 ng/ml). Equivalent amounts of cell lysates were immunoprecipitated with monoclonal anti-PI 3-kinase antibodies, (a) Immune complexes were assayed for PI 3-kinase activity by measuring the incorporation of [y- P]ATP into Ptdlns. The 32  position of the migration of PtdlnsP (PIP) and the origin of chromatography are indicated, (b) Spots corresponding to PtdlnsP were excised and analyzed by liquid scintillation counting and the values obtained are expressed relative to control (480 cpm). Shown is one offivesimilar experiments.  90  0  0  1  0.1  1  10  100  ng/ml  Figure 24. Dose dependence of PI 3-kinase activation in response to LPS. (a) Monocytes were incubated for 10 min with the indicated concentrations of LPS and assayed for PI 3-kinase activity as in Figure 23. (b) Lysates corresponding to control (lanes 1 and 4), or LPS treated cells (1 ng/ml LPS: lanes 2 and 5; 100 ng/ml: lanes 3 and 6) were immunoprecipitated with normal mouse serum (lanes 1-3) or monoclonal anti PI 3-kinase antibody (lanes 4-6). Immunoprecipitated material was separated by polyacrylamide gel electrophoresis and immunoblotted with rabbit anti-PI 3-kinase. The positions of p85 and molecular weight markers are indicated. Data are representative of three experiments.  91  7. LPS treatment activates pSJ/S^y  7  and promotes its transient association with PI 3 kinase  As reported above, LPS brings about the activation of several tyrosine kinases, among them p53/56> . It has been observed that PI 3-kinase may associate with and be activated by /  w  a variety of both receptor and non-receptor tyrosine kinases (86). Taken together, these findings indicated the possibility that activation of PI 3-kinase in LPS-treated M<)>s might involve its association with activated ^SZ/S^y".  In fact, when lysates from anti-lyn immunoprecipitates  were examined in the in vitro PI 3-kinase assay, increased enzymatic activity was detected in samples prepared from LPS-treated cells (Figure 25). The association between p53/56> and /  77  PI 3-kinase exhibited atimecourse similar to that of PI 3-kinase activation per se (e.g. see Figure 23). Maximal activity was observed after 5-10 min of LPS treatment and by 40 min evidence of association was no longer present  When anti-lyn immunoprecipitates were  subjected to immunoblotting using an anti-PI 3-kinase rabbit serum a band corresponding to the M 85,000 regulatory subunit was not detected (data not shown). This indicated that the r  amount of enzyme that associated with p53/56> was only a small percentage of the total PI /  77  3-kinase present in the cell and this was below the sensitivity of the detection system. As discussed above, when pSZ/Sb ^ 1  immunoprecipitates were examined for Lyn  phosphotransferase activity in an immune complex kinase assay, the enzyme was found to be more active in LPS treated samples when compared to control samples (Figures 10 and 11). The kinetics of Lyn activation showed that this response was maximal at 10 min and this coincided with its maximal association with PI 3-kinase and with PI 3-kinase activation per se (Figure 25). Taken together, these results indicate that LPS is able to bring about the  92 coordinate activation of p53/56^ and PI 3-kinase and that these enzymes become transiently //7  associated for a period of time that corresponds to the duration of PI 3-kinase activation. Further evidence to suggest that activation of PI 3-kinase is dependent upon its association with activated p53/56 y" is provided by thefindingthat inhibition of Lyn activity ,  with the tyrosine kinase inhibitor herbimycin A abrogates activation of PI 3-kinase (Figure 26). Addition of herbimycin to PI 3-kinase immunoprecipitates preparedfromLPS-treated cells immediately before assay did not affect the lipid-kinase activity, indicating that the effect of herbimycin on intact cells was unlikely related to a direct effect on PI 3-kinase.  93  a  0  1  2  3  4  5  6  7  8  •  9  +-PIP  10  b  Figure 25. Association of p53/56> with PI 3-kinase. Cells were either left untreated (lanes I /  77  and 6) or treated with 100 ng/ml LPS (lanes 2-5, 7-10) for 5 (lanes 2 and 7), 10 (lanes 3 and 8), 20 (lanes 3 and 9) or 40 min (lanes 5 and 10). (a) Cell lysates were prepared as described and equivalent amounts of protein were used for immunoprecipitation with normal rabbit serum (lanes 1-5) or Lyn antiserum (lanes 6-10). Immunoprecipitates were assayed for PI 3-kinase activity using Ptdlns as substrate as described, (b) The autoradiogram was analyzed by densitometry, and the background signals from lanes 1 -5 were subtracted individually from the corresponding signals in lanes 6-10. The difference between lane 6 and lane 1 was given an activity value of 1, and the other activity values shown reflect the effects of LPS on PI 3kinase associated with p5J/5^ . m  Shown is one of three experiments with similar results.  95  Figure 26. Inhibition of PI 3-kinase activity by herbimycin A. U937 cells were pretreated with 5 ng/ml of herbimycin (lanes 4-6; solid bars) or with a similar volume of Me2SO (lanes I3, empty bars) for 4 h before any stimulation. Cells were then left untreated (lanes 1 and 4) or were stimulated with either 100 ng/ml (lanes 2 and 5), or 1 ng/ml (lanes 3, 6 and 7) of LPS for 5 min. Cell extracts were prepared and equivalent amounts of protein were used for (a) immunoprecipitation with anti-Lyn antibodies.  Immune-complex kinase assays were  performed as described in Figure 10. Bands corresponding to lyn were excised, radioactivity was measured by scintillation counting and activity was expressed as compared to control cells. (b) Parallel immunoprecipitations with monoclonal anti-PI 3-kinase antibodies were performed and PI 3-kinase activity was detected as described in Figure 23. Spots corresponding to PtdlnsP were excised and analyzed by liquid scintillation counting and expressed relative to control.  96 8. Tyrosine phosphorylation state of the M 85,000 subunit of PI 3-kinase r  The M 85,000 regulatory subunit of PI 3-kinase is known to become phosphorylated r  by a variety of tyrosine kinases in vitro. Furthermore, PI 3-kinase may be detected in antiphosphotyrosine immunoprecipitates prepared from different cell types following agonist treatment Whereas this implicated tyrosine phosphorylation of p85 in enzyme activation, the latter does not strictly correlate with higher levels of tyrosine phosphorylation of the regulatory subunit To examine whether p85 becomes phosphorylated on tyrosine in response to LPS in M<|>s, anti-PI 3-kinase immunoprecipitates were reprobed with 4G10. No detectable tyrosine phosphorylation of p85 was observed in either control or treated samples (data not shown). This negative result might be explained by the fact that the amount of tyrosine phosphorylated p85 brought down with the anti-PI 3-kinase antibody represented only a minor fraction of the total enzyme in the immunoprecipitates. To maximize the potential for detecting tyrosine phosphorylation of p85, cell lysates were immunoprecipitated with 4G10. As shown in Figure 27a, treatment of monocytes with LPS brought about an increase in the amount of PI 3-kinase activity that could be immunoprecipitated with anti-phosphotyrosine antibody 4G10. When parallel 4G10 immunoprecipitates were immunoblotted with 4G10, several proteins exhibited enhanced phosphorylation in response to LPS (Figure 27b). When this blot was stripped and reprobed with a polyclonal antibody specific for p85 it was found that p85 did not comigrate with any of the tyrosine phosphorylated bands previously detected (Figure 27c). This results indicates that tyrosine phosphorylation of the regulatory subunit of PI 3-kinase is not necessary for its activation in LPS-treated monocytes.  