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Tissue heterogeneity in the production of tumor necrosis factor (TNF) by rat macrophages
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HEAT SHOCK ALTERS CELL RESPONSE TO TUMOR NECROSIS FACTOR. D. I. Kusher and L. Ft. Goodinq. Emory University School of Medicine, Atlanta, GA 30322. TNF kills cells by a process that involves receptor binding followed by triggering of cell death (by an unknown mechanism). Most cells protect themselves from TNF killing by a TNF-induced protein synthetic response. We wondered if cells undergoing a stress response (e.g. heat shock response), which influences the synthesis of cellular proteins itself, would impact a cell’s response to TNF. We found that C3HA mouse fibroblasts and human embryo lung cells (HEL299), which are made sensitive to TNF by inhibition of protein synthesis with anisomycin (70 and 90% lysis, respectively, as determined by 5’Cr-release cytotoxicity assays), remain resistant to TNF + anisomycin following heat shock (20 & 30% lysis, respectively). LM mouse fibroblasts, which are spontaneously sensitive to TNF (90% lysis), are also made resistant to TNF lysis by heat shock (20% lysis). When exposure to sodium arsenite is used as an alternative means of inducing heat shock, identical changes in TNF sensitivity are observed. We used “‘1-TNF binding assays to examine what effect heat shock has on cell surface receptors. We found that for LM, C3HA and HELZ99 cells “‘1-TNF did bind specifically to its receptors following a hyperthermic exposure. These data support a role for heat shock in the protection of cells from TNF-mediated cell killing, and that the TNF surface receptors are not lost following heat treatment. Perhaps, heat shock is part of a mechanism in stressed cells that prevents their destruction by inflammatory mediators during their iecovery from damage. Supported by grants CA40266, Al26035 and CA46219 from the NIH.
DETECTIOll AND PROPERTIES OF IL-1 RECEPTORS IN A MURINE OSTEOBLASTIC CELL LIE!E. A.L. Laborde, S.E. Truesdell, and J.A. Shelly. The Upjohn Company, Kalamazoo, MI 49001. The murine osteoblastic cell line, MC3T3-El, is a homogeneous population known to respond to IL-l. Al though the biologicial effects of IL-1 on these cells are documented, there has been no report regarding the presence and characteristics of IL-1 receptors on MC3T3-El cells. Our studies indicate the presence of IL-1 receptors and define some characteristics of this receptor. Both IL-la and IL-16 bind to receptors on the cell surface. The receptor is saturable with equilibrium binding being achieved by 4 hrs at 25°C. The receptor has a dissociation constant (KD) of Ix~O-‘~M with 6500 receptors per cell. Treatment with acidic buffers distinguishes surface bound from internalized ligand and azide inhibits internal accumulation of IL-l, suggesting that internalization of the ligand occurs via receptor-ligand complex internationalization. Maximum levels of internal IL-1 are achieved by 3 hrs at 37°C. After this time, both the internal pool of IL-1 and surface bound IL-1 decline, indicating loss of surface receptors. Increasing levels of TCA-soluble radioactivity with time as compared to TCAprecipitable activity suggests intracellular degradation of the internalized ligand. These initial studies define the properties of the IL-1 receptor on MC3T3-El cells and support the use of this cell line as a system for understanding the role of IL-l in bone remodelling.
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CYTOKINE INTERACTION IN REGULATION OF ACUTE PHASE PROTEIN SYNTHESIS. I. Kushner, A. Mackiewicz, M.K. Ganapathi, and D. Schultz. Case Western Reserve Univ. at MetroHealth Med. ctr., Cleveland, OH 44109. A number of cytokines, alone or in combination, may influence synthesis of plasma proteins in hepatocyte-derived model systems. These studies were undertaken to further our understanding of how acute phase protein changes in the human hepatoma cell lines Hep 38 or Hep G2 cells are regulated by various combinations of cytokines. Decrease in albumin and increase in al-antichymotrypsin synthesis were induced by IL-6, IL-l, and TGFO; the combination of IL-6 with either IL-l or TGFB was additive. al-protease inhibitor synthesis could be induced by IL-6 and TGFO, but not by IL-l; the combination of IL-6 and TGFR was additive. Fibrinogen synthesis was up regulated by IL-6 and down regulated by IL-l or TGFB; addition of either IL-l or TGFR inhibited the inducing effect of IL-6. None of these 3 cytokines alone caused more than modest changes in induction of C-reactive protein (CRP) or serum amyloid A (SAA) but the combination of IL-6 plus IL-l led to significant increase in CRP and SAA synthesis in Hep 38 cells; TGFD did not substantially influence this induction. The negative acute phase protein alphafetoprotein was down regulated by both IL-6 and TGFB; the combination was additive. Synthesis of al-acid glycoprotein and haptoglobin was induced by IL-6; this response was not affected by TGFR. These studies further our understanding of how cytokine interactions regulate the acute phase response.
