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Lactate dehydrogenase-elevating virus-induced polioencephalomyelitis in immunosuppressed mice: Cytokines in the spinal cord
184
P08.12 THE EFI~CT OF GROWTH HORMONE ON THYMIC FUNCTION IN OLD AGE MAY BE MEDIATED BY ZINC POOL E. Mocchegiani, L. Santarelli, D. Bulian, N. Pabris. Ctr. Immanol. INRCA, Ancona Italy. Experimental am/clinical evidences sugge,st that thymic endocrine activity, as measured by the plasma level of thymulin (Zn-FTS), is modulated by the neuroendocrine network, in particular by growth hormone (GH). GH treatment in old mice is able to increase the reduced thymic size and to recover the low thymic function. Also in adult men with GH-deficiency, a GH treatment is effective in recovering the reduced active thymu[in (ZE-FFS) levels to values of age matched controls. Direct mechanisms of action of GH on tbymic functions have been suggested dBe to the presence of specific GH-receptors into the thymus. However, without excluding direct actions of GH and taking into account the pivotal role played by zinc for neuruendoorine regulation, data obtained in adult GH-deficient men, suggest as possible involvement of zinc, since it is found reduced in GH-deficient men and increased after GH-trestment. In old mice a GH-treatment for I month (2ttg/b. w., twice week of rhGH) increases the negative crude zinc balance (-1,2 mE/day/mouse) in old mice to positive values (+1,0 mE/day/mouse) similar to those ones observed in young mice (+ 1,4 mE/day/mouse). Such findings might suggest a possible involvement of zinc pool in GH effect on thymic function.
P03.13 INIX)MEll,lACIN AND D PRODUCTION BY MiCE ~
P14.11 LACTATE DEHYDROGENASE-ELEVATINGVlRU,S-INDUCED POUOENCEPHALOMYELITISIN IMMUNOgUPPIREg~-"~MICE: CYTOKINES IN THE SPINALCORD. ph Montevne (1), C.J M Sindio (2), =L-P~C ~ , ~ r (J~ (1) Unit of Experimental Medicine, U.C.L., and (2) Laboratory of NeurochBm~ry, U.C.L., B e a m . Lactate dehydrogenase-elevating virus (LDV) induces a polioencephalomyelltls in C58 and AKR mice. The development of the paralytic d b e a u oorrelatN with old age, ~ of immune o ~ l e n ~ , end genetic predlepoaltion w~th presence of endogenous mudna leukemia virus (MuLV). It hembeefl d ~ e d that ~ reEU#Sfrom the loss of some T cell~but this protectlVB cellular subl~0pul~ion remains to be charactedzed. We investigated the role of the cellular immune response in this system by replacing Ihe ¢laasioal irradiation of the mice or the cyclophosphamide injection by more restricted forms of immunosupprassion. We also studied the cellist r s ~ by an~yzin0 the cytokine gene expression in the spinal cord by revBrse~ P.C.R. The role of the T lymphecytas was first sUggBStedby the pose~ility to reproduce this model in micB Imrmmosuppreued by Injection of a monoclonal antt-Thy-1 antibody. The effeof of injection of other antilymphocyte an~k~clt~ such as an antt-OD4 monoclmmt m',g~.,~i i~ now investigated. Messages coding for IFN-g, IL-12 and other cytokines were shown in the spinal cord and their kinetics were dstBrminsd and compared with messages found in immunocompetent mice.
P08.13 ~
E
INHIBIT NITRIC OXIDE CELL CUI.TUREg.
A NOVEL NEURONAL AUTOANTIGEN PRESENT IN THE LIMBIC SYSTEM, RETINA, AND ADRENAL
F, Molina-Hol~ado. A. LLed6, C, Sandi and C. Guaza Neural P l a t y Department, Cajel Institute, CSIC, Madrid, Spain
Moore. P.M. and Carlock, L.R.