9 7  b  a  200-  c m  200-  1  Figure 27. PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Human M<|>s were either left untreated (1) or treated with 100 ng/ml LPS for 10 min (2). Cell lysates were prepared and immunoprecipitated with 4G10. (a) Immune complexes were either assayed for PI 3-kinase activity as described previously or were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with (b) polyclonal anti-PI 3-kinase or (c) 4G10. The positions of Ptdlns/', molecular weight markers and p85 are indicated (n=2).  98 9. Activation of PKC-C is phosphatidylinositol 3-kinase-dependent  Recent evidence has implicated a role for PI 3-kinase metabolites in a signaling cascade leading to activation of PKC (124). To examine the potential involvement of PI 3-kinase in LPS-induced activation of PKC-C, cells were incubated with the PI 3-kinase inhibitors wortmannin or LY294002, prior to the addition of LPS. As shown in Figure 28A, when used at concentrations known to be relatively selective for inhibition of PI 3-kinase, both inhibitors markedly attenuated activation of PKC-£ induced by LPS in peripheral blood monocytes. This effect was also observed in PKd; immunoprecipitated from THP-1 cells. Preincubation of the cells with 32 uM LY294002 or 100 nM wortmannin for 30 min resulted in the abrogation of LPS-stimulated PKC-C activity (Figure 28B).  '  The requirement for PI 3-kinase for activation of PKC-£ was also examined in cells transfected with a dominant negative mutant of p85 (Ap85). The expression of p85 in the different populations of U937 cells was examined (Figure 29). Lysates from non-transfected U937 cells (lane 1), and cells transfected with either Ap85 (lane 2), or with wild-type p85 (lane 3) were separated by PAGE and immunoblotted with anti-p85 antibodies. The results indicate that equivalent amount of p85 are expressed in these cell populations. Stable transfection with Ap85 resulted in a significant reduction in both basal and LPSstimulated PI 3-kinase activity (Figures 30a, b). In contrast, cells transfected with wild-type p85 showed the expected increase in PI 3-kinase activity in response to LPS stimulation, cells and PKC-C, immunoreactivity correlated with the peak kinase activity.  99  A  LPS  -  +  . +  Wortmannin  -  -  -  -  +  +  LY294002  _  +  +  +  100  Figure 28. Abrogation of LPS-induced activation of PKC-t; by the PI 3-kinase inhibitors, wortmannin or LY294002.  (A) Human monocytes were preincubated with 50 nM  wortmannin (n = 4), 16 uM LY294002 (n = 2), or vehicle for 15 min. Cells were then treated with 100 ng/ml of LPS or medium alone for an additional 15 min. Detergent lysates were prepared and fractionated by Mono Q chromatography. MBP kinase activities were determined in fractions (5 pi)fromeach cell preparation as described in Figure 11. (B) THP-1 cells were preincubated with either 100 nM wortmannin, 32 uM LY294002 or vehicle for 20 min prior to addition of LPS (1 ng/ml, 10 min). Lysates were immunoprecipitated with antiPKC-i; antibodies and MBP-kinase activity was measured in the immunoprecipitates as described in the legend to Figure 15C (n=2).  101  97-  69»  1  2  3  Figure 29. Expression of p85 in U937 cells. Fifty micrograms of whole cell lysates from untransfected (1), Ap85-transfected (2) and Wp85-transfected (3) U937 cells were electrophoresed and immunoblotted with polyclonal antibodies against the p85 subunit of PI 3kinase. Migration of molecular mass marker proteins are shown in kDa.  102  •  •  PIP  •  •  •  •  -< Ori  0  100  0  100  ng/mL  Ap85  Wp85  Ap85  Wp85  Figure 30. PI 3-kinase activity in U937 cells transfected with either wild-type, bovine p85a or dominant negative mutant, Ap85a. Cells were stimulated with 100 ng/ml LPS or medium alone followed by detergent lysis. (A) Lysates were immunoprecipitated with anti-PI 3-kinase antibody and phosphatidylinositol kinase activity was assayed as described in Materials and Methods. Radioactivity observed at the origin reflects residual, water-soluble "P-labeled material in the samples, the amount of which was not relevant to the results. (B) Spots corresponding to PtdlnsP (PIP) were excised and analyzed by scintillation counting. Activity is expressed as compared to control (untreated) cells transfected with wild-type p85a (Wp85). Results presented arefromone of two independent experiments with similar results.  103  To assess the effects of Ap85 on activation of PKC-£ by LPS, lysates of transfected cells were analyzed using Mono Q chromatography. As shown in Figure 31 A, activation of PKC-£ by LPS was abrogated in cells transfected with Ap85 while cells expressing wild-type p85 showed activation of PKC-C in response to LPS (Figure 31B). Figure 31C demonstrates that PKC-C was expressed in Ap85 transfected; no significant difference in the relative abundance of PKC-C in two additional experiments was observed.  104  Ap85  Wp85  P K C < -  32  35  36  37  41  32  34  Fraction #  35  36  40  105  Figure 31. Attenuation of LPS-induced activation of PKC-C, by a dominant negative mutant (Ap85) of the p85 subunit of PI 3-kinase. Quiescent, transfected U937 cells were treated either with LPS (1 ng/ml, 15 min) or medium alone and lysed in FPLC extraction buffer. Detergent lysates were fractionated by Mono Q chromatography as described in Materials and Methods. Aliquots (5 ul) of each fractionfromcells transfected with Ap85 (A) or with wildtype p85 (Wp85) (B) were assayed for MBP kinase activity in triplicate. The standard deviation of each data point was <20% of the mean. (C) Aliquots of fractions from LPStreated cells were analyzed by immunoblotting with anti-PKC-i; antibodies. The results shown are from one of four experiments with similar results.  