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The E1A proteins of adenovirus render infected cells susceptible to TNF cytolysis
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INTERFERON-y (IFN-Y)-INDUCED CYTOTOXICITY FIBROBLASTS: POTENTIATION BY LIPOPOLYSACCH~!DE 1% INTERLEUKIN-1 (IL-11 OR TUMOR NECROSIS FACTOR (TW). R. Dijkmans, J. Van Daanne, H. Heremans, A. Billiau. Rega Insti= tute, Unlversitv of Leuven (Belqiuml. IFN-y can be cytolytic for -normal mouse fibroblasts isolated from embryonic or adult tissue. This cytotoxicity is transcriptionand translation-dependent, thus suggesting involvement of a suicide-like mechanism. The dose required for cytotoxicity is higher than that needed for antiviral effect or macrophage activation but is reduced 10 to lOOfold by co-treatment of the cells with TNF and/or IL-l, two cytokines that, by themselves are not toxic for these cells. Bacterial LPS, which also by itself has no effect on the viability of mouse fibroblasts, similarly stimulates cell suicide induced by IFN-y. The effect was observed in cultures that were virtually free of non-fibroblastoid cells. Inclusion of antibodies directed against TNF-cr or IL-la in the culture medium did not block the cytotoxic effect of combined IFN-y + LPS treatment. In addition to LPS, heat-killed Gramnegative (Escherichia coli) but also Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) were found to enhance IFN-y-induced cell death. These findings suggest that IFN-y formed in vivo during infectious processes may directly aggravate tissue destruction.
THE ElA PROTEINS OF ADENOVIRUS RENDER INFECTED CELLS SUSCEPTIBLE TO TNF CYTOLVSIS. P. J. Duerksen-Huahes. W. S. M. Wold and L. FL Goodina. Emory University, Atlanta. GA 30322 and St. Louis University School of Medicine, St. Louis, MO 63110. Adenovirus encodes a 14.7K protein which inhibits cell killing by TNF. This is presumably necessary for the virus because, at least under conditions used in v&o, Ad infection makes normally resistant cells sensitive to TNF killing. To determine which viral function(s) was/were responsible for inducing the TNF susceptibility, cells were infected with a panel of viral double mutants, lacking both the 14.7 kD gene and another part of the viral genome. The infected cells were then treated with TNF, and the sensitivity determined with a "Cr release assay. Experiments with hydroxyurea and I-beta-D-arabinofuranosylcysteine showed that the sensitivity function mapped to an early region. TNF lysed cells infected by mutants which lacked regions E3, E4, ElB, and VA-RNA. Thus, none of these regions are necessary to produce TNF sensitivity. However, when cells were infected with a mutant lacking the 14.7K gene plus the EIA region, dL312, TNF was unable to lyse the cells. Thus, ElA is necessary to induce sensitivity to TNF. The ElA region encodes hvo overlapping proteins of 243 and 289 amino acids, both of which can induce TNF susceptibility. Thus, the domain present in 289R but lacking in 243R, which activates transcription of specific cellular genes during virus infection, is not responsible for TNF susceptibility. We are currently testing other ElA mutants to determine which of the other ElA ascribed functions, e.g. enhancer repression, growth factor induction. ras cooperation, correlates with induction of TNF sensitivity. This work was supported by grants CA08544 (PDH). CA24710 (WSMW), and CA40266, A126035, and CA48219 (LRG) from the NIH.
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DIFFERENTIAL REGULATION OF IL-1 BY IFN> AND IL-4. R.P. Donnelly Center for Biologics Evaluation MD
PRODUCTION IN HUMAN MONOCYTES and T.L. Gerrard. FDA 6 Research, NIH, Bethesda,
20892.
A number of T cell-derived cytokines, including IFNJ and IL-4, have been shown to regulate monocytelmacrophage function. ha~se MHc class II antigen (HLA-DR) expression and the ability to produce IL-l are two elements which influence the overall antigen-presenting capacity oE monocytes, we evaluated rHuIFN6 and rHuIL-4 for their ability to regulate these properties in normal human monocytes. Monocytes were prepared by counter-current centrifugal elutriation and demonstrated to be>90% pure as assessed by Giemsa and non-specific esterase staining. Treatment with IFNd or IL-4 significantly increased the basal expression of HLA-DR in human monocytes as determined by flow cytometric analysis after staining with HLA-DR-specific monoclonal antibodies. HOWeVer, unlike IFNr which potentiated monocyte IL-1 production induced by lipopolysaccharide (LPS), IL-4 inhibited IL-l production if added prior to or concomitant with LPS. This inhibitory effect was apparent with IL-4 These findings suggest concentrations as low as 1 ngfml. that the relative levels of IFNd and IL-4 mav reeulate the state of monocyte activation. Moreover, IL-4 may specifically function to prevent overexpression of IL-l by monocytes during the later stages of a cell-mediated immune response.
