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Expression of CD21, the receptor for C3d and EBV, by nerve cells
161
P06.05 'FAILURE TO TRANSFER MULTIPLE SCLEROSIS (MS) INTO SCID MICE BY INTRAPERITONEAL (I.P.) INJECTION OF PBLs FROM MS PATIENTS R, Furlan 1,2, G. Martino 1,2, E. Brambilla 1, L. Moiola 1,2, M. Castellano 1, V. Casadei 1,2, L. Pogliani1, V. Martinelli2, B. Colombo =, L. M.E. Grimaldi t,2 1Neumimmunology Unit - DIBIT and ~'Dept. of Neurology, Univ. of Milano, San Raffaele Scientific Institute, Milano, Italy. INTRODUCTION: Intracembrst transplantation of lymphocytes from patients with MS into severe combined immunodeflcienoy (SCID) mice has been reported to reproduce demyelinating lesions typical of MS. The validity of these studies has been disputed. MATERIALS AND METHODS: PBLs from 7 patients with MS were transplanted by I.P. injection into 28 SCID mice (MSSCID). Seven animals transplanted with PBLs from 2 healthy subjects (huSCID) were used as controls. Animals were sacnficed between 2 and 8 weeks alter transplantation. RESULTS: PCR analysis aimed to the HLA-DQ region showed presence of human cells in neural tissues of MS-SCID mice. Immunocytochemical analysis showed that the majority of cells present in the brain of MS-SCID were T-cells and resided in the meningeal spaces or choroid plexuses. Isolated human cells were tound in the immediate submeningeal cerebral parenchyma. However, human lymphocytes were also found in brains of 2/7 hu-SCID mice. No signs or symptoms of neurological impairment were observed in MS-SCID. CONCLUSIONS: Our results indicate that T-calls from MS patients can reach the central nervous system of SCID mice when I.P. injected. However, no immuno-pathological signs of neurological impairment can be observed in this model. In our hands the I.P. MS-SCID mouse is not a valid tool for studies on MS immunopathology.
P03.04 EXPRESSION OF THE TERMINAL PATHWAY OF COMPLEMENT BY HUMAN GLIAL CELLS. P GASQUEa, M FONTAINEb and BP MORGANa •i University of Wales College of Medicine, Med. Biochem. dept, Heath park, CARDIFF, CF44XN, OK. b) INSERM U78, BP 73, F76233, Bois guillaume, France In previous workj'2` we have described the expressionof the alternative pathway components (C3, factors B, H, I) and the classical pathway components (Clq, Clr, Cls, CINH, C4, C2) of complement by three human astroglioma cell lines as a model for primary glial ceils. Our current work aims to examine whether glial cells can synthesisc a complete complement system. Using Western blotting, ELISA, ]mmunoprecipitation and PCR techniques we now demonstrate that these same cell lines produce all components and regulators of the terminal pathway (C5, C6, C7, C8, C9, S protein, clusteriu) and are capable of generating the .cytotoxic membrane attack complex (MAC). Complement expression is regulated by cytokines, specifically gamma-IFN, and TNF-alpha. We are currently seeking to confirm the .synthesis of complement components in vivo using RT-PCR techniques in normal and inflamed brain. These studies will provide further evidence of the role of complement in the pathology of CNS disease. I (}asqtl¢,P. Julen, N, ]~]l~lxko.A.M, Picot. C., Mauser. C, Chauzy. C,, Ripoche, J,. & M. Fontaille. 1992 .L hmm~noL, 149, 1381-1387 2 Gal~lUe,P., I~l~lko. A. Leg~dec. J., Manger, C, Sdloull, M T & M Fonlain¢,1993, .L Biol Chem. 268. 25068-25074
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Pl1.03
IL-10 IS I N T R A T H E C A L L Y SYNTHESIZED IN HIVI N D U C E D / R E L A T E D CNS DISEASES, BUT IT IS NOT P R O D U C E D BY HIV-INFECTED M O N O N U C L E A R CELLS.