Intruducthan: Nitnc Oxide (NO) is released from different cell types. There is evidence for an inducible focm of NO wnthase (NOS) in estrogl~l cells, that shares a number of similarities with the inducible form of c y c ~ . In the Ixseant study we have evaluated the effects of indomethsoin and dexantofhasona in the release of nitrite (NO2-) from estrocytes after induction v,4thLP~. Methode: Primary Ufrocytes cultures were prep~'ed from neonat~ mice to routinely contain >95% GFAP positive a~ro~al c e l l s . . ~ were cultured in DMEM media containing 1/Jg/ml of LPS (E. Coli) for t8 hours, NO2- analysis in cell culture medium wae pedoened by Gr~'-,,,,,sreaction. 1~4~ltl: LPS induced an increase in the rsleaso of NO2-, that was inhibited in a concentrationdependent manner by coir,,~dmtion with L-NAME (10pM-lmM). Indomethacin at high doses (20pM) was also able to reduce NO~oproduclion induced by LPS, A maximal dose of dexamethasons (1.u~) consistently i:xeve~ed LPS induction of NO2-. Con~uoform: As in other cell types, in utrocytes dexamethesonB is able to inhibit NO2production ir~_uc~LJ_by LPS. Wealsosoggest an interaction between NOS and the cycioes'tganese pathways. Supported by grants from the DGCYT (PM 92-0Of 4), Spain Cytokinss in the Brain. Concerted Action Program for the EEC
Introduction: Identifying the autoantigons of spontaneously occurring antineuronal antibodies in murine and human s y e t m l c lupus erythematoaus will provide interesting biologic information and may elucidate pathogenic mechanisms of disease. Materials and ~ : Brain plasma membrane specific autoantibodieB identified a 1.2 kb oDRA of previously unrecognized sequence from a brain expression library. Northern analysis identified a 13 kb transcript in brain but no other organs. Using digoxygenin transcribed riboprobes we performed in situ hybridization on brain sections and other organs. Rezultst In situ hybridization revealed transcript in the l£mblc system, few other brain areas and small amounts in the adrenal gland and retina. Conclueionst Transcript for a brain plaBfna membrane peptide reactive w~th zpontaoeou01y o~curring antineuronal antibodies is present primarily in the limbic syste*n but also to some extent in the retina and adrenal gland. This localization may explain some of the symptoms in neuro-SLE.
P09.08 ROLE OF CELLULAR INFLAMMATION IN DERMATOMYOSITIS
P03.14 INTERLEUKIN-4MODULATESMHC CLASS H EXPRESSION AND NITRIC OXIDESYNTHESISBY RATASTROCY'rES
~;, Monaco I , G.L. Zanusso2, B. Bonetti 2, G. Tomelleri2, G. Pasinetti 3, N. Rizzuto2 llnst, of Neurology, Univ. of Padova, Italy, 2 Dept. of Neuxology, Univ. of V e ~ e . Italy. 3 Univ. of Southern California. Los Angeles, USA
E. MORGAand P. HEUSCHLING Neuroimmanoiogte& Inflammation.CRP - Sant~,Luxembourg Introduction: Cerebral astrocyles can be brought in rive and in vitro to adopt a • ~ m c e ~ Yaletwl.dan ~ ~ ~ class It expressian, increased ¢ytokJse production and syodm~ of an inducible nitric oxide (NO)-synthase.These chaaSesare c~.tsicaily mediated by baclecial LPS and inflammatory c/retinas like y-interferon (T-IFN)and ltlmor necrosis factor-el. N e ~ , like nee-epinephrine, and several cytokines like transforminggrowthfactm-l~and interlanldwl[~have bean shown to down-regulate some of these pl~motyptcstchange& In/edeedtin4 (IL-4), a T ceibdedved Jmwth factor, has recentlybeen ~ to act on ceeebrulastmcytes.We studied the ability of IL-4 to modalate tha exwessim of la moleculesand the symhe~s of NO radiculs on cultmedrst aslrocytesin the presence~ al~maceof ~IFN. Mstenlets i Mstkocls: Class It expression is mmlsmedby flow cytometry. NO synthesis is estimatedby the Gaieasmethod. Results In the ab~see of ~ll[~l, IL.4 redeces c o n c e n l x a l i o a ~ t l y the basal la exweseim, tbe degreeof significance~ ~ at S U/eL ¥-IFNugegulstes the exprenim of cam rl ~ bni this enact is....~..,~velLmd~ued by n.-4. These propertiesof 11.-4 are ~ by indmaethacin (IO°M). 7.1FN triggers the production of an inducible form of NO-systhase in astrogliat cells. A coincubation with IL-4 up-~eBLdutasfltis preduclio~ as visealiml by the quantification of NO2"in the cultm,e sepemat~t. Conclusion: These results indicate that IL-4 can act as a modalator of some immunologicaland inflammatoryfanctioasof astrocytes.