TISSUE HETEROGENEITY IN THE PRODUCTION OF TUMOR NECROSIS FACTOR (TNF) BY.nATs MACROPHAGES. & & sk'n . . L. e . Gardner. Rutgers University, Piscataway, NJ 08854 The ability of different tissue macrophages to a cytokine with potent cytotoxic and produce TNF, immunoregulatory activity was compared. Wacrophages, (Kupffer isolated from rats by liver perfusion lavage (peritoneal, PW and alveolar cells, KC), macrophages, PIM) or tissue disruption (pulmonary interstitial macroohaaes. PIM1. were cultured in the presence of lipopblysaccharihe (LPS) and/or gamma interferon (IFN). TNF activity in macrophage assayed by cytotoxicity towards supernatants was actinomycin D sensitized L929 cells. Unstimulated liver and lung macrophages were found to produce TNF immediately after plating large amounts of reaching a maximum within 2 h for PAN and PIM and 4macrophages produced 3-4 times 6 h for KC. Liver more TNF than PIM. In contrast, unstimulated PM did not produce TNF even after 48 h incubation. Treatment of cultured KC, PAN or PM with LPS (100 rig/ml, 24 h) stimulated the cells to produce additional maximal stimulation of TNF prodTNF. In contrast, uction by cultured PIM required the presence of both LPS and IFN (100 U/ml). These results suggest that macrophages from different tissues are heterogeneous with respect to their ability to produce TNF.
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TUMOR NECROSIS FACTOR a LTNFa) CAN PARTICIPATE IN INDUCTION OF HUMAN SERUM AMYLOID A IS&). I. Kushner, D. Schultz, M.K. Ganapathi. Case Western Reserve Univ. at MetroHealth Med. Ctr., Cleveland, OH 44109. Induction of the human acute phase proteins C-reactive protein (CRP) and SAA in the human hepatoma cell line Hep 3B can be accomplished by conditioned medium (CM) from LPS-stimulated human monocytes or by the combination of interleukin-6 (IL-6) and interleukin-1 (IL-l). A mixture of antibodies to rIL-la and rIL-1R reduced CRP induction by CM by about 85%, and had a comparable effect on SAA induction. Since TNFa shares many functional activities with IL-l, we explored the possibility that TNFa may influence induction of CRP and SAA. This cytokine alone had no effect on induction of these acute phase proteins in Hep 3B cells, but the combination of IL-6 and TNFa led to induction of SAA: maximal response was 25-50% of that seen with IL-6 and IL-l. The SAA response to IL-6 and IL-l could not be augmented by the addition of TNFa. In contrast, there was minimal, if any, CRP response to the combination of IL-6 and TNFa. The finding that TNFa in combination with IL-6 led to SAA induction in Hep 3B cells indicates that TNFa can participate in induction of SAA in cells of human origin. In addition, these findings suggest that control of CRP and SAA synthesis involves different sets of cytokines.
Modulation of Generation of Human Adherent Lymphokine-activated Killer (A-LAK) Cells by LPhenylalanine Methyl Ester (PME). Kam H. L-sung. E. I. du Pont de Nemours & Co., Glenolden, PA 19036. It was recently reported that natural killer cells adhere to plastic flasks after (NW interleukin 2 (IL-2) activation and they are termed A-LAK cells. We have demonstrated that depletion of monocytes from peripheral blood mononuclear cells (PBMC) by PME allows genegation of LAK cells at We have studied high cell density (>5 X 10 /mL). the generation of A-LAK cells from PBMC treated with PME. PBMC wer 2 treated with 5 mM PME and cultured at 2 X 10 /mL with rIL-2 for 1 day in plastic flasks. Nonadherent (NA) cells were removed and cells adherent to flasks were cultured with media containing rIL-2 for 8 - 12 days. A-LAK cells were more cytotoxic than NA-LAK cells against K562 cells. also more and Raji target They are proliferative. They have the phenotype of NK/LAK cells (Leu 19+). Partial depletion of monocytes by effects on A-LAK cell 1 mM PME shows similar However, higher cellular expansions generation. Therefore, a few monocytes are were observed. necessary for optimal A-LAK cellular expansion. PME treatment of PBNC provides an efficient means to generate A-IAK cells for adpotive immunotherapy.
