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Cytokines and neuronal degeneration in cerebellar mutant mice
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CYTOKINES AND NEURONAL DEGENERATION IN CEREBELLAR MUTANT MICE. B. Kopmels*, A. Bakalian”, N. Delhaye-Bouchaud”, D. Fradelizi”, J. Mariani” and E.E. Wollman’. * Laboratoire d’Immunolo@e, URA CNRS 1154, Institut Gustave Roussy! 94805 Villejuif, France. Instltut des Neurosclences, URA1199, 9 Qua1 St. Bernard, 75252 Paris Cedex 05, France. We recently reported an abnormal production of IL1 in peripheral macrophages of mutant mice that exhibit patterns of neuronal degeneration in the cerebellum (Kopmels et al., 1990). After in vitro activation by LPS, these macrophages hyperexpress IL-IS mRNA and hyperproduce IL-1 protein, in comparison with (t /+) controls. In the present study, we investigate in the staggerer mutant mice, if this dysregulation is specific for IL-la or if it reflects an hyperexcitability state of these macrophages. The hyperexpression is present whatever the time of LPS stimulation even as short as 15 minutes, the classical period being of 4 hours. The hyperinducibility of (sg/sg) macrophages is observed even with very low doses of LPS (0,Ol g/ml) and reaches a maximum for 5 g/ml LPS. Synthetic molecules as muramyl dipeptides are efficient to reveal the IL-18 hyperexpression. Moreover, an hyperexpression of 3 other cytokines, TNF IL-1 and IL-6 is also detected in LPS-stimulated macrophages of mutant mice. We conclude that the (sg/s ) macrophages are in a state of general hyperexcitability compared to $ + /+ ) ones.
CHOLERA TOXIN STRONGLY PROMOTES ANTIGEN- PRESENTATION IN VIVO AND IN VITRO BY STIMULATION OF ILl: N. Lvcke . A. Bromanda and I. Hplmerea. Depnf Medical Microbiology and Immunology. University of G6tetorg ,%I13 46 Gdtebag. Sweden. Cholera toxin (CT) is an effective mucasd adjuvant that enhanceslocal gut mucosal IgA immune responsesby~0.fold ormore.ln apreviousrepxtusing .dloreactiveTcells we provided evidence for that CT p~tentiaresantigen presentation by increasing ca-stimulation through U-l production and in particular cell-associated L-la. In this study we have investigated CT% enhancingeffect further using the IL-1 reactive clonal. conalbumin-specific,T cell line ,DlO.G4.1 (H-2k) as indicator system. We found that CT-eeatment in vitro of normal peritonealmacmphages strongly augmentedthecapaciry topresent the soluble antigen,conalbumin,to the DlO.G4.1 cells. This effect by CT was U-la dependentBSdemonsvated by the snong inhibition of DlO.G4.1 proliferation in the presence of a polyclonal lL.-la-spsitic antisemm. We next investigated whether antigen-presentingcells(APC)expased to~invivothmughpaoraladminisUation also squired an enhancedcapacity u1pesent antigen.When APC were isolatedfrom Peyer's Patches 24h following oralC!T-eearmentweobser~ed e.3.6 fold highercapacify 10presemcanalbnminto DlO.G4.1 as compared fo APC from no&T-@eated mice. Moreover. APC with several-fold incredTceIlaiggerhg abilLy could alsabeisolatedhommesmrericlymphnodes orevenfrom the spleen24hfollowing pemralfl-administration Thea-potentiated APC werenon-adherent cells,Thy 1.2 negative.Mac1 negative snd most probably B cells. Another population in the mucusal tissuethat has beendemonsbatedto present antigento T cells isepithelislcels. We therefore used aratepithelial cell line lEC 17,pulsed for 24h with or withoutCT,to sttiyifthesecels WouldpresentallaantigentoMHC incompadbleTce1l.vinvitro. Also in this systemCTpatentiatedthe antigen-presenting capacity3.fold. Studiesareundenuayta examineifthis effectby CTanepirhelalcellshas functionalimplications for antigen-presentation in the gut immune system and by what mechainsm CT induces enhtmced AK-function in epilhelial cells.