Dermatomyositis (DM) is an inflammatory myopathy characterized by perimysial and perivascular infiltrates. Previous studies explain muscle neerusis as a consequence of microvessel involvement following deposition of immune complexes and terminal complement complex (TCC) in the wall of ,perimysial vanules. We investigated the role of humornl and cellular factors in 6 muscle biopsies from patients with active DM. By immunocytoehemistry, deposition of TCC was seen in the walls of arterioles and capillaries as well as in fibers und~rgo/nl| n ~ i s ; TCC, however, co-localized with Sprot¢/n and clasterin, thus anggesdn~ that TCC may be funotiomally inactive. Increased cxp~ssion of VCAM and ICAM-I was observed in andothelia and macrophaBas ( M # ) a t sites of exudmiun of mono¢Ttes a~l CD4 l ~ o c y t e s . In addition, MHC-II molecules were up-regulated on endothelia and APCs. These observations s u g l ~ t that a T-cetl-mediated, MHC-ll-restr/¢W,d process plays a seminal role in DM. Supported in part by a grant from Telethon to S. Monaco.
P08.12 THE EFI~CT OF GROWTH HORMONE ON THYMIC FUNCTION IN OLD AGE MAY BE MEDIATED BY ZINC POOL E. Mocchegiani, L. Santarelli, D. Bulian, N. Pabris. Ctr. Immanol. INRCA, Ancona Italy. Experimental am/clinical evidences sugge,st that thymic endocrine activity, as measured by the plasma level of thymulin (Zn-FTS), is modulated by the neuroendocrine network, in particular by growth hormone (GH). GH treatment in old mice is able to increase the reduced thymic size and to recover the low thymic function. Also in adult men with GH-deficiency, a GH treatment is effective in recovering the reduced active thymu[in (ZE-FFS) levels to values of age matched controls. Direct mechanisms of action of GH on tbymic functions have been suggested dBe to the presence of specific GH-receptors into the thymus. However, without excluding direct actions of GH and taking into account the pivotal role played by zinc for neuruendoorine regulation, data obtained in adult GH-deficient men, suggest as possible involvement of zinc, since it is found reduced in GH-deficient men and increased after GH-trestment. In old mice a GH-treatment for I month (2ttg/b. w., twice week of rhGH) increases the negative crude zinc balance (-1,2 mE/day/mouse) in old mice to positive values (+1,0 mE/day/mouse) similar to those ones observed in young mice (+ 1,4 mE/day/mouse). Such findings might suggest a possible involvement of zinc pool in GH effect on thymic function.