INCREASXO EfPRESSION OF FceRII/CD23 ON RAT ALvNaLAN NAcRoPNAGEs AFrEN ANymEN czImam3 AND IL-4 STIWJLATION. B.DUGAS, V.LAGENTE, E.BOICHOT, M.CAPRON*, J.M.MENCIA-HUERTA and P.BRAQUET. Institut Henri Beaufour, Les Ulis, France and *Institut Pasteur, Lille, France. The possible modulation of FceRII/CD23 expression on alveolar macro?ahases (AM) rats followino -- in _., from sensitized viva antigen challenge and --in vitro incubation with IL-4 was Gstiqated. Brown NOLWW rats (ZOO-250 9) were sensitized by two bvalbumin aerosols (OA, i%), at 4; h intervals and with a booster administration (1% aerosol) at day 14. At day 21, control and sensitized rats were challenged or not with CA (or solvent) by aerosol and after 24 h, AM were obtained by bronchoalveolar lavages and the expression of FccRII/CD23 antigen "as measured by flow cytometry using the BBIO monoclonal antibody. No expression of FccRII/CD23 on AM from non sensitized rats challenged or not with OA, "as observed. 85% of the AM from sensitized rats After OA challenge, expressed Fc~R11/CD23 antigen, compared to 40% after saline challenge. AM from non-sensitized rats were stimulated & vitro with recombinant murine IL-4 (l-100 U/ml) and for defined time intervals. AM only expressed FcsRII/CD23 after IL-4 stimulation, with a maximum of 40-50 % with the dose of 1OC U/ml and after 48 h. These results suggest that IL-4 midht play a role in the late phase of allergic reaction through the regulation of AM FczRII/C!D23 expression.
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TRANSFORMING GROWTH FACTOR-BETA 1 (TGF AND T LYMPHOCYTES REGULATE THE GROWTH OF DAY 14 CFU-GM F ROM PERIPHERAL BLOOD (PB). D.C. Doolev. P. Law, American Red Cross, Holland Laboratories. Rockville. MD. 20855. In this itudy, we eiamined the effect of TGF on da 14 CFU-GM from PB. Two PB cell populations were used: BAull cells MNC depleted of T cells and monocytes) and null cells f (B/nul cells treated with Camoath-1M monoclonal antibodv and Enrichments fo'r PB CFU-GM were 17-fold and
AUGMENTATION OF ENDOTOXIN-INDUCED NECROSIS OF TUMORS AND PRIMING OF MACROPHAGES FOR TUMOR NECROSIS FACTOR PRODUCTION BY GROWTH HORMONE. C.K. Edwards. III. R.M. Lorence, R.J. Walter. K.W. Kellev, L.M. Yunqer. and J.A. Greaaer. Dept. of Animal Sciences, Univ. of Illinois, Urbana 61801; and Depts. of Surgery, Univ. of Illinois and Cook County Hospital, Chicago, IL 60612 We have previously demonstrated that growth hormone (GH) is a potent activator of macrophages in vivo. To test for b w production of tumor necrosis factor (TNF), we assessed hemorrhagic necrosis of TNF-sensitive murine Meth A fibrosarcomas. Native, pituitary-derived porcine GH (133 ug/mouse/day, n = 8) or control medium (placebo, 200 ul/mouse/day, n = 10) was injected subcutaneously for 7 days into Balb/c mice having Meth A tumors. On the last day of treatment, 2 yg of lipopolysaccharide (LPS) were administered intraperitoneally to each mouse. The extent of necrosis and the total tumor size were measured 48 hours later and a ratio of these sizes calculated for each animal (N/T ratio). Treatment of tumor-bearing mice with GH significantly (~~0.05) increased the degree of LPS-induced necrosis of tumors (N/T ratio = 0.41 f 0.10) compared to the placebo/LPStreated mice (N/T ratio = 0.09 ? 0.05). This result confirms and extends our previous findings which demonstrated that treatment of hypophysectomized Fats with GH primes macrophages in vivo for TNF release. [Supported by ACS Ill. Div. (JAG), NIH AG-06246 and USDA 86-CRCR-1-2003 (KWK)]
be blocked b a n&utralizing antibody to TGF: We previously reported ti(,t CFU-GM growth could be enhanced by co-cultivation with unstimulated autologous T cells. T cells stimulated CFU-GM growth even in the oresence of TGF. Enhancement was not sue to binding of TGF by theft cells. T cells stimulated all mveloid colonies to an eaual extent,