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DEMONSTRATION OF LOCAL INDUCTION OF HIGH TNFa LEVELS BY LPS AND LPSl ANTI-LPS IMMUNE COMPLEXES IN CHRONIC PSEUDOMONAS AERUGINOSA LUNG INFECTION. Gitte Kronborg, Anders Fomsgaard, Morten B. Hansen. Dpt. of Clinical Microbiology and Dpt. of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark. TNF, a major inflammatory cytokine, was detected in sputum samples from 10 patients with cystic fibrosis (CF) and chronic P.aeruainosa lung infection. In ELISA the amounts of TNF measured varied from O-900 pg/ml and in a WEHI cell cytotoxicity bioassay from 150-5600 pg/ml. In contrast, all plasma samples taken at the same time were negative in both systems. We have measured very high titers of anti-LPS antibodies and high amounts of circulating immune complexes (IC)s, containing P.aeruainom LPS and lgG2, in serum and sputum from the same
ANTIMALARIALS INHIBIT THE ANTIVIRAL ACTIVI-IY OF INTERFERON IN MICE. R.K. Maheshwari. J. Sklarsh. D. Bhartiva, and V. Srikantan, Uniformed Services University of the Health Sciences, Bethesda, MD 20814. Studies reported here demonstrate that anti-malarials (chloroquine, primaquine, pyrimethamine, sulfadoxine, and quinine sulfate) enhances the semliki forest and encephalomyocarditis virus replication in mice. It is possible that these antimalarials inhibit the endogenous production of interferon (IFN), which may be responsible for the enhancement of virus virulence. We have therefore, treated mice with IFN or polyinosinic-polycytidylic acid (Poly I:C) a potent inducer of IFN. Both IFN and Poly I:C protected mice against these virus infections. However, treatment with these antimalarials abrogated the antiviral activity of IFN and Poly I:C as evident by a decrease in the survivors and mean survival time. These results may have clinical implications especially with the use of IFN against viral infections in malaria endemic areas. Secondly, the widespread use of these antimalarials may predispose the population to enhanced viral infections including j-&/ in malaria endemic areas. In fact, the spread of AIDS has been especially rapid in the areas of tropical Africa that have a high incidence of malaria and CHL has been frequently used in the chemotherapy of malaria.
group of patients. We made ICs ofP.aeruainoa LPS and antiLPS from CF patients -in different ratios of LPS/ anti-LPS and stimulated MNCs. The TNF response in the supernatants increased with lower amounts of anti-LPS in the ICs, indicating a neutralizing effect of the antibodies. Thus, the higher load of unbound LPS in the lungs could be responsible for the induction of local TNF production. The LPS induced TNF may contribute to the local inflammation and tissue damage, which isthe major course of mortality in these patients.
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ELEVATED ENDOGENOUS IL-6 IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE): A PUTATIVE ROLE IN PATHOGENESIS. M. Linker-Israeli, D.J. Wallace. J Prehn. T. Ozeri-Chen and J.R. Klinenberq. Cedars-Sinai Medical Center, Los Angeles, CA 90048. To identify the factors that support spontaneous IgG production in SLE, we studied IL-6, a cytokine promoting B cell differentiation. Endogenous IL-6 levels in serum of SLE pts were measured with the B9 assay. Elevated serum levels of IL-6 (~~0.05) were found in 36/39 pts. 19/19 pts with active disease had detectable IL-6 and the average titer was higher (p=O.OOS) than that of inactive SLE (N=20) which was higher (~~0.05) than in healthy controls (N=ll). In cytoplasmic RNA extracted from freshly isolated PBMC of 7 SLE pts, IL-6 mRNA levels were 5-17.5 fold higher than normal (~~0.05). IL-6 was also detected in 3/3 kidney biopsies of pts with kidney disease. In short-term cultures of SLE PBMC, neutralizing antibodies to IL-6, TNF or IL-1 decreased spontaneous IgG production by 30.1+4.3X, 27+5.7% and 30+10.2% respectively. Anti-TNF treatment was associated with decreased IL-6 content of cultures (18.321 u/ml vs 53.2214.9 u/ml untreated) and exogenous IL-6 reversed the anti-TNF inhibition. In contrast, neutralization of endogenous IL-4 increased IgG production by 44.7+14.5X, and anti-IL-4 increased IL-6 to 155 u/ml. Thus, TNF and IL-4 effects on spontaneous IgG production in SLE may be attributed to modulation of IL-6 production or activity. Together, these results support the concept that SLE B cell hyperactivity is maintained by dysregulation of endogenous cytokines and suggest that IL-6 in particular has an important role in SLE pathogenesis.
