- Email: [email protected]
Secretion of tumor necrosis factor by human monocytes and protein kinase C (PKC)
SECOND
INTERNATIONAL
WORKSHOP
ON CYTOKINES
/ 85
13
76
THE
THE EFFECTS OF CYTOKINES ON RECEPTOR EXPRESSION AND PHAGOCYTOSIS BY HUMAN NEUTROPHILS. J.Ogle, G.Noel, M.Sramkoski, C.Oqle and J.Alexander. Univ. Cincinnati Medical Center and Shriners Burns Institute, Cincinnati, OH 45219. The cytokines TNFC(, GM-CSF, G-CSF, PDGF, TGFB, IL-1 and IL2 were studied for their effects on CF.1 expression and phagocytosis of C3b-IgG-coated microspheres. The effects of the cytokines on these parameters were compared to the effect of FMLP used as an optimal, positive control. TNFa, GM-CSF, G-CSF and IL-1 125%a-75%6) upregulated CR1 on neutrophils as well as or slightly better than FMLP. PDGF, TGF and IL-2 had no effect on CR1 expression. TNFa and IL-1 enhanced phagocytosis but not to the extent that FMLP did. There was considerable individual variation among different sources of neutrophils in their response to these cytokines. Despite the large upregulation of CR1 mediated by GM-CSF and G-CSF, these cytokines had very little or no effect on phagocytosis. Nevertheless, the CR1 upregulation caused by these cytokines manifested itself by an increase in the total number of cell-associated microspheres (mostly externally bound). PDGF, TGFB and IL-2 were without The observation of receptor upregulaeffect on phagocytosis. tion by mediators without concomitant enhancement of phagocytosis is very unusual and may have implications regarding different intracellular pools of CR1 and different mechanisms of phagocytosis by neutrophils.
LYMPHOCYTE CHEMOKINETIC RESPONSE IS MEDIATED THROUGH ACTIVATION OF PROTEIN KINASE C. Robin G McFadden, Kenneth E
Vickers, Laurence J Fraher. Lawson Research Institute, St Joseph’s Hospital, London, Ontario, CANADA N6A 4V2 We have performed experiments to investigate the transmembrane signalling process responsible for in vitro lymphocyte migration (LyM). We have studied rat splenic and human peripheral blood lymphocyte response in a nitrocellulose filter mic-rocheflotaxis chamber assay. Neither dibutyryl CAMP- (lO_ 5-10 I4 M) nor adenylate cyclase activators (10 s-10 I4 M forskolin or .Ol-100 rig/ml parathyroid hormone) had any effect on LyM, but a potent positive chemokinetic response Was seen with- activators of protein kinase C (PK-C). Phorbol ester (TPA 10 7 M) incre-ased LyM to 274 f 26% of control and dioctanoylglycerol 10 * M The novel PK-C stimulated LyM to 283 f 19% of control. activator_ N-(6-phenylhexyl)-5-chloro-l-naphthalenesulfonamide (SC-9 10 9 M) increased LyM to 311 f 17% of control values. In addition, the chemokinetic response to lymphocyte chemo -attractant fact_or (LCF) and bradykinin (BK) Was completely inhibited by 10 s M l-(S-isoquinoline sulphonyl) -2-methyl piperazine (H7), an inhibitor of PK-C. The PK-A inhibitor N-(Z-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004 10-o M) and inhibitors of eicosanoid synthesis (nordihydroguaiaretic acid and indomethacin, 10-s M) could not block the response to LCF or -6K. None of the enzyme inhibitors prevented LyM by 10 e M colchicine. We conclude that stimulation of LyM is mediated through activation of PK-C.
71
74 SECRETION
OF TUMOR NECROSIS FACTOR BY HUMAN MONOCYTES AND
PROTEIN KINASE C (PKC) V. SANGUEDOLCE, C. CAPO, P. BONGRAND and J.L. MEGE Lab. Immunol, Hapit. Ste Marguerite,13277 MARSEILLE-FRANCE. Tumor Necrosis factor alpha (TNF& ) is secreted by macrophages in response to various agonists, but the mechanisms leading to its release are unclear. Human monocytes are stimulated for 18 hours with PMA (50 rig/ml), LPS (20 mcg/ml) or opsonized zymosan (1.2 mg/ml). Supernatants are assayed for the presence of TNF by the L929 fibroblast lytic assay.When needed, cytotoxic activity is neutralized by an excees of antibody anti human TNFa( or checked by using a RIA assay. Opsonized zymosan and LPS induced a release of TNF
15 ROLE OF TRANSGLUTAMINASE CATALYZED REACTIONS IN GROWTH FACTOR-DEPENDENT PROLIFERATION OF HUMAN B-LYMPHOCYTES. C. G. Sahasrabuddhe.