EXPRESSION OF CD21, THE RECEPTOR FOR C3d AND EBV, BY NERVE CELLS.
P. Gallo.*, A. De Rossi *, S. Sivieri *, L. Rinaldi *, A.M. Ferrarini *, L.Chle¢o-Bianchi *, and B.Tavolato * Institute of Neurofogy-Second Neurologlc Clinic * and Institute of Oncology*, University of Padova, via E.Vendraminl,7 - 36137 Padova, ITALY.
Interleukin 10 (IL-IO) was detected by ELISA in the cerebroapinal fluid (CSF) from HIV-infacted patients with HIV-related ancephantis/teukoencephalopathy (HIVE) or with opportunistic intectiona (cryptococcal meningifia), but not in neurologically aaymptornatic patients. This finding suggeate a role for this immunosuppreaaive cytokine in 1) favouring the intrathecal virus spread and 2) increasing the auscapfibllity of HIV-infacted patients to develop chronic opportuniitic CNS infections. Since the infrathecal source of IL-IO could be Hiv-infacted mononuclear cells invading the CN8, the effect of HIV infection on IL-lO production by cultures/co-culturea of monocyte, T and B lymphocytea was atudled. HIV infection did not induced IL-IO syntheais/release by these calla. Moreover, the constitutive lowlevel production of IL-tO by EBV-immortalized B cell lines was completely suppressed by HIV infection. Our date suggest that the cellular source of intrathecally synthesized IL-IO in HIV-infacted patients are likely uninfacted IL-IO producing cells (a.g., reactive microgltal cells).
P. GASQUE°, C. MAUGERb, P CHANb, MT. SCHOUFTb, M. DIEPdCI-I~, B.P MORGAN° & M, FONTAINEb a) UWCM,Med. Biochem,Heath park, Cardiff,CF44XN, UK.;b) INSERMU78, BP73, F76233, Bois Guillaumecedex, France.; c) Institutefor hygiene,hmsbruk, A6010, Austria. During these last years mm~erous studies have shown that cells from the CNS express munerous inunune fimetions. Astrocyte seems to be the major residem thununocoml~etent cell in the brain, lmvingthe the capacity to present antigen, to synthesise rations cytokines, and to secrete proinflanmmtoryproducts. Usingdiffereuthuman astrogliomacell lines, we lmve shown that astweyte can produce all components of the complement, and recently we have extended this investigationto the study of cotuplement receptor expression. Of the multiple receptors for C3 fragmants, ouly CD2I is expressed by human astrogliomacell lines (T98, T193, T109, Ul lSMG). We have not deteetod the expression of CD35 (CRI), CD11b/CDI8 (CR3) or CDI Ic./CDlg (CR4) on these cells using FACS, coufocal laser microscopy, westorn blot, mid RTPCR to detect nlRNA. For each r~:eptor we have used at least two Mahs -Pab, and in the case of CD21, we have used eight Mabs anti-huCR2. The glial CR2 seems to be identical to the lymphocyte CR2, 140 to 145 kDa m nou-reducmg conditions, and can biod polymeric C3d. These observations are ill agreement with the expression of an OKB7 bindiugmembrane protein detected ill vitro an human fetal astrocytos Expression of CR2 on astrocytos would explain the frequent infection of the CNS by EBV and perhaps implicatethis agent in braindiseases, includingMS
W05.05
W11.04
QUALITATIVE AND QUANTITATIVE DIFFERENCES IN HSP60 AND TCR ?6 EXPRESSION IN MURINE CHRt)NIC.RELAPSING EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE).