PHYSIOLOGICAL HYPORESPONSIvENESe TO INFLUENZA VIRUS IS INDUCED BY dsP.NA AND ASSOCIATED WITH ABSENCE OF ANTIVIRAL ACTIVITY. J.Maide, M.Kimura-Takeuichi +g@ J.Krueoer Univ. Tennessee. Hamhis. TN 38163 We are~inv&tig$ting the relationship of viral-derived dsRNA to the triggering of cytokines driving constitutional symptoms in acute influenza. Rabbits were injected IV at 24 hr intervals as follows: Day l--control egg allantoic fluid: Day 2--influenza virus (2 x 10' LD, A/PR/8/34) m 2.5 pg/kg poly[rI*rC] (synthetic dsRNA); Day 3--virus as on Day 2. Sleep activity and fever (detected as elevated brain temperature) were monitored continuously for 72 hr. serum samoles were taken iust before iniections itime 01. 4 and 24 hr after injections-and were ass&d for &ivFrai'activity (AVA) in X-13 cells challenged with VSV. Day 2 challenge with virus or poly[rI*rC] resulted in excess sleep and fever over the first 7 hr. manifested 4 hr earlier following poly(rI.rC]. Day 3 viral challenge resulted in sleep and fev& patterns similar to baselin; (hyporesponsiven&) reaardless of challenoe twx on Da" 2. AVA titers on DLY 2 w&e below detectable-limits at t&e 0 or at 24 hr, but i hr AVA titers following virus were 351 + 179 and following poly[rI*rC] were 2600 + 536 IU/ml serum. All Day 3 serum samples were negative for AVA. This model demonstrates that poly(rI.rC], which we have shown induces sleep and fever responses indistinguishable from viral-associated dsRNA, can induce physiological hypfxesponsiveness indistinguishable from that induced by virus itself. Because cytokines such as interferon-a/R/r, tumor necrosis factor-a/0, and interleukin-6 can induce the AVA we measured here, and all of these cytokines can be induced by virus and by poly[rI.rC], it is unknown as to which cytokinee are involved in the observed hyporesponsiveness phenomena.
INTERNATIONAL
WORKSHOP
ON CYTOKINES
/ 511
367
370
CYTOKINES AND NEURONAL DEGENERATION IN CEREBELLAR MUTANT MICE. B. Kopmels*, A. Bakalian”, N. Delhaye-Bouchaud”, D. Fradelizi”, J. Mariani” and E.E. Wollman’. * Laboratoire d’Immunolo@e, URA CNRS 1154, Institut Gustave Roussy! 94805 Villejuif, France. Instltut des Neurosclences, URA1199, 9 Qua1 St. Bernard, 75252 Paris Cedex 05, France. We recently reported an abnormal production of IL1 in peripheral macrophages of mutant mice that exhibit patterns of neuronal degeneration in the cerebellum (Kopmels et al., 1990). After in vitro activation by LPS, these macrophages hyperexpress IL-IS mRNA and hyperproduce IL-1 protein, in comparison with (t /+) controls. In the present study, we investigate in the staggerer mutant mice, if this dysregulation is specific for IL-la or if it reflects an hyperexcitability state of these macrophages. The hyperexpression is present whatever the time of LPS stimulation even as short as 15 minutes, the classical period being of 4 hours. The hyperinducibility of (sg/sg) macrophages is observed even with very low doses of LPS (0,Ol g/ml) and reaches a maximum for 5 g/ml LPS. Synthetic molecules as muramyl dipeptides are efficient to reveal the IL-18 hyperexpression. Moreover, an hyperexpression of 3 other cytokines, TNF IL-1 and IL-6 is also detected in LPS-stimulated macrophages of mutant mice. We conclude that the (sg/s ) macrophages are in a state of general hyperexcitability compared to $ + /+ ) ones.