Departments of Clinical Immunology and Molecular Pathology, Univ. Texas, M.D. Anderson Cancer Center, Houston, TX 77030. The specific bichemical mechanisms underlying the process of lymphocyte activation and triggering of cell proliferation by mitogenic factors have not been completely elucidated. Our recent studies suggest that transglutaminase (TGase), a Ca+*-dependent enzyme that catalyzes the covalent conjugation of polyamines to proteins, may have an important role in driving human B-lymphocytes through cell cycle. Inhibitors of TGase, such as monodansylcadaverlne and I(5aminopentyl)3-phenylthiourea, specifically blocked the growth factor (BCGF) dependent S-phase entry of human B lymphocytes in a dose and time dependent manner. This inhibitor-mediated growth arrest of B lymphocytes was reversible and was caused by arrest of the cells in Gl-phase of the cell cycle as deterimined by FACS analysis. TGasecatalyzed post-translational modification of protein(s) seems to be critical for pushing the cells from Gl to S-phase. lmmunoblot analysis, using a monospeciflc antibody against tissue TGase, revealed that lymphocytic TGase is an 65 Kd enyme, present in the cytosolic fraction of B-lymphocytes. In vitro studies suggest that at least two protiens (16 Kd and 56 Kd), present in the cytosolic fraction of B-lymphocytes, can serve as substrates for tissue TGase Supported by NIH grants CA 36751 and GM 35463.
Cytosolic Calcium is Elevated in Small Cell Lung Carcinoma Cell Lines by Human Recombinant IL-1 B. LW. Palaszv&& S Mahmoud and T.W. Moodv George Washington University Medical Center.Washington, DC 20037. Interleukin-1 Alpha and Beta (IL-la and gl have been shown to be involved in multiple physiological systems, but its actual role in these sytems is not well understood. Small Cell Lung Carcinoma Cell Lines can be stimulated to grow by the peptide Bombesin (BN). Because IL-1 is widely distributed throughout the body and Lung Carcinogenesis has an inflammatory component associated with it. we scanned Oat-cell carcinoma cell lines for the presence or absence of the IL-1 receptor. The Small Cell Lung Carcinoma lines, 592 and 345 were found to possess specific membrane receptors for IL-l using radiolabelled IL-l g. When radiolabelled IL-1 g was covalently cross-linked to its receptor, several bands were resolved upon SDS-Gel electrophoresis and autoradiography. A major band at P 9.5 kD was observed as well as minor bands at c 120 kD. When 345 cells were assayed for calcium responses (Fura-2). we observed a consistent rise in calcium flux specifically attributable to the addition of IL-lg. This response is dose dependent and is maximal at 1.4 nM. EGTA addition indicated that the calcium flux induced by IL-l& is primarily associated with external pools as opposed to internal pool mobilization by BN. We are currently investigating the possible role of IL-l in the growth of Small Cell Lung Carcinoma and determining the significance of these observed calcium responses.
78 DIFFERENTIATION RELATED EXPRESSION OF TYPE IV COLLAGENASE IN INTERLEUKIN 6 STIMULATED MYELOID LEUKEMIA Ml CELLS. D. H. P-d R. Reid Division of Cytokine Biology, CBER, FDA and Laboratory of Developmental Biology and Anomalies, NIDR, NIH, Bethesda, MD 20892. Tissue macrophages originate in the bone marrow and reach their target tissues by egression from the blood stream and traversing connective tissue barriers like the basement membrane. Since type IV collagen is an important structural component of this membrane, we studied whether the induction of type IV collagenase is related lo the process of differentiation of myeloid cells to mature macrophages. The murine myeloid leukemia Ml cells when stimulated with interleukin 6 differentiate to mature macrophages as quantified by sequential expression of surface markers, induction of enzymes and morphological changes from blast cells 10 mature macrophages. Polycarbonate filters coated with an extract of basement membrane components (Matrigel) were used for invasion assays. Stimulated Ml cells reached full differentiation after 72 hours as determined by expression of Fc receptors, induction of lysozyme and morphology. In parallel, the amount of type IV collagenase in the supernatants of the cells and the cells invasion capacity continuously increased up to 72 hours. Zymographic analysis revealed a type IV collagenase with a molecular weight of 92kDa. Since type IV collagen is the only collagen found in Matrigel, our results suggest that the induction of type IV collagenase provides the mature macrophages but not the undifferentiated cells with the ability to traverse the basement membrane.