106 D. EFFECTS OF LPS ON p2/  ras  AND THE V A V PROTOONCOGENE  p21 * (Ras) is a small G-protein which has been found to be positioned upstream of /a  MAPK as well as PI 3-kinase in some systems (141,155,156). The protein is present in cells coupled to either GDP or GTP. Growth factor stimulation results in an increase in GTP-Ras with a corresponding decrease in GDP-Ras and this is usually expressed as a change in the ratio of GTP-Ras to GDP-Ras. When PBM<|)S were examined, no consistent results were obtained to indicate that Ras activation was induced by LPS (data not shown). The guanine nucleotide status of p21  ras  is controlled by exchange proteins. The  product of the protooncogene vav has been reported to fulfill this function in cells of lymphocytic origin. In response to agonists, Vav is rapidly phosphorylated on tyrosine which activates the guanine nucleotide exchange activity of the protein. As shown in Figure 32A, when anti-Vav immunoprecipitates were electrophoresed and blotted, two immunoreactive bands of 90 and 104 kDa were observed with anti-Vav antibodies (arrow heads). When the blots were stripped and reprobed with 4G10 (Figure 32B), only a band comigrating with the fastest migrating band in the anti-Vav blot was detected. However, treatment with LPS did not bring about detectable increase in tyrosine phosphorylation of this protein. Taken together, these results indicate that p2f  ras  and  Vav are not involved in LPS signaling in human M<|>s.  Figure 32. Tyrosine phosphorylation of p97  V3y  \n PBM<|>s. PBM(|>s were treated with 100  ng/ml of LPS for the indicatedtimes,or were left untreated ("C"). Whole cell lysates were prepared and Vav was immunoprecipitated using Vav-antiserum. Immune complexes were separated in a 7.5% polyacrylamide gel and immunoblotted with anti-Vav antiserum (A). The arrows indicate bands of 95 and 98 kDa recognized by the anti-vav antibodies. After stripping of antibodies, the blots were re-probed with 4G10 for the detection of phosphotyrosine residues (B). The immunoreactive band denoted by the arrow in panel B, comigrates with the lower band in panel A.  108 E. PI 3-KINASE AND LPS-ELICITED FUNCTIONAL RESPONSES /. Effects of PI 3-kinase inhibitors on LPS-induced enhancement of cytokine mRNA levels  As reviewed above, the effect of LPS on M<)>s are pleiotropic. Amongst the most profound responses Induced by LPS Is the Induction of cytokine production (3,8). To examine whether cytokine induction by LPS requires activation of PI 3-kinase, a set of templates able to recognize mRNA for eight different monokines was used (Table 1). Treatment of THP-1 -WT (CD14 positive) cells with either 100 ng/ml or 1 ug/ml LPS for 3 h resulted in increased levels of mRNA for IL-I p, and TNF-a (Figure 31). Prolonged exposure of the gels allowed detection of enhanced mRNA levels for IL-1a as well. Pretreatment of cells with PI 3-kinase inhibitors, either wortmannin (100 nM), or LY294002 (32 uM), had no detectable effects on induction of cytokine mRNA levels by LPS. Treatment of undifferentiated U937 cells with LPS did not result in increased mRNA levels of any of the cytokines present in the H-14 set (data not shown). Thus, it was not possible to examine the effects of transfection of U937 cells with Ap85 on cytokine mRNA production.  2. PI 3-kinase and LPS-induced adhesion of M<l>s  The ability of M<|>s to adhere to substrates and to other cells is important for many of their functional activities. THP-1 -WT cells expressing CD14, were used to study the role of PI 3-kinase in LPS-induced adherence. Overnight treatment of THP-1 -WT cells with 1 ug/ml LPS resulted in increased adherence of cells to uncoated microtitre wells as compared to cells incubated with medium alone (34B vs. 34A). This effect was dependent on the LPS receptor, CD 14, as pretreatment with 10 ng/ml of anti-CD 14 markedly reduced the number of cells attached to the wells (Figure 34C and D). Treatment with 100 nM wortmannin (Figure 34E)  109 or 16 uM LY294002 (Figure 34F) for 30 min prior to LPS stimulation resulted in a marked reduction in LPS-induced adherence as compared to control, LPS-treated cells (Figure 34B). Quantitative resultsfromthis experiment are shown in Figure 34G . A parallel adhesion assay was performed using fibronectin as substrate and adherence was quantitated spectrophotometrically based upon retention of crystal violet dye. As seen in Figure 35, overnight treatment of THP-1 cells with LPS resulted in increased adherence of the cells to fibronectin. As described for adherence of cells directly to plastic, treatment of cells with the PI 3-kinase inhibitors resulted in a marked reduction of LPS-induced adherence of cells to fibronectin-coated wells (Figures 34 and 35). In contrast, adherence induced in response to PMA was unaffected by the presence of wortmannin.  110  IL-la~  TGF-P-*  a  b  c  a  b  c  + Wrt  a  b  c  a  b  c  +LY  Figure 33. Effect of PI 3-kinase inhibitors on mRNA levels of different cytokines. Quiescent THP-1 -WT cells were incubated with vehicle alone or with 100 nM wortmannin (+ Wrt) or 32 uM LY294002 ( + Ly) for 30 min, and then were either left untreated (a), or treated with 0.1 (b) or 1 ng/ml of LPS (c). Total RNA was extracted and P-labeled cytokine RNA probes 32  were used to detect specific, endogenous mRNAs in a ribonuclease protection assay as described under Materials and Methods.  111  112  *  *  •  m  M  *  t  c  *%•  *  te »  1  It  f*  #  ft «  * * *  9 -  »  f•• • •  • •  4 «.  *  •  •  -i.  •  114  G 750  Figure 34. LPS-induced adherence of THP-1-WT cells is PI 3-kinase dependent (A) THP-1WT cells were incubated with vehicle (A, B, C), with 10 ng/ml anti-CD 14 antibodies (C, D), with 100 nM wortmannin (E), or with 16 uM LY294002 (F). After 30 min, medium (A, C) or 1 ug/ml LPS (B, D, E, F) were added to the wells and incubated overnight Non-adherent cells were removed by rinsing with HBSS and the wells were observed using an inverted microscope at 200X magnification (n=2). (G) Adherent cells per field (minimum of 3 fields counted at 200X magnification) were counted for each of the treatments described in A-F (Ab = anti-CD 14, Ly = LY294002, W = wortmannin).  115  2.0  = I  1.5H  X  a* *a» O T3  ra  LO  i.oH  • Med • PMA ^ 0 . 1 LPS LPS • 10 LPS  v/i <U  <  0.5 0.0  a Wort-  Figure 35. THP1-WT cells were added to wells coated withfibronectin.Cells were then incubated either with vehicle or with wortmannin prior to addition of either 50 nM PMA or the indicated concentrations (ng/ml) of LPS overnight  Adherent cells were quantified  spectrophotometrically (570 nm) after staining with crystal violet as described in Materials and Methods (n=2).  