CHOLERA TOXIN STRONGLY PROMOTES ANTIGEN- PRESENTATION IN VIVO AND IN VITRO BY STIMULATION OF ILl: N. Lvcke . A. Bromanda and I. Hplmerea. Depnf Medical Microbiology and Immunology. University of G6tetorg ,%I13 46 Gdtebag. Sweden. Cholera toxin (CT) is an effective mucasd adjuvant that enhanceslocal gut mucosal IgA immune responsesby~0.fold ormore.ln apreviousrepxtusing .dloreactiveTcells we provided evidence for that CT p~tentiaresantigen presentation by increasing ca-stimulation through U-l production and in particular cell-associated L-la. In this study we have investigated CT% enhancingeffect further using the IL-1 reactive clonal. conalbumin-specific,T cell line ,DlO.G4.1 (H-2k) as indicator system. We found that CT-eeatment in vitro of normal peritonealmacmphages strongly augmentedthecapaciry topresent the soluble antigen,conalbumin,to the DlO.G4.1 cells. This effect by CT was U-la dependentBSdemonsvated by the snong inhibition of DlO.G4.1 proliferation in the presence of a polyclonal lL.-la-spsitic antisemm. We next investigated whether antigen-presentingcells(APC)expased to~invivothmughpaoraladminisUation also squired an enhancedcapacity u1pesent antigen.When APC were isolatedfrom Peyer's Patches 24h following oralC!T-eearmentweobser~ed e.3.6 fold highercapacify 10presemcanalbnminto DlO.G4.1 as compared fo APC from no&T-@eated mice. Moreover. APC with several-fold incredTceIlaiggerhg abilLy could alsabeisolatedhommesmrericlymphnodes orevenfrom the spleen24hfollowing pemralfl-administration Thea-potentiated APC werenon-adherent cells,Thy 1.2 negative.Mac1 negative snd most probably B cells. Another population in the mucusal tissuethat has beendemonsbatedto present antigento T cells isepithelislcels. We therefore used aratepithelial cell line lEC 17,pulsed for 24h with or withoutCT,to sttiyifthesecels WouldpresentallaantigentoMHC incompadbleTce1l.vinvitro. Also in this systemCTpatentiatedthe antigen-presenting capacity3.fold. Studiesareundenuayta examineifthis effectby CTanepirhelalcellshas functionalimplications for antigen-presentation in the gut immune system and by what mechainsm CT induces enhtmced AK-function in epilhelial cells.
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DEMONSTRATION OF LOCAL INDUCTION OF HIGH TNFa LEVELS BY LPS AND LPSl ANTI-LPS IMMUNE COMPLEXES IN CHRONIC PSEUDOMONAS AERUGINOSA LUNG INFECTION. Gitte Kronborg, Anders Fomsgaard, Morten B. Hansen. Dpt. of Clinical Microbiology and Dpt. of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark. TNF, a major inflammatory cytokine, was detected in sputum samples from 10 patients with cystic fibrosis (CF) and chronic P.aeruainosa lung infection. In ELISA the amounts of TNF measured varied from O-900 pg/ml and in a WEHI cell cytotoxicity bioassay from 150-5600 pg/ml. In contrast, all plasma samples taken at the same time were negative in both systems. We have measured very high titers of anti-LPS antibodies and high amounts of circulating immune complexes (IC)s, containing P.aeruainom LPS and lgG2, in serum and sputum from the same
ANTIMALARIALS INHIBIT THE ANTIVIRAL ACTIVI-IY OF INTERFERON IN MICE. R.K. Maheshwari. J. Sklarsh. D. Bhartiva, and V. Srikantan, Uniformed Services University of the Health Sciences, Bethesda, MD 20814. Studies reported here demonstrate that anti-malarials (chloroquine, primaquine, pyrimethamine, sulfadoxine, and quinine sulfate) enhances the semliki forest and encephalomyocarditis virus replication in mice. It is possible that these antimalarials inhibit the endogenous production of interferon (IFN), which may be responsible for the enhancement of virus virulence. We have therefore, treated mice with IFN or polyinosinic-polycytidylic acid (Poly I:C) a potent inducer of IFN. Both IFN and Poly I:C protected mice against these virus infections. However, treatment with these antimalarials abrogated the antiviral activity of IFN and Poly I:C as evident by a decrease in the survivors and mean survival time. These results may have clinical implications especially with the use of IFN against viral infections in malaria endemic areas. Secondly, the widespread use of these antimalarials may predispose the population to enhanced viral infections including j-&/ in malaria endemic areas. In fact, the spread of AIDS has been especially rapid in the areas of tropical Africa that have a high incidence of malaria and CHL has been frequently used in the chemotherapy of malaria.
group of patients. We made ICs ofP.aeruainoa LPS and antiLPS from CF patients -in different ratios of LPS/ anti-LPS and stimulated MNCs. The TNF response in the supernatants increased with lower amounts of anti-LPS in the ICs, indicating a neutralizing effect of the antibodies. Thus, the higher load of unbound LPS in the lungs could be responsible for the induction of local TNF production. The LPS induced TNF may contribute to the local inflammation and tissue damage, which isthe major course of mortality in these patients.