Interactions between EAE and Theiler's v i r u s i n f e c t i o n in the CNS o f P L / J mice. A. GATRILL, P. TONKS, D. WRAITH & A.A. NASH. Dept. Veterinary Pathology, Royal (Dick) School o f Vet. Studies, Edinburgh, U.K, and Dept. Pathology, University o f Cambridge, U.K. Animal models for multiple sclerosis include an autoimmune model, experimental allergic encephalomyelitis (EAE), and models of CNS virus infection such as Theiler's murine e n c e p h a l o m y e l i t i s v i r u s (TMEV) which provokes an i m m u n o p a t h o l o g i c a l d e m y e l i n a t i n g disease. T cell responses and immunopathology have been well studied in both these models. PL/J (H-2 u) mice are highly s u s c e p t i b l e to d e m y e l i n a t i n g p a t h o l o g y in both T M E V infection and EAE. We have examined the effect o f an u n d e r l y i n g p e r s i s t e n t CNS TMEV infection upon the outcome of EAE induced in PL/J mice by p r i m i n g with spinal cord homogenate. TMEV-infected a n i m a l s displayed the same time of onset, incidence and severity of EAE as that seen in u n i n f e c t e d animals. H o w e v e r , in c o n t r a s t to uninfected animals, which undergo relapsing and remitting clinical signs, infected mice generally failed to recover fully from the first round of paralysis. The results of immunohistological studies to determine the distribution of TMEV in the CNS o f these animals in relation to demyelinating lesions will be presented.
Y-L. Gao, C.F. Brosnan and C.S. Rainc. Dept. of Pathology, Albert Einstein Coil Medicine, Bronx, NY, USA Introduction: In both multiple sclerosis (MS) and EAE, the disease may take an acute exacerbating-remitting course or may develop a chronic course. We have proposed that heat shock proteins (hap) may function as additional targets of the immune response and perhaps contribute to the development of chronic disease. Methods: With immunocytochemistry, FACS analysis and Western blots, we assessed hsp6(I and "76 TCR expression in the chronic-relapsing mouse model of EAE. Results: Normal mouse spinal cord showed hsp60 located to mitochondria. In animals with acute EAE, lesions were clearly defined by increased expression of hsp00. Within the lesion, strong immunoreactivity for hsp60 was detected on perivascular inflammatory cells, and in oligodendrocytes and astrocytes. In chronic EAE, extensive immunoreactivity for hsp60 was found in submeningeal locations, and this correlated with increased expression in Western blots. "t6 T cells were rare in acute EAE lesions hut were more frequent in chronic EAE. Furthermore, '76 T cells showed a higher frequency of CD8 expression. Conclusion: The development of autoimmunc-directed inflammation in the CNS is associated with qualitative and quantitative differences in hs|~50 expression, and an increased frequency of '76 T cells. These may contribute to the development of the chronic disease. Supported by NS11920, ( NS08952, ( NS31919 ( a nd NMSS 10ql-H-8.
P06.05 'FAILURE TO TRANSFER MULTIPLE SCLEROSIS (MS) INTO SCID MICE BY INTRAPERITONEAL (I.P.) INJECTION OF PBLs FROM MS PATIENTS R, Furlan 1,2, G. Martino 1,2, E. Brambilla 1, L. Moiola 1,2, M. Castellano 1, V. Casadei 1,2, L. Pogliani1, V. Martinelli2, B. Colombo =, L. M.E. Grimaldi t,2 1Neumimmunology Unit - DIBIT and ~'Dept. of Neurology, Univ. of Milano, San Raffaele Scientific Institute, Milano, Italy. INTRODUCTION: Intracembrst transplantation of lymphocytes from patients with MS into severe combined immunodeflcienoy (SCID) mice has been reported to reproduce demyelinating lesions typical of MS. The validity of these studies has been disputed. MATERIALS AND METHODS: PBLs from 7 patients with MS were transplanted by I.P. injection into 28 SCID mice (MSSCID). Seven animals transplanted with PBLs from 2 healthy subjects (huSCID) were used as controls. Animals were sacnficed between 2 and 8 weeks alter transplantation. RESULTS: PCR analysis aimed to the HLA-DQ region showed presence of human cells in neural tissues of MS-SCID mice. Immunocytochemical analysis showed that the majority of cells present in the brain of MS-SCID were T-cells and resided in the meningeal spaces or choroid plexuses. Isolated human cells were tound in the immediate submeningeal cerebral parenchyma. However, human lymphocytes were also found in brains of 2/7 hu-SCID mice. No signs or symptoms of neurological impairment were observed in MS-SCID. CONCLUSIONS: Our results indicate that T-calls from MS patients can reach the central nervous system of SCID mice when I.P. injected. However, no immuno-pathological signs of neurological impairment can be observed in this model. In our hands the I.P. MS-SCID mouse is not a valid tool for studies on MS immunopathology.