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HEAT SHOCK ALTERS CELL RESPONSE TO TUMOR NECROSIS FACTOR. D. I. Kusher and L. Ft. Goodinq. Emory University School of Medicine, Atlanta, GA 30322. TNF kills cells by a process that involves receptor binding followed by triggering of cell death (by an unknown mechanism). Most cells protect themselves from TNF killing by a TNF-induced protein synthetic response. We wondered if cells undergoing a stress response (e.g. heat shock response), which influences the synthesis of cellular proteins itself, would impact a cell’s response to TNF. We found that C3HA mouse fibroblasts and human embryo lung cells (HEL299), which are made sensitive to TNF by inhibition of protein synthesis with anisomycin (70 and 90% lysis, respectively, as determined by 5’Cr-release cytotoxicity assays), remain resistant to TNF + anisomycin following heat shock (20 & 30% lysis, respectively). LM mouse fibroblasts, which are spontaneously sensitive to TNF (90% lysis), are also made resistant to TNF lysis by heat shock (20% lysis). When exposure to sodium arsenite is used as an alternative means of inducing heat shock, identical changes in TNF sensitivity are observed. We used “‘1-TNF binding assays to examine what effect heat shock has on cell surface receptors. We found that for LM, C3HA and HELZ99 cells “‘1-TNF did bind specifically to its receptors following a hyperthermic exposure. These data support a role for heat shock in the protection of cells from TNF-mediated cell killing, and that the TNF surface receptors are not lost following heat treatment. Perhaps, heat shock is part of a mechanism in stressed cells that prevents their destruction by inflammatory mediators during their iecovery from damage. Supported by grants CA40266, Al26035 and CA46219 from the NIH.
DETECTIOll AND PROPERTIES OF IL-1 RECEPTORS IN A MURINE OSTEOBLASTIC CELL LIE!E. A.L. Laborde, S.E. Truesdell, and J.A. Shelly. The Upjohn Company, Kalamazoo, MI 49001. The murine osteoblastic cell line, MC3T3-El, is a homogeneous population known to respond to IL-l. Al though the biologicial effects of IL-1 on these cells are documented, there has been no report regarding the presence and characteristics of IL-1 receptors on MC3T3-El cells. Our studies indicate the presence of IL-1 receptors and define some characteristics of this receptor. Both IL-la and IL-16 bind to receptors on the cell surface. The receptor is saturable with equilibrium binding being achieved by 4 hrs at 25°C. The receptor has a dissociation constant (KD) of Ix~O-‘~M with 6500 receptors per cell. Treatment with acidic buffers distinguishes surface bound from internalized ligand and azide inhibits internal accumulation of IL-l, suggesting that internalization of the ligand occurs via receptor-ligand complex internationalization. Maximum levels of internal IL-1 are achieved by 3 hrs at 37°C. After this time, both the internal pool of IL-1 and surface bound IL-1 decline, indicating loss of surface receptors. Increasing levels of TCA-soluble radioactivity with time as compared to TCAprecipitable activity suggests intracellular degradation of the internalized ligand. These initial studies define the properties of the IL-1 receptor on MC3T3-El cells and support the use of this cell line as a system for understanding the role of IL-l in bone remodelling.
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CYTOKINE INTERACTION IN REGULATION OF ACUTE PHASE PROTEIN SYNTHESIS. I. Kushner, A. Mackiewicz, M.K. Ganapathi, and D. Schultz. Case Western Reserve Univ. at MetroHealth Med. ctr., Cleveland, OH 44109. A number of cytokines, alone or in combination, may influence synthesis of plasma proteins in hepatocyte-derived model systems. These studies were undertaken to further our understanding of how acute phase protein changes in the human hepatoma cell lines Hep 38 or Hep G2 cells are regulated by various combinations of cytokines. Decrease in albumin and increase in al-antichymotrypsin synthesis were induced by IL-6, IL-l, and TGFO; the combination of IL-6 with either IL-l or TGFB was additive. al-protease inhibitor synthesis could be induced by IL-6 and TGFO, but not by IL-l; the combination of IL-6 and TGFR was additive. Fibrinogen synthesis was up regulated by IL-6 and down regulated by IL-l or TGFB; addition of either IL-l or TGFR inhibited the inducing effect of IL-6. None of these 3 cytokines alone caused more than modest changes in induction of C-reactive protein (CRP) or serum amyloid A (SAA) but the combination of IL-6 plus IL-l led to significant increase in CRP and SAA synthesis in Hep 38 cells; TGFD did not substantially influence this induction. The negative acute phase protein alphafetoprotein was down regulated by both IL-6 and TGFB; the combination was additive. Synthesis of al-acid glycoprotein and haptoglobin was induced by IL-6; this response was not affected by TGFR. These studies further our understanding of how cytokine interactions regulate the acute phase response.