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INTERFERON-y (IFN-Y)-INDUCED CYTOTOXICITY FIBROBLASTS: POTENTIATION BY LIPOPOLYSACCH~!DE 1% INTERLEUKIN-1 (IL-11 OR TUMOR NECROSIS FACTOR (TW). R. Dijkmans, J. Van Daanne, H. Heremans, A. Billiau. Rega Insti= tute, Unlversitv of Leuven (Belqiuml. IFN-y can be cytolytic for -normal mouse fibroblasts isolated from embryonic or adult tissue. This cytotoxicity is transcriptionand translation-dependent, thus suggesting involvement of a suicide-like mechanism. The dose required for cytotoxicity is higher than that needed for antiviral effect or macrophage activation but is reduced 10 to lOOfold by co-treatment of the cells with TNF and/or IL-l, two cytokines that, by themselves are not toxic for these cells. Bacterial LPS, which also by itself has no effect on the viability of mouse fibroblasts, similarly stimulates cell suicide induced by IFN-y. The effect was observed in cultures that were virtually free of non-fibroblastoid cells. Inclusion of antibodies directed against TNF-cr or IL-la in the culture medium did not block the cytotoxic effect of combined IFN-y + LPS treatment. In addition to LPS, heat-killed Gramnegative (Escherichia coli) but also Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) were found to enhance IFN-y-induced cell death. These findings suggest that IFN-y formed in vivo during infectious processes may directly aggravate tissue destruction.
THE ElA PROTEINS OF ADENOVIRUS RENDER INFECTED CELLS SUSCEPTIBLE TO TNF CYTOLVSIS. P. J. Duerksen-Huahes. W. S. M. Wold and L. FL Goodina. Emory University, Atlanta. GA 30322 and St. Louis University School of Medicine, St. Louis, MO 63110. Adenovirus encodes a 14.7K protein which inhibits cell killing by TNF. This is presumably necessary for the virus because, at least under conditions used in v&o, Ad infection makes normally resistant cells sensitive to TNF killing. To determine which viral function(s) was/were responsible for inducing the TNF susceptibility, cells were infected with a panel of viral double mutants, lacking both the 14.7 kD gene and another part of the viral genome. The infected cells were then treated with TNF, and the sensitivity determined with a "Cr release assay. Experiments with hydroxyurea and I-beta-D-arabinofuranosylcysteine showed that the sensitivity function mapped to an early region. TNF lysed cells infected by mutants which lacked regions E3, E4, ElB, and VA-RNA. Thus, none of these regions are necessary to produce TNF sensitivity. However, when cells were infected with a mutant lacking the 14.7K gene plus the EIA region, dL312, TNF was unable to lyse the cells. Thus, ElA is necessary to induce sensitivity to TNF. The ElA region encodes hvo overlapping proteins of 243 and 289 amino acids, both of which can induce TNF susceptibility. Thus, the domain present in 289R but lacking in 243R, which activates transcription of specific cellular genes during virus infection, is not responsible for TNF susceptibility. We are currently testing other ElA mutants to determine which of the other ElA ascribed functions, e.g. enhancer repression, growth factor induction. ras cooperation, correlates with induction of TNF sensitivity. This work was supported by grants CA08544 (PDH). CA24710 (WSMW), and CA40266, A126035, and CA48219 (LRG) from the NIH.
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DIFFERENTIAL REGULATION OF IL-1 BY IFN> AND IL-4. R.P. Donnelly Center for Biologics Evaluation MD
PRODUCTION IN HUMAN MONOCYTES and T.L. Gerrard. FDA 6 Research, NIH, Bethesda,
20892.
A number of T cell-derived cytokines, including IFNJ and IL-4, have been shown to regulate monocytelmacrophage function. ha~se MHc class II antigen (HLA-DR) expression and the ability to produce IL-l are two elements which influence the overall antigen-presenting capacity oE monocytes, we evaluated rHuIFN6 and rHuIL-4 for their ability to regulate these properties in normal human monocytes. Monocytes were prepared by counter-current centrifugal elutriation and demonstrated to be>90% pure as assessed by Giemsa and non-specific esterase staining. Treatment with IFNd or IL-4 significantly increased the basal expression of HLA-DR in human monocytes as determined by flow cytometric analysis after staining with HLA-DR-specific monoclonal antibodies. HOWeVer, unlike IFNr which potentiated monocyte IL-1 production induced by lipopolysaccharide (LPS), IL-4 inhibited IL-l production if added prior to or concomitant with LPS. This inhibitory effect was apparent with IL-4 These findings suggest concentrations as low as 1 ngfml. that the relative levels of IFNd and IL-4 mav reeulate the state of monocyte activation. Moreover, IL-4 may specifically function to prevent overexpression of IL-l by monocytes during the later stages of a cell-mediated immune response.