116 X. DISCUSSION  Mononuclear phagocytes undergo many physiological and functional changes in response to LPS, these responses being initiated through CD 14, a LPS cell surface receptor. The importance of this receptor in M<> | activation is manifest by the findings that overexpression of CD 14 leads to hypersensitivity to endotoxic shock in mice (157) and M<> js derived from CD 14-knockout mice do not express TNF-a, IL-lp, and interferon-inducible protein-10 genes after exposure to low concentrations of LPS (158). One of the most rapid events reported after exposure of M<|)S to endotoxin is the increased tyrosine phosphorylation of proteins (69,70). These changes are apparently initiated through CD 14, as antibodies against CD 14 are able to block LPS-induced protein tyrosine phosphorylation (71). Working with freshly isolated peripheral blood  Mcjis,  we have been able  to demonstrate the increased tyrosine phosphorylation of a range of proteins after exposure to LPS (Figure 6). Similar effects were observed using the monocytic cell lines THP-1 and U937 (data not shown). These responses are rapid, with increased tyrosine phosphorylation being apparent as early as 2 min and returning to near basal levels by 30-40 min. Increased protein tyrosine phosphorylation is elicited in peripheral blood M<|>s with concentrations of LPS in the picogram/ml range reaching a plateau at 10 to 100 ng/ml LPS (data not shown). These changes are due, at least in part, to increased tyrosine kinase activity in LPS-treated cells. This conclusion is based in part upon renaturation kinase assays, in which it was shown that renaturable tyrosine kinases become activated in response to LPS (Figure 7). The proteins with the highest activity observed in whole cell lysates appeared as three bands in the 49-70 kDa range. In addition, when ^53/56^" was immunoprecipitated from equal amounts of whole cell  117 lysates, Increased in vitro kinase activity was found in peripheral blood M<j>s, as well as in U937 cells (Figures 8 and 9) after treatment with LPS. Stefanova* and coworkers have also reported that LPS-activated peripheral blood M<> js have increased protein tyrosine kinase activity. Specifically they observed activation of v58/64 , pSI/S^W, and pd^-fr (80). In a similar hck  manner, Beaty and workers (159) have shown increased Hck and Lyn kinase activity after LPS treatment of human monocytes. The involvement of these tyrosine kinases in downstream events is reinforced in this study by the fact that tyrosine kinase inhibitors are able to block the LPS-induced TNF-a and IL-6 production. Increased protein tyrosine kinase activity was also observed to be associated with CD 14 in LPS-treated cells (Figure 8). Although the tyrosine kinases associating with CD14 were not specifically identified, the apparent molecular weights of these enzymes were in the range of the Src-kinases. These observations could be related to increased amounts of enzymes becoming associated with CD 14 in response to LPS. On the other hand, the amounts of tyrosine kinases associated with CD 14 could be the same in control and LPS treated monocytes, but the increased kinase activity observed could result from enhanced specific activities of the enzymes in LPS-treated samples. Stefanova* and coworkers also demonstrated that pSJ/So^y"/ but not ^58/6^ or p69 - 8 , associates with CD 14 (80). The amounts of pSJ/S^" associated c  f  r  with CD 14 in both resting and LPS-stimulated cells was similar, but Lyn activity was increased in LPS-treated samples. This favors the argument that LPS induces changes in the specific activities of the Src kinases.  Taken together, thesefindingsindicate that $53/56^"  constitutively associates with CD 14 and its activity is modified by LPS. Lyn and other Src kinases have been shown to play an important role in the immune response. Mice homozygous for a disruption at the Lyn locus show defects in B cell and mast  118  cell functions implying an obligatory role for Lyn in cell signaling (160).  In addition,  macrophages from doubly homozygous hck'/'-fgr"/" mice, show impaired phagocytosis and increased susceptibility to infection with Listeria monocytogenes, indicating that either hck, fgr or both are required for effective host defense (161).  Despite these observations, the  importance of Lyn and other Src kinases in M<> | signaling has been questioned recently. Thus, M<|>s derivedfrommice deficient in Lyn, Hck, and Fgr, do respond to LPS if they are incubated with LPS and IFN-y for 6-48 h (84) suggesting that these kinases may be dispensable for LPS responses. However, these cells were not examined for their functional responses to LPS after short term exposure (e.g., <2 h). This is an important caveat, since while M(j>s derived from Src-triple knockout mice expressed neither mRNA nor proteins corresponding to these enzymes under basal conditions, it is possible that prolonged exposure to LPS and IFN-y may have led to the induction of other tyrosine kinases, including other members of the Src family. This possibility is supported by studies referred to above (78) in which prolonged incubation of M<|)S with LPS and IFN^ resulted in induction of the Src kinases, Hck and Lyn. Moreover, in the Lyn"-Hck"-Fgr"-cells, treatment with the general tyrosine kinase inhibitor, herbimycin, inhibited nitrite production induced by LPS and IFN-y indicating continued dependency upon tyrosine kinases for signaling, the possibility remains that Lyn, Hck and Fgr may be required for rapid responses to LPS, but that with longer term exposure to LPS and IFN-y, their absence may be compensated for by the induction of other tyrosine kinases. The association of tyrosine kinases with GPI-linked molecules to is not limited to CD 14, as several GPI-anchored proteins have been shown to associate with tyrosine kinases of the Srcfamily (83). The function of GPI anchors in proteins is still speculative. In the case of integral membrane proteins, the interaction of their cytoplasmic domains with cytoskeletal components  119 has the potential to influence their mobility. However, no such constraints apply to GPIanchored proteins which results in their having increased mobility in the plane of the membrane. This feature may be important for their interaction with other transmembrane proteins with the capacity to transduce signals into the cell. In this regard, we observed the association of CD 14 with a tyrosine phosphorylated protein of approximately 170 kDa (PY 170) in phosphotyrosine immunoblots of CD 14 immunoprecipitates from LPS-treated cells (Figure 9). It is unlikely that this protein is a protein tyrosine kinase perse, since no protein of similar size was observed either when CD 14 immune complexes were subjected to in vitro kinase assays (Figure 8), or when whole cell lysates were analyzed by renaturation kinases assays (Figure 7). The identity of this protein has not been established. However, among the potential candidates, and because of similarity in molecular mass, we examined the possibility of it being the insulin receptor substrate-1 (IRS-1). IRS-1 is a protein without tyrosine kinase activity that becomes tyrosine phosphorylated when insulin binds to its receptor (162). IRS-1 is able to bind both to the insulin receptor and to intracellular proteins, thereby serving to propagate extracellular signals inside the cell. No immunoreactivity was observed when CD 14immunoprecipitates, positive for PY170, were examined with antibodies raised against IRS-1 (data not shown). Nevertheless, the distinct possibility exists that PY170 is involved in relaying signals inside the cell after LPS binds to CD 14. In murine M(j>s, amongst the most prominently tyrosine phosphorylated bands appearing after LPS treatment are a series