TISSUE HETEROGENEITY IN THE PRODUCTION OF TUMOR NECROSIS FACTOR (TNF) BY.nATs MACROPHAGES. & & sk'n . . L. e . Gardner. Rutgers University, Piscataway, NJ 08854 The ability of different tissue macrophages to a cytokine with potent cytotoxic and produce TNF, immunoregulatory activity was compared. Wacrophages, (Kupffer isolated from rats by liver perfusion lavage (peritoneal, PW and alveolar cells, KC), macrophages, PIM) or tissue disruption (pulmonary interstitial macroohaaes. PIM1. were cultured in the presence of lipopblysaccharihe (LPS) and/or gamma interferon (IFN). TNF activity in macrophage assayed by cytotoxicity towards supernatants was actinomycin D sensitized L929 cells. Unstimulated liver and lung macrophages were found to produce TNF immediately after plating large amounts of reaching a maximum within 2 h for PAN and PIM and 4macrophages produced 3-4 times 6 h for KC. Liver more TNF than PIM. In contrast, unstimulated PM did not produce TNF even after 48 h incubation. Treatment of cultured KC, PAN or PM with LPS (100 rig/ml, 24 h) stimulated the cells to produce additional maximal stimulation of TNF prodTNF. In contrast, uction by cultured PIM required the presence of both LPS and IFN (100 U/ml). These results suggest that macrophages from different tissues are heterogeneous with respect to their ability to produce TNF.
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TUMOR NECROSIS FACTOR a LTNFa) CAN PARTICIPATE IN INDUCTION OF HUMAN SERUM AMYLOID A IS&). I. Kushner, D. Schultz, M.K. Ganapathi. Case Western Reserve Univ. at MetroHealth Med. Ctr., Cleveland, OH 44109. Induction of the human acute phase proteins C-reactive protein (CRP) and SAA in the human hepatoma cell line Hep 3B can be accomplished by conditioned medium (CM) from LPS-stimulated human monocytes or by the combination of interleukin-6 (IL-6) and interleukin-1 (IL-l). A mixture of antibodies to rIL-la and rIL-1R reduced CRP induction by CM by about 85%, and had a comparable effect on SAA induction. Since TNFa shares many functional activities with IL-l, we explored the possibility that TNFa may influence induction of CRP and SAA. This cytokine alone had no effect on induction of these acute phase proteins in Hep 3B cells, but the combination of IL-6 and TNFa led to induction of SAA: maximal response was 25-50% of that seen with IL-6 and IL-l. The SAA response to IL-6 and IL-l could not be augmented by the addition of TNFa. In contrast, there was minimal, if any, CRP response to the combination of IL-6 and TNFa. The finding that TNFa in combination with IL-6 led to SAA induction in Hep 3B cells indicates that TNFa can participate in induction of SAA in cells of human origin. In addition, these findings suggest that control of CRP and SAA synthesis involves different sets of cytokines.
Modulation of Generation of Human Adherent Lymphokine-activated Killer (A-LAK) Cells by LPhenylalanine Methyl Ester (PME). Kam H. L-sung. E. I. du Pont de Nemours & Co., Glenolden, PA 19036. It was recently reported that natural killer cells adhere to plastic flasks after (NW interleukin 2 (IL-2) activation and they are termed A-LAK cells. We have demonstrated that depletion of monocytes from peripheral blood mononuclear cells (PBMC) by PME allows genegation of LAK cells at We have studied high cell density (>5 X 10 /mL). the generation of A-LAK cells from PBMC treated with PME. PBMC wer 2 treated with 5 mM PME and cultured at 2 X 10 /mL with rIL-2 for 1 day in plastic flasks. Nonadherent (NA) cells were removed and cells adherent to flasks were cultured with media containing rIL-2 for 8 - 12 days. A-LAK cells were more cytotoxic than NA-LAK cells against K562 cells. also more and Raji target They are proliferative. They have the phenotype of NK/LAK cells (Leu 19+). Partial depletion of monocytes by effects on A-LAK cell 1 mM PME shows similar However, higher cellular expansions generation. Therefore, a few monocytes are were observed. necessary for optimal A-LAK cellular expansion. PME treatment of PBNC provides an efficient means to generate A-IAK cells for adpotive immunotherapy.