INCREASXO EfPRESSION OF FceRII/CD23 ON RAT ALvNaLAN NAcRoPNAGEs AFrEN ANymEN czImam3 AND IL-4 STIWJLATION. B.DUGAS, V.LAGENTE, E.BOICHOT, M.CAPRON*, J.M.MENCIA-HUERTA and P.BRAQUET. Institut Henri Beaufour, Les Ulis, France and *Institut Pasteur, Lille, France. The possible modulation of FceRII/CD23 expression on alveolar macro?ahases (AM) rats followino -- in _., from sensitized viva antigen challenge and --in vitro incubation with IL-4 was Gstiqated. Brown NOLWW rats (ZOO-250 9) were sensitized by two bvalbumin aerosols (OA, i%), at 4; h intervals and with a booster administration (1% aerosol) at day 14. At day 21, control and sensitized rats were challenged or not with CA (or solvent) by aerosol and after 24 h, AM were obtained by bronchoalveolar lavages and the expression of FccRII/CD23 antigen "as measured by flow cytometry using the BBIO monoclonal antibody. No expression of FccRII/CD23 on AM from non sensitized rats challenged or not with OA, "as observed. 85% of the AM from sensitized rats After OA challenge, expressed Fc~R11/CD23 antigen, compared to 40% after saline challenge. AM from non-sensitized rats were stimulated & vitro with recombinant murine IL-4 (l-100 U/ml) and for defined time intervals. AM only expressed FcsRII/CD23 after IL-4 stimulation, with a maximum of 40-50 % with the dose of 1OC U/ml and after 48 h. These results suggest that IL-4 midht play a role in the late phase of allergic reaction through the regulation of AM FczRII/C!D23 expression.
I89
192
TRANSFORMING GROWTH FACTOR-BETA 1 (TGF AND T LYMPHOCYTES REGULATE THE GROWTH OF DAY 14 CFU-GM F ROM PERIPHERAL BLOOD (PB). D.C. Doolev. P. Law, American Red Cross, Holland Laboratories. Rockville. MD. 20855. In this itudy, we eiamined the effect of TGF on da 14 CFU-GM from PB. Two PB cell populations were used: BAull cells MNC depleted of T cells and monocytes) and null cells f (B/nul cells treated with Camoath-1M monoclonal antibodv and Enrichments fo'r PB CFU-GM were 17-fold and
AUGMENTATION OF ENDOTOXIN-INDUCED NECROSIS OF TUMORS AND PRIMING OF MACROPHAGES FOR TUMOR NECROSIS FACTOR PRODUCTION BY GROWTH HORMONE. C.K. Edwards. III. R.M. Lorence, R.J. Walter. K.W. Kellev, L.M. Yunqer. and J.A. Greaaer. Dept. of Animal Sciences, Univ. of Illinois, Urbana 61801; and Depts. of Surgery, Univ. of Illinois and Cook County Hospital, Chicago, IL 60612 We have previously demonstrated that growth hormone (GH) is a potent activator of macrophages in vivo. To test for b w production of tumor necrosis factor (TNF), we assessed hemorrhagic necrosis of TNF-sensitive murine Meth A fibrosarcomas. Native, pituitary-derived porcine GH (133 ug/mouse/day, n = 8) or control medium (placebo, 200 ul/mouse/day, n = 10) was injected subcutaneously for 7 days into Balb/c mice having Meth A tumors. On the last day of treatment, 2 yg of lipopolysaccharide (LPS) were administered intraperitoneally to each mouse. The extent of necrosis and the total tumor size were measured 48 hours later and a ratio of these sizes calculated for each animal (N/T ratio). Treatment of tumor-bearing mice with GH significantly (~~0.05) increased the degree of LPS-induced necrosis of tumors (N/T ratio = 0.41 f 0.10) compared to the placebo/LPStreated mice (N/T ratio = 0.09 ? 0.05). This result confirms and extends our previous findings which demonstrated that treatment of hypophysectomized Fats with GH primes macrophages in vivo for TNF release. [Supported by ACS Ill. Div. (JAG), NIH AG-06246 and USDA 86-CRCR-1-2003 (KWK)]
be blocked b a n&utralizing antibody to TGF: We previously reported ti(,t CFU-GM growth could be enhanced by co-cultivation with unstimulated autologous T cells. T cells stimulated CFU-GM growth even in the oresence of TGF. Enhancement was not sue to binding of TGF by theft cells. T cells stimulated all mveloid colonies to an eaual extent,