of 40-45 kDa proteins. These have been demonstrated to be the p42 and p44 isoforms of MAP kinase (69). In human blood M<|)S, we have consistently observed increased tyrosine phosphorylation of proteins with apparent molecular masses in the 40-45 kDa range. This led us to investigate the possibility that some of these may be members  120 of the MAPK family. As shown in Figure 13A, anion exchange chromatography of lysates from LPS treated peripheral blood M^s, showed two peaks of increased MBP-kinase activity. Immunoblotting with anti-erkl antibodies demonstrated the presence of p42 and p44 MAPKs and tyrosine phosphorylation of these proteins was markedly enhanced in LPS treated cells. Further evidence to support the conclusion that Mono Q peak one corresponded to p42/44 MAPKs was obtained based upon substrate specificity of the two peaks of MBP-kinase activity detected. Only Mono Q peak 1 was able to efficiently phosphorylate a peptide substrate specific for members of the MAPK family. As seen in Figure 14B, peak two showed almost negligible activity towards this substrate even though it was more efficient than peak one in phosphorylating MBP (Figure I4A). These findings support the conclusion that LPS treatment of human, peripheral blood mononuclear cells brings about the activation of two members of the MAPK family, MAPK1 and MAPK2. The importance of MAPK in  cell signaling is  strengthened by the finding that MAPKs become activated in M<> j s, not only in response to LPS (163,164), but also in response to.GM-CSF (163) and engagement of the TNF receptor (165). Furthermore, other cell types have also been shown to respond to LPS with activation of MAPK, including endothelial cells (72) and neutrophils (139). In addition to p42 and p44 MAPKs, a p38 MAPK isoform is also activated in response to LPS in M<|>s, neutrophils (139) and in 70Z/3 cells transfected with CD 14 (140). The activation of this isoform in our system cannot be excluded as antibodies specific for p38 MAPK were not used to probe Mono Q fractions. It is interesting to note, however, that a band of approximately 38-40 kDa appeared to be tyrosine phosphorylated and showed weak immunoreactivity with erk-CT antibodies (Figure 13). The importance of this enzyme in M<j>  121 signal transduction is indicated by the finding that a family of cytokine-suppressive antiinflammatory drugs are potent and selective inhibitors of this enzyme (166). Work done in this laboratory (167) and presented here (Figures 13A, 15A, 16A, and 17A), indicated that the second and later eluting Mono Q peak (peak two) of LPS-activated MBP kinase activity might be accounted for by an apparently calcium-independent isoform of PKC. The evidence supporting this includes: (i) reactivity of peak fractions with anti-pan PKC antibodies in immunoblots of a protein with an apparent subunit M of approximately 80,000r  81,000, (ii) immunoprecipitation of peak two MBP kinase activity with anti-pan PKC antibodies, (iii) sensitivity of this kinase to inhibition by a PKC inhibitor peptide, and (iv) detection of stimulated activity in the presence of 2 mM EGTA (free calcium below 10"& M) and in the absence of diacylglycerol and phosphatidylserine. The identification and mechanism of activation of this PKC is discussed below. Thefindingsthat Src-family tyrosine kinases are activated in LPS-treated M<|>s raised the question of potential downstream effectors and one possibility is the lipid kinase, PI 3-kinase. Concentrations of PI 3-kinase metabolites, such as Ptdlns3,4,5P3, are low or undetectable in resting cells and increase rapidly and transiently in response to various agonists. In this regard, activation of both receptor and non-receptor, cytosolic tyrosine kinases (168,169), including p53/56> (91,170,171) have been linked to increases in PI 3-kinase metabolites. The /  77  finding that p53/56> is activated in M<|>s incubated with LPS, therefore, supported the /  77  possibility that PI 3-kinase is a downstream effector in this system. In fact, the results of the present study show that PI 3-kinase is activated in LPS-treated human M<|>s. This conclusion is based both upon the detection of elevated levels of PI 3-kinase metabolites in vivo in LPS-treated peripheral blood M<|>s and U937 cells, as well as increased  122  enzymatic activity assayed in vitro in PI 3-kinase immunoprecipitates. Detailed analysis of phosphoinositide levels done using LPS-treated U937 cells revealed that both Ptdlns3P and Ptdlns3,4,5P3 are increased over basal levels (Figure 20 and Table 2). No significant changes were observed in the other phosphoinositides analyzed. Increased levels of these metabolites could be a consequence of either higher PI 3-kinase activity or decreased activity of phosphomonoesterases resulting in the accumulation of these phospholipids. Increased specific activity of PI 3-kinase observed in immunoprecipitatesfromLPS-treated cells as compared to control cells (Figures 23 and 24) is consistent with the first possibility. This conclusion is also supported by the finding that LPS-induced increases in levels of Ptdlns3,4,5P3 were completely abrogated when cells were pre-incubated with the PI 3-kinase inhibitor wortmannin (Figure 21). Many responses of M<|>s to physiological concentrations of LPS are believed to involve the cell surface molecule CD14 (14). In the present study, PI 3-kinase activation in peripheral blood M<|)S was observed using LPS concentrations in the range of 10-100 picogram/ml. Thus, the concentrations of LPS that bring about activation of PI 3-kinase are similar to those required to induce M<t>s functional responses through CD 14.  Moreover, incubation of cells with  antibody 3C10, specific for CD14 and which neutralizes M(|> responses to LPS, abrogated LPSinduced-increases in Ptdlns3,4,5P3 levels (Figure 22), indicating that activation of PI 3-kinase byLPSisCDH-dependenL In other systems, such as antigen receptor cross-linking in B cells, PI 3-kinase is known to interact physically with tyrosine phosphorylated p53/56>'' and this is associated with /  f,  increased lipid-kinase activity (170). Thus, the finding that p53/56^ is activated in LPS/  /7  treated M(> j s is of particular interest and the results presented in Figure 24 identify LPS signaling  123 in M<|>s as another system in which PI 3-kinase and p53/56>' transiently associate leading to /  increased lipid kinase activity.  /7  Furthermore, treatment of cells with the PTK inhibitor  herbimycin A abrogates the LPS-induced increase in PI 3-kinase activity (Figure 26). This effect was not a result of a direct inhibition of PI 3-kinase, since in vitro incubation of herbimycin A with PI 3-kinase, immunoprecipitated from LPS-treated cells, did not affect lipid kinase activity (Figure 26c). The association of p85 with other tyrosine phosphorylated proteins is not excluded, however. As reported by Stefanovi and coworkers (80) LPS brings about the activation of Lyn, Hck, and Fgr in M<J>s. The PI 3-kinase activity observed in phosphotyrosine immunoprecipitates could be the results of the association of p85 with any, or all, of these tyrosine kinases, as well as with any protein that became their target after activation by LPS. Following agonist treatment, PI 3-kinase is known to associate not only with tyrosine kinases, but also with other tyrosine phosphorylated proteins. Reports in the literature show that, in some cases, these associations appear to be mediated through SH2 domains in the p85 regulatory subunit of the enzyme. For example, phosphotyrosine containing peptides derived from insulin receptor substrate-1 (89,172), polyoma virus middle t antigen and platelet derived growth factor receptor (173) activate PI 3-kinase from 3T3-L1 adipocytes or from CHO cells in vitro. Other mechanisms, either alternative or complementary, that mediate these protein-protein interactions appear to involve SH3 domains from either p59 5 or /  77  p53/56> binding to proline rich regions in p85. This binding also appears to upregulate PI 3,  77  kinase activity (91).  Thus, it is possible that either or both of these interactions with  vSZ/^y may account for activation of PI 3-kinase in LPS treated Ntys. 1  124 Three potential tyrosine phosphorylation sites have been mapped in the regulatory subunit of PI 3-kinase and phosphorylation of one or more of these tyrosine residues has been proposed as an alternative mechanism for enzyme activation. In contrast, there are multiple reports of activation of PI 3-kinase in the absence of changes in tyrosine phosphorylation of p85 [reviewed in (86)]. The results presented in this thesis indicate that there is no apparent change in tyrosine phosphorylation of p85 in LPS-treated cells (Figure 27). Failure to detect a change in tyrosine phosphorylation of p85 could be explained by the fact that increased PI 3kinase activity in the anti-p85 immunoprecipitates resulted from only a minor fraction of the enzyme being tyrosine phosphorylated. This might have been below the sensitivity of the detection system used thereby contributing to a false negative result To maximize the chance of detecting tyrosine phosphorylated p85, 4G10 immunoprecipitates were preparedfromLPStreated cells to provide material enriched in phosphotyrosine containing proteins. Even under these conditions, p85 was not observed to comigrate with any of the tyrosine phosphorylated proteins detected in parallel Western blots prepared with anti-phosphotyrosine antibody (Figure 24b, c). Taken together, these results indicate that tyrosine phosphorylation of the regulatory subunit is not necessary for activation of the enzyme in response to LPS. Although p85 was not observed to be tyrosine phosphorylated in 4G10 immunoprecipitates from LPS-treated cells, relative to control cells these samples contained more PI 3-kinase protein (Figure 27b). This presumably indicates that PI 3-kinase associates with phosphotyrosine containing proteins (p53/56> at least and perhaps others) preferentially in LPS-treated cells. As discussed above, /  77  these protein-protein interactions likely contribute to enzyme activation. In Schizosaccharomyces pombe, Ras has been shown to be involved in regulating PI 3kinase (174). In addition, Ras has been reported to be activated in LPS treated human  125 monocytes (144). However, we have been unable to demonstrate that LPS activation of M<|>s promotes increased GTP loading of Ras (data not shown). Similarly, work done by Buscher and coworkers in LPS-treated (I ng/ml) BAC-I,2F5 macrophages showed stimulation of MAP kinase occurring independently of Ras activation (143). Guanine nucleotide exchangers such as Vav, are responsible for the appearance of GTP-Ras in activated T cells (175). This 97 kDa protein is a common target of protein tyrosine kinases (176,177), and in T cells Gulbins and coworkers reported that tyrosine phosphorylation of Vav correlates with increased nucleotide exchange activity (175).  When Vav immunoprecipitates were analyzed for tyrosine  phosphorylation by immunoblotting, we were unable to detect any difference between the control and LPS treated cells (Figure 32B). Taken together, these results indicate that Ras activation may not be involved in the LPS-dependent activation of PI 3-kinase. The mechanisms for regulation of p85/p110 in mammalian cells are diverse. For instance, the fMLP receptor in neutrophils is known to be coupled to heterotrimeric G-proteins, and this chemoattractant peptide causes rapid activation of p85/p110 PI 3-kinase in neutrophils (87,88,101,113).  Furthermore, Matsuo etal. (178) demonstrated that  concanavalin A treatment of THP-1 cells results in PI 3-kinase activation through dual signaling pathways: one G-protein-coupled and the second phosphotyrosine-related.  In addition to  p85/p110 PI 3-kinase, another PI 3-kinase family member consisting of a single polypeptide chain, plOOy, has been described. This isoform is present in the human leukemia cell lines U937 and K562 (93) and becomes activated by heterotrimeric, G protein-linked receptors. Thus, while it appears that activation of PI 3-kinase by LPS in human monocytes occurs independent of Ras, based upon inhibitor studies (Figure 26), it appears to be regulated by tyrosine-kinase dependent mechanisms and the role of G proteins remains to be examined.  126 Increased activity of PI 3-kinase in human monocytes incubated with LPS results in elevated levels of Ptdlns3,4,5P3 as well as Ptdlns3,4P in U937 cells (Figure 20). The exact 2  roles for these and other metabolites of PI 3-kinase are still not completely clear. Transient increases in the concentrations of these metabolites in response to activation by different agonists, however, does implicate their function as second messengers. Several PH-domain containing proteins have the ability to bind to D3-phosphorylated phosphoinositides [reviewed in (98)]. Among these, Akt/PKB has been reported to bind Ptdlns(3,4)P resulting in 2  activation of the enzyme (105). Furthermore, treatment of cells with the PI 3-kinase inhibitor wortmannin results in inhibition of PKB activity, suggesting that activation of PKB is an event occurring downstream of PI 3-kinase (103). MAP kinase is another potential target of the action of PI 3-kinase. Treatment of COS-7 and CHO cells with wortmannin or LY294002, or overexpression of a dominant negative mutant of PI 3-kinase results in attenuation of MAP kinase activation through G proteins (1 79). Other potential targets of activated PI 3-kinase are members of the PKC family. PKC-S has been found to associate with PI 3-kinase following cytokine stimulation (180). It has also been reported that the inositol phospholipids Ptdlns3,4P and Ptdlns3,4,5P3 have the capacity 2  to activate calcium-independent isoforms of PKC in vitro (109,124). As discussed above, the work reported here indicates that LPS treatment of human monocytes results in the activation of a form of PKC that is active in the absence of calcium and lipid co-factors. The activation of this form of PKC was not limited to monocytes from peripheral blood, as it was also observed in the monocytic cells lines THP-1 and U937 (Figures 16A, 16C and 17A).  