P03.13 INIX)MEll,lACIN AND D PRODUCTION BY MiCE ~
P14.11 LACTATE DEHYDROGENASE-ELEVATINGVlRU,S-INDUCED POUOENCEPHALOMYELITISIN IMMUNOgUPPIREg~-"~MICE: CYTOKINES IN THE SPINALCORD. ph Montevne (1), C.J M Sindio (2), =L-P~C ~ , ~ r (J~ (1) Unit of Experimental Medicine, U.C.L., and (2) Laboratory of NeurochBm~ry, U.C.L., B e a m . Lactate dehydrogenase-elevating virus (LDV) induces a polioencephalomyelltls in C58 and AKR mice. The development of the paralytic d b e a u oorrelatN with old age, ~ of immune o ~ l e n ~ , end genetic predlepoaltion w~th presence of endogenous mudna leukemia virus (MuLV). It hembeefl d ~ e d that ~ reEU#Sfrom the loss of some T cell~but this protectlVB cellular subl~0pul~ion remains to be charactedzed. We investigated the role of the cellular immune response in this system by replacing Ihe ¢laasioal irradiation of the mice or the cyclophosphamide injection by more restricted forms of immunosupprassion. We also studied the cellist r s ~ by an~yzin0 the cytokine gene expression in the spinal cord by revBrse~ P.C.R. The role of the T lymphecytas was first sUggBStedby the pose~ility to reproduce this model in micB Imrmmosuppreued by Injection of a monoclonal antt-Thy-1 antibody. The effeof of injection of other antilymphocyte an~k~clt~ such as an antt-OD4 monoclmmt m',g~.,~i i~ now investigated. Messages coding for IFN-g, IL-12 and other cytokines were shown in the spinal cord and their kinetics were dstBrminsd and compared with messages found in immunocompetent mice.
P08.13 ~
E
INHIBIT NITRIC OXIDE CELL CUI.TUREg.
A NOVEL NEURONAL AUTOANTIGEN PRESENT IN THE LIMBIC SYSTEM, RETINA, AND ADRENAL
F, Molina-Hol~ado. A. LLed6, C, Sandi and C. Guaza Neural P l a t y Department, Cajel Institute, CSIC, Madrid, Spain
Moore. P.M. and Carlock, L.R.
Intruducthan: Nitnc Oxide (NO) is released from different cell types. There is evidence for an inducible focm of NO wnthase (NOS) in estrogl~l cells, that shares a number of similarities with the inducible form of c y c ~ . In the Ixseant study we have evaluated the effects of indomethsoin and dexantofhasona in the release of nitrite (NO2-) from estrocytes after induction v,4thLP~. Methode: Primary Ufrocytes cultures were prep~'ed from neonat~ mice to routinely contain >95% GFAP positive a~ro~al c e l l s . . ~ were cultured in DMEM media containing 1/Jg/ml of LPS (E. Coli) for t8 hours, NO2- analysis in cell culture medium wae pedoened by Gr~'-,,,,,sreaction. 1~4~ltl: LPS induced an increase in the rsleaso of NO2-, that was inhibited in a concentrationdependent manner by coir,,~dmtion with L-NAME (10pM-lmM). Indomethacin at high doses (20pM) was also able to reduce NO~oproduclion induced by LPS, A maximal dose of dexamethasons (1.u~) consistently i:xeve~ed LPS induction of NO2-. Con~uoform: As in other cell types, in utrocytes dexamethesonB is able to inhibit NO2production ir~_uc~LJ_by LPS. Wealsosoggest an interaction between NOS and the cycioes'tganese pathways. Supported by grants from the DGCYT (PM 92-0Of 4), Spain Cytokinss in the Brain. Concerted Action Program for the EEC
Introduction: Identifying the autoantigons of spontaneously occurring antineuronal antibodies in murine and human s y e t m l c lupus erythematoaus will provide interesting biologic information and may elucidate pathogenic mechanisms of disease. Materials and ~ : Brain plasma membrane specific autoantibodieB identified a 1.2 kb oDRA of previously unrecognized sequence from a brain expression library. Northern analysis identified a 13 kb transcript in brain but no other organs. Using digoxygenin transcribed riboprobes we performed in situ hybridization on brain sections and other organs. Rezultst In situ hybridization revealed transcript in the l£mblc system, few other brain areas and small amounts in the adrenal gland and retina. Conclueionst Transcript for a brain plaBfna membrane peptide reactive w~th zpontaoeou01y o~curring antineuronal antibodies is present primarily in the limbic syste*n but also to some extent in the retina and adrenal gland. This localization may explain some of the symptoms in neuro-SLE.