369
372
ELEVATED ENDOGENOUS IL-6 IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE): A PUTATIVE ROLE IN PATHOGENESIS. M. Linker-Israeli, D.J. Wallace. J Prehn. T. Ozeri-Chen and J.R. Klinenberq. Cedars-Sinai Medical Center, Los Angeles, CA 90048. To identify the factors that support spontaneous IgG production in SLE, we studied IL-6, a cytokine promoting B cell differentiation. Endogenous IL-6 levels in serum of SLE pts were measured with the B9 assay. Elevated serum levels of IL-6 (~~0.05) were found in 36/39 pts. 19/19 pts with active disease had detectable IL-6 and the average titer was higher (p=O.OOS) than that of inactive SLE (N=20) which was higher (~~0.05) than in healthy controls (N=ll). In cytoplasmic RNA extracted from freshly isolated PBMC of 7 SLE pts, IL-6 mRNA levels were 5-17.5 fold higher than normal (~~0.05). IL-6 was also detected in 3/3 kidney biopsies of pts with kidney disease. In short-term cultures of SLE PBMC, neutralizing antibodies to IL-6, TNF or IL-1 decreased spontaneous IgG production by 30.1+4.3X, 27+5.7% and 30+10.2% respectively. Anti-TNF treatment was associated with decreased IL-6 content of cultures (18.321 u/ml vs 53.2214.9 u/ml untreated) and exogenous IL-6 reversed the anti-TNF inhibition. In contrast, neutralization of endogenous IL-4 increased IgG production by 44.7+14.5X, and anti-IL-4 increased IL-6 to 155 u/ml. Thus, TNF and IL-4 effects on spontaneous IgG production in SLE may be attributed to modulation of IL-6 production or activity. Together, these results support the concept that SLE B cell hyperactivity is maintained by dysregulation of endogenous cytokines and suggest that IL-6 in particular has an important role in SLE pathogenesis.
PHYSIOLOGICAL HYPORESPONSIvENESe TO INFLUENZA VIRUS IS INDUCED BY dsP.NA AND ASSOCIATED WITH ABSENCE OF ANTIVIRAL ACTIVITY. J.Maide, M.Kimura-Takeuichi +g@ J.Krueoer Univ. Tennessee. Hamhis. TN 38163 We are~inv&tig$ting the relationship of viral-derived dsRNA to the triggering of cytokines driving constitutional symptoms in acute influenza. Rabbits were injected IV at 24 hr intervals as follows: Day l--control egg allantoic fluid: Day 2--influenza virus (2 x 10' LD, A/PR/8/34) m 2.5 pg/kg poly[rI*rC] (synthetic dsRNA); Day 3--virus as on Day 2. Sleep activity and fever (detected as elevated brain temperature) were monitored continuously for 72 hr. serum samoles were taken iust before iniections itime 01. 4 and 24 hr after injections-and were ass&d for &ivFrai'activity (AVA) in X-13 cells challenged with VSV. Day 2 challenge with virus or poly[rI*rC] resulted in excess sleep and fever over the first 7 hr. manifested 4 hr earlier following poly(rI.rC]. Day 3 viral challenge resulted in sleep and fev& patterns similar to baselin; (hyporesponsiven&) reaardless of challenoe twx on Da" 2. AVA titers on DLY 2 w&e below detectable-limits at t&e 0 or at 24 hr, but i hr AVA titers following virus were 351 + 179 and following poly[rI*rC] were 2600 + 536 IU/ml serum. All Day 3 serum samples were negative for AVA. This model demonstrates that poly(rI.rC], which we have shown induces sleep and fever responses indistinguishable from viral-associated dsRNA, can induce physiological hypfxesponsiveness indistinguishable from that induced by virus itself. Because cytokines such as interferon-a/R/r, tumor necrosis factor-a/0, and interleukin-6 can induce the AVA we measured here, and all of these cytokines can be induced by virus and by poly[rI.rC], it is unknown as to which cytokinee are involved in the observed hyporesponsiveness phenomena.