Detection of  activity in the absence of cofactors is consistent with PKC-^, and immunoblotting of fractions from Mono Q peak two indicated the presence of PKC-< in these samples (Figures 15B , 16B  127 and 17B). PKC-< activity immunoprecipitated from LPS-stimulated THP-1 cells also showed increased in vitro kinase activity as compared with the activity present in immunoprecipitates from control, untreated cells (Figure 15C). Furthermore, LPS-enhanced MBP kinase activity could be removed by immunoadsorption with anti-PKC-^ (Figure 17). Prior analyzes of PKC expression in blood monocytes, U937 cells, and HL-60 cells indicated the presence of PKC-a, pi, -pn, -e,  and 6 isoforms (118,130,131,181). In U937 cells, PKC-a, -p, and -e elute  from Mono Q at or below 320 mM NaCI, white PKC-< elutes at -460 mM NaCI (118). The somewhat earlier elution of PKC-C (370-410 mM NaCI) observed in the present study most likely reflects procedural differences related to detergent solubilization and variations in elution buffers. The evidence presented indicating that PKC< is activated by LPS is of potential interest since previous studies concerned with the role of PKC in LPS cell signaling in M<|>s have been inconsistent For example, it has been reported that treatment of human monocytes with LPS brings about translocation of PKC-a and PKC-p isoforms to the membrane fraction (182). In contrast, incubation of murine macrophages with LPS was not observed to activate PKC in the presence of calcium, phosphatidylserine and diacylglyceror (128). Studies from this laboratory also found no evidence for LPS-induced translocation of Ca/phospholipid dependent-PKC enzymatic activity to the membrane fraction of human monocytes (P. Herrera, R. Brownsey and N. Reiner, unpublished data). Two of the three anti-PKC-^ antibodies used in this study were raised against a peptide (SEFEGFEYINPLLLSAEESV) corresponding to amino acids 573-592 present in the COOHterminus of the kinase. This exact sequence is not found in any of the other known PKC isoforms (183). The closely related PKCi contains a similar COOH-terminal sequence  128 (SEFEGFEYINPLLMSAEECV) differing at only two amino acids and is detected with antibodies to the COOH-terminus of PKC-^. However, it is unlikely that the major immunoreactive band detected in this study was PKC-i, since this isoform is known to migrate at 65 kDa (184), consistently lower than the -80-85 kDa protein observed in this study. The third antibody used (Upstate Biotechnology) is reported not to cross-react with PKC-a, PKC-i or PKC-A,. It has also been reported that antibodies directed against the COOH-terminus of PKC-C react with a C a  2+  and phorbol ester-sensitive PKC isoforms (185). However, the kinase detected  and characterized in this thesis was only weakly activated by a combination of PMA, C a , and 2+  PS and had sustained activity in the absence of exogenous lipids (Figure 18). These findings preclude the notion that it may be a member of either the cPKC or nPKC subfamilies and indicates strongly that it is PKC-C. PKC isoforms have been observed to display different substrate specificity profiles and to some extent the results obtained are influenced by the specific assay conditions (118,154,186). In the present study, it was found that MBP, peptide e, and S6 peptide were equally efficient substrates for monocyte PKC-C,. In contrast, protamine sulfate, histone, and kemptide were relatively poor substrates (Figure 18).  These findings are consistent with  previous studies in which PKC-i; phosphorylated MBP and peptide substrates with equal efficiency (109), but showed lower activity against histone and kemptide (118,123). While the data contrast somewhat with other results indicating that peptide e may be a more efficient substrate for PKC-C, than MBP (123,154), these comparisons are complicated by species differences as well as different assay conditions. Activator and cofactor requirements for PKC isoforms have served as the basis for a general classification scheme for these kinases. Members of the cPKC and nPKC subgroups  129 have generally been found to require the presence of PS and diacylglycerol for activation and exhibit little activity in the absence of these factors (187,188). The cPKC members also require the presence of Ca . In contrast, human PKG-£ exhibits activator- and cofactor2+  independent activity which is not increased by the addition of PS (118). The biochemical characteristics of LPS-activated monocyte PKC-C, reported in this paper (Figure 18) are consistent with previous studies. The findings presented contrast somewhat, however, with studies of PKC-C, in other species. For example, rat and bovine homologues display activatorand cofactor-independent activity which is significantly enhanced by the addition of PS [reviewed in (122)]. The mechanisms leading to the activation of PKC-C, in cells in response to external stimuli are not fully understood. As discussed above, the involvement of PI 3-kinase in the regulation of nPKC and aPKC isoforms has been implicated by studies showing a stimulatory effect of the PI 3-kinase products Ptdlns3,4P and PIP3 in vitro (109,124). Mizukami and 2  coworkers found an in vivo correlation between activation of PKC-C, and PI 3-kinase. These authors reported that after ischemia of cardiac tissue, PKC-C, is translocated to the nucleus and treatment with wortmannin prevents this translocation in a dose dependent manner (189). In addition, the PI 3-kinase products Ptdlns3,4P and Ptdlns3,4,5P3 show a stimulatory effect on 2  the in vitro activity of aPKCs (109,124). The role of PI 3-kinase in LPS-induced activation of monocyte PKC-^ was examined in this thesis using two different approaches. The first approach involved the use of two structurally unrelated PI 3-kinase inhibitors. LPS-induced activation of PKC-C, was abrogated by both wortmannin and LY294002 (Figure 28A and 28B). The effects of wortmannin are considered to be relatively specific for PI 3-kinase at concentrations similar to those used in this study (50-100 nM). However the compound has  130 been shown to inhibit phospholipase A with an IC50 similar to that previously reported for PI 2  3-kinase (190). The structurally unrelated compound, LY294002, has been shown to have inhibitory effects on PI 3-kinase by a different mechanism (191). Moreover, LY294002 shows no inhibitory effects on other lipid kinases or on several protein kinases, including PKC and MAP kinase (191). The findings that both compounds exhibit inhibitory effects on LPSinduced activation of PKC-<, therefore, support the argument that PI 3-kinase is involved in the regulation of PKC-cj in this system. The second approach to examine the role of PI 3-kinase in regulating PKC-C activation involved