INTERNATIONAL
WORKSHOP
ON CYTOKINES
/ 85
13
76
THE
THE EFFECTS OF CYTOKINES ON RECEPTOR EXPRESSION AND PHAGOCYTOSIS BY HUMAN NEUTROPHILS. J.Ogle, G.Noel, M.Sramkoski, C.Oqle and J.Alexander. Univ. Cincinnati Medical Center and Shriners Burns Institute, Cincinnati, OH 45219. The cytokines TNFC(, GM-CSF, G-CSF, PDGF, TGFB, IL-1 and IL2 were studied for their effects on CF.1 expression and phagocytosis of C3b-IgG-coated microspheres. The effects of the cytokines on these parameters were compared to the effect of FMLP used as an optimal, positive control. TNFa, GM-CSF, G-CSF and IL-1 125%a-75%6) upregulated CR1 on neutrophils as well as or slightly better than FMLP. PDGF, TGF and IL-2 had no effect on CR1 expression. TNFa and IL-1 enhanced phagocytosis but not to the extent that FMLP did. There was considerable individual variation among different sources of neutrophils in their response to these cytokines. Despite the large upregulation of CR1 mediated by GM-CSF and G-CSF, these cytokines had very little or no effect on phagocytosis. Nevertheless, the CR1 upregulation caused by these cytokines manifested itself by an increase in the total number of cell-associated microspheres (mostly externally bound). PDGF, TGFB and IL-2 were without The observation of receptor upregulaeffect on phagocytosis. tion by mediators without concomitant enhancement of phagocytosis is very unusual and may have implications regarding different intracellular pools of CR1 and different mechanisms of phagocytosis by neutrophils.
LYMPHOCYTE CHEMOKINETIC RESPONSE IS MEDIATED THROUGH ACTIVATION OF PROTEIN KINASE C. Robin G McFadden, Kenneth E
Vickers, Laurence J Fraher. Lawson Research Institute, St Joseph’s Hospital, London, Ontario, CANADA N6A 4V2 We have performed experiments to investigate the transmembrane signalling process responsible for in vitro lymphocyte migration (LyM). We have studied rat splenic and human peripheral blood lymphocyte response in a nitrocellulose filter mic-rocheflotaxis chamber assay. Neither dibutyryl CAMP- (lO_ 5-10 I4 M) nor adenylate cyclase activators (10 s-10 I4 M forskolin or .Ol-100 rig/ml parathyroid hormone) had any effect on LyM, but a potent positive chemokinetic response Was seen with- activators of protein kinase C (PK-C). Phorbol ester (TPA 10 7 M) incre-ased LyM to 274 f 26% of control and dioctanoylglycerol 10 * M The novel PK-C stimulated LyM to 283 f 19% of control. activator_ N-(6-phenylhexyl)-5-chloro-l-naphthalenesulfonamide (SC-9 10 9 M) increased LyM to 311 f 17% of control values. In addition, the chemokinetic response to lymphocyte chemo -attractant fact_or (LCF) and bradykinin (BK) Was completely inhibited by 10 s M l-(S-isoquinoline sulphonyl) -2-methyl piperazine (H7), an inhibitor of PK-C. The PK-A inhibitor N-(Z-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004 10-o M) and inhibitors of eicosanoid synthesis (nordihydroguaiaretic acid and indomethacin, 10-s M) could not block the response to LCF or -6K. None of the enzyme inhibitors prevented LyM by 10 e M colchicine. We conclude that stimulation of LyM is mediated through activation of PK-C.
71
74 SECRETION
OF TUMOR NECROSIS FACTOR BY HUMAN MONOCYTES AND
PROTEIN KINASE C (PKC) V. SANGUEDOLCE, C. CAPO, P. BONGRAND and J.L. MEGE Lab. Immunol, Hapit. Ste Marguerite,13277 MARSEILLE-FRANCE. Tumor Necrosis factor alpha (TNF& ) is secreted by macrophages in response to various agonists, but the mechanisms leading to its release are unclear. Human monocytes are stimulated for 18 hours with PMA (50 rig/ml), LPS (20 mcg/ml) or opsonized zymosan (1.2 mg/ml). Supernatants are assayed for the presence of TNF by the L929 fibroblast lytic assay.When needed, cytotoxic activity is neutralized by an excees of antibody anti human TNFa( or checked by using a RIA assay. Opsonized zymosan and LPS induced a release of TNF
15 ROLE OF TRANSGLUTAMINASE CATALYZED REACTIONS IN GROWTH FACTOR-DEPENDENT PROLIFERATION OF HUMAN B-LYMPHOCYTES. C. G. Sahasrabuddhe.