P09.08 ROLE OF CELLULAR INFLAMMATION IN DERMATOMYOSITIS
P03.14 INTERLEUKIN-4MODULATESMHC CLASS H EXPRESSION AND NITRIC OXIDESYNTHESISBY RATASTROCY'rES
~;, Monaco I , G.L. Zanusso2, B. Bonetti 2, G. Tomelleri2, G. Pasinetti 3, N. Rizzuto2 llnst, of Neurology, Univ. of Padova, Italy, 2 Dept. of Neuxology, Univ. of V e ~ e . Italy. 3 Univ. of Southern California. Los Angeles, USA
E. MORGAand P. HEUSCHLING Neuroimmanoiogte& Inflammation.CRP - Sant~,Luxembourg Introduction: Cerebral astrocyles can be brought in rive and in vitro to adopt a • ~ m c e ~ Yaletwl.dan ~ ~ ~ class It expressian, increased ¢ytokJse production and syodm~ of an inducible nitric oxide (NO)-synthase.These chaaSesare c~.tsicaily mediated by baclecial LPS and inflammatory c/retinas like y-interferon (T-IFN)and ltlmor necrosis factor-el. N e ~ , like nee-epinephrine, and several cytokines like transforminggrowthfactm-l~and interlanldwl[~have bean shown to down-regulate some of these pl~motyptcstchange& In/edeedtin4 (IL-4), a T ceibdedved Jmwth factor, has recentlybeen ~ to act on ceeebrulastmcytes.We studied the ability of IL-4 to modalate tha exwessim of la moleculesand the symhe~s of NO radiculs on cultmedrst aslrocytesin the presence~ al~maceof ~IFN. Mstenlets i Mstkocls: Class It expression is mmlsmedby flow cytometry. NO synthesis is estimatedby the Gaieasmethod. Results In the ab~see of ~ll[~l, IL.4 redeces c o n c e n l x a l i o a ~ t l y the basal la exweseim, tbe degreeof significance~ ~ at S U/eL ¥-IFNugegulstes the exprenim of cam rl ~ bni this enact is....~..,~velLmd~ued by n.-4. These propertiesof 11.-4 are ~ by indmaethacin (IO°M). 7.1FN triggers the production of an inducible form of NO-systhase in astrogliat cells. A coincubation with IL-4 up-~eBLdutasfltis preduclio~ as visealiml by the quantification of NO2"in the cultm,e sepemat~t. Conclusion: These results indicate that IL-4 can act as a modalator of some immunologicaland inflammatoryfanctioasof astrocytes.
Dermatomyositis (DM) is an inflammatory myopathy characterized by perimysial and perivascular infiltrates. Previous studies explain muscle neerusis as a consequence of microvessel involvement following deposition of immune complexes and terminal complement complex (TCC) in the wall of ,perimysial vanules. We investigated the role of humornl and cellular factors in 6 muscle biopsies from patients with active DM. By immunocytoehemistry, deposition of TCC was seen in the walls of arterioles and capillaries as well as in fibers und~rgo/nl| n ~ i s ; TCC, however, co-localized with Sprot¢/n and clasterin, thus anggesdn~ that TCC may be funotiomally inactive. Increased cxp~ssion of VCAM and ICAM-I was observed in andothelia and macrophaBas ( M # ) a t sites of exudmiun of mono¢Ttes a~l CD4 l ~ o c y t e s . In addition, MHC-II molecules were up-regulated on endothelia and APCs. These observations s u g l ~ t that a T-cetl-mediated, MHC-ll-restr/¢W,d process plays a seminal role in DM. Supported in part by a grant from Telethon to S. Monaco.