experiments in which the effects of transfection with a dominant negative mutant (DNM) of PI 3-kinase (Ap85) on PKC-C activity was analyzed. As described in the Materials and Methods section, the mutant Ap85 subunit lacks a region of the protein involved in binding to the catalytic subunit of the enzyme. However, Ap85 retains both SH2 domains and the SH3 domain of the wild type enzyme, allowing it to interact with other signaling molecules, for example tyrosine kinases and possibly others. Thus, coexpression of Ap85 with native enzyme results in competition between the proteins for binding to critical signaling molecules. Consequently, the native pi 10 catalytic subunit fails to interact with regulatory proteins and impaired PI 3-kinase signaling occurs. When the activity of PI 3-kinase was examined in U937 cells that had been transfected with either Ap85 or bovine wild type p85 (Wp85), it was observed that cells transfected with the dominant negative mutant protein showed decreased in vitro activity as compared to Wp85-transfected U937 cells (Figure 30). Furthermore, treatment of cells with LPS failed to increased the enzymatic activity of PI 3-kinase, whereas Wp85-transfectants showed LPSdependent responses similar to those observed in non-transfected cells. Immunoblotting of  131 whole cell lysatesfromnon-transfected, Ap85- and Wp85-transfected U937 cells showed similar levels of expression of p85 in the three cell types. The fact that no overexpression of p85 was detected, could be explained by the low levels of expression that U937 cells show when transfected with foreign DNA (192). These results indicate that although there is no apparent overexpression of p85 in either of the two transfectants, expression of native p85 in the DNM is not impaired, and lower PI 3-kinase activity is not a result of diminished amount of enzyme present in the cell. Expression of Ap85 in U937 cells impaired LPS-induced activation of PKC-C (Figure 31B) providing additional support for the argument that PI 3-kinase is an upstream activator of PKC-^. An important question arisingfromthese observations is how PKC-C maintains its activation following exposure to Ptdlns3,4,5P3 produced by activated PI 3-kinase. One possibility is that Ptdlns3,4,5P3 induces a change in the phosphorylation state of the PKC-C^ which sustains its activity until it becomes dephosphorylated by a cellular phosphatase. Changes in PKC-C could also involve association with other proteins resulting in enhancement of its activity. The importance of these signaling events in regulating the functions of M<|>s as immunologically competent cells is an issue of extreme interest As discussed earlier, LPS is capable of eliciting a wide variety of responses in these cells, including the production and secretion of many growth factors and cytokines (193,194). We explored the hypothesis that PI 3-kinase may be important for the induction of cytokines production in human M<J>s. Using RNase protection assays to measure mRNA levels for different cytokines in U937 cells, we were unable to detect significant increases in any of eight different cytokines. Thus it was not possible to examine the effects of transfection of Ap85 in the LPS-elicited responses of U937  132 cells. THP-WT cells, however, showed increased mRNA levels for IL-1 a, IL-1 p and TNF-a in response to LPS. Treatment of THP-1 -WT cells with the PI 3-kinase inhibitors LY294002 and wortmannin, while inhibiting PI 3-kinase and PKC-C activities, failed to inhibit LPS-induced increases of cytokine mRNA (Figure 33). These findings indicate that PI 3-kinase is not involved in signaling events leading to increased cytokine production by M<|>s. We cannot exclude completely the possibility that PI 3-kinase is involved in regulating cytokine production. Even though mRNA levels for these cytokines are not impaired after treatment with PI 3-kinase inhibitors, the expression or secretion of these proteins could be affected, consistent with posttranscriptional regulation. In comparison to resting cells, activated rAfys show increased adhesive properties which are vital for the many of their functions, including tumoricidal activity. As PI 3-kinase appears to be involved in cytoskeletal rearrangements in other cells from the immune system (115), we examined the effects of PI 3-kinase on the adhesive capacity of M(|>s. CD 14-positive THP-1 -WT cells growing in suspension showed increased adhesiveness to culture dishes (Figure 34) or fibronectin (Figure 35) when treated with LPS. After pretreatment with either wortmannin or LY294002, they showed marked reductions in LPS-induced adherence. PMA is an agent commonly used for differentiation of U937 and THP-1 cells and it causes the cells to become tightly adherent to plastic surfaces. When THP-1 cells were incubated with the PI 3-kinase inhibitors before the addition of PMA, no effect on PMA-induced adherence was observed. These results indicate that in THP-1 cells, the effects of the PI 3-kinase inhibitors on the adhesive properties of the cells are selective and likely related to inhibition of PI 3-kinase activation in response to LPS. Furthermore, the data show that LPS-induced, but not PMA-  133 induced cell adherence appears to be PI 3-kinase dependent These findings are consistent with the observation that PMA does not activate PI 3-kinase in M<> js (data not shown). These studies concerned with adherence were carried out using a human monocytic cell line. Experiments to determine whether LPS-induced adherence is similarly regulated in peripheral blood monocytes are in progress. However, the fact that many of the signaling events reported in this thesis were comparable in THP-1, U937 cells, and in peripheral blood monocytes, suggests that changes in monocyte adherence in response to LPS are likely to be PI 3-kinase dependent in the latter cells. Several questions emanating from the studies reported in this thesis warrant further investigation. For example, the involvement of PKC-i; in LPS-induce, macrophage adherence remains to be elucidated. Work done by Derman and coworkers showed that Ptdlns3,4,5P3 increases the motility of fibroblasts and suggested the involvement of a calcium-independent isoform of PKC in this process (195). The use of specific inhibitors of PKC-t, or transfection of cells with a dominant negative mutant of the enzyme could help establish the exact role PKC£ plays in the downstream events initiated by LPS activation. In addition, both tyrosine kinaseas well as G protein-dependent pathways for the activation of PI 3-kinase have been demonstrated (85-87). Whether one or both of these is involved in LPS cell signaling is an important question. Notably, G proteins have been shown to participate in LPS-induced responses in a macrophage cell line (196). In conclusion, this thesis examined LPS signaling in M<}>s and the relationship of activated pathways to functional responses. 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