Departments of Clinical Immunology and Molecular Pathology, Univ. Texas, M.D. Anderson Cancer Center, Houston, TX 77030. The specific bichemical mechanisms underlying the process of lymphocyte activation and triggering of cell proliferation by mitogenic factors have not been completely elucidated. Our recent studies suggest that transglutaminase (TGase), a Ca+*-dependent enzyme that catalyzes the covalent conjugation of polyamines to proteins, may have an important role in driving human B-lymphocytes through cell cycle. Inhibitors of TGase, such as monodansylcadaverlne and I(5aminopentyl)3-phenylthiourea, specifically blocked the growth factor (BCGF) dependent S-phase entry of human B lymphocytes in a dose and time dependent manner. This inhibitor-mediated growth arrest of B lymphocytes was reversible and was caused by arrest of the cells in Gl-phase of the cell cycle as deterimined by FACS analysis. TGasecatalyzed post-translational modification of protein(s) seems to be critical for pushing the cells from Gl to S-phase. lmmunoblot analysis, using a monospeciflc antibody against tissue TGase, revealed that lymphocytic TGase is an 65 Kd enyme, present in the cytosolic fraction of B-lymphocytes. In vitro studies suggest that at least two protiens (16 Kd and 56 Kd), present in the cytosolic fraction of B-lymphocytes, can serve as substrates for tissue TGase Supported by NIH grants CA 36751 and GM 35463.
Cytosolic Calcium is Elevated in Small Cell Lung Carcinoma Cell Lines by Human Recombinant IL-1 B. LW. Palaszv&& S Mahmoud and T.W. Moodv George Washington University Medical Center.Washington, DC 20037. Interleukin-1 Alpha and Beta (IL-la and gl have been shown to be involved in multiple physiological systems, but its actual role in these sytems is not well understood. Small Cell Lung Carcinoma Cell Lines can be stimulated to grow by the peptide Bombesin (BN). Because IL-1 is widely distributed throughout the body and Lung Carcinogenesis has an inflammatory component associated with it. we scanned Oat-cell carcinoma cell lines for the presence or absence of the IL-1 receptor. The Small Cell Lung Carcinoma lines, 592 and 345 were found to possess specific membrane receptors for IL-l using radiolabelled IL-l g. When radiolabelled IL-1 g was covalently cross-linked to its receptor, several bands were resolved upon SDS-Gel electrophoresis and autoradiography. A major band at P 9.5 kD was observed as well as minor bands at c 120 kD. When 345 cells were assayed for calcium responses (Fura-2). we observed a consistent rise in calcium flux specifically attributable to the addition of IL-lg. This response is dose dependent and is maximal at 1.4 nM. EGTA addition indicated that the calcium flux induced by IL-l& is primarily associated with external pools as opposed to internal pool mobilization by BN. We are currently investigating the possible role of IL-l in the growth of Small Cell Lung Carcinoma and determining the significance of these observed calcium responses.
78 DIFFERENTIATION RELATED EXPRESSION OF TYPE IV COLLAGENASE IN INTERLEUKIN 6 STIMULATED MYELOID LEUKEMIA Ml CELLS. D. H. P-d R. Reid Division of Cytokine Biology, CBER, FDA and Laboratory of Developmental Biology and Anomalies, NIDR, NIH, Bethesda, MD 20892. Tissue macrophages originate in the bone marrow and reach their target tissues by egression from the blood stream and traversing connective tissue barriers like the basement membrane. Since type IV collagen is an important structural component of this membrane, we studied whether the induction of type IV collagenase is related lo the process of differentiation of myeloid cells to mature macrophages. The murine myeloid leukemia Ml cells when stimulated with interleukin 6 differentiate to mature macrophages as quantified by sequential expression of surface markers, induction of enzymes and morphological changes from blast cells 10 mature macrophages. Polycarbonate filters coated with an extract of basement membrane components (Matrigel) were used for invasion assays. Stimulated Ml cells reached full differentiation after 72 hours as determined by expression of Fc receptors, induction of lysozyme and morphology. In parallel, the amount of type IV collagenase in the supernatants of the cells and the cells invasion capacity continuously increased up to 72 hours. Zymographic analysis revealed a type IV collagenase with a molecular weight of 92kDa. Since type IV collagen is the only collagen found in Matrigel, our results suggest that the induction of type IV collagenase provides the mature macrophages but not the undifferentiated cells with the ability to traverse the basement membrane.