P03.04 EXPRESSION OF THE TERMINAL PATHWAY OF COMPLEMENT BY HUMAN GLIAL CELLS. P GASQUEa, M FONTAINEb and BP MORGANa •i University of Wales College of Medicine, Med. Biochem. dept, Heath park, CARDIFF, CF44XN, OK. b) INSERM U78, BP 73, F76233, Bois guillaume, France In previous workj'2` we have described the expressionof the alternative pathway components (C3, factors B, H, I) and the classical pathway components (Clq, Clr, Cls, CINH, C4, C2) of complement by three human astroglioma cell lines as a model for primary glial ceils. Our current work aims to examine whether glial cells can synthesisc a complete complement system. Using Western blotting, ELISA, ]mmunoprecipitation and PCR techniques we now demonstrate that these same cell lines produce all components and regulators of the terminal pathway (C5, C6, C7, C8, C9, S protein, clusteriu) and are capable of generating the .cytotoxic membrane attack complex (MAC). Complement expression is regulated by cytokines, specifically gamma-IFN, and TNF-alpha. We are currently seeking to confirm the .synthesis of complement components in vivo using RT-PCR techniques in normal and inflamed brain. These studies will provide further evidence of the role of complement in the pathology of CNS disease. I (}asqtl¢,P. Julen, N, ]~]l~lxko.A.M, Picot. C., Mauser. C, Chauzy. C,, Ripoche, J,. & M. Fontaille. 1992 .L hmm~noL, 149, 1381-1387 2 Gal~lUe,P., I~l~lko. A. Leg~dec. J., Manger, C, Sdloull, M T & M Fonlain¢,1993, .L Biol Chem. 268. 25068-25074
P l l .02
Pl1.03
IL-10 IS I N T R A T H E C A L L Y SYNTHESIZED IN HIVI N D U C E D / R E L A T E D CNS DISEASES, BUT IT IS NOT P R O D U C E D BY HIV-INFECTED M O N O N U C L E A R CELLS.
EXPRESSION OF CD21, THE RECEPTOR FOR C3d AND EBV, BY NERVE CELLS.
P. Gallo.*, A. De Rossi *, S. Sivieri *, L. Rinaldi *, A.M. Ferrarini *, L.Chle¢o-Bianchi *, and B.Tavolato * Institute of Neurofogy-Second Neurologlc Clinic * and Institute of Oncology*, University of Padova, via E.Vendraminl,7 - 36137 Padova, ITALY.
Interleukin 10 (IL-IO) was detected by ELISA in the cerebroapinal fluid (CSF) from HIV-infacted patients with HIV-related ancephantis/teukoencephalopathy (HIVE) or with opportunistic intectiona (cryptococcal meningifia), but not in neurologically aaymptornatic patients. This finding suggeate a role for this immunosuppreaaive cytokine in 1) favouring the intrathecal virus spread and 2) increasing the auscapfibllity of HIV-infacted patients to develop chronic opportuniitic CNS infections. Since the infrathecal source of IL-IO could be Hiv-infacted mononuclear cells invading the CN8, the effect of HIV infection on IL-lO production by cultures/co-culturea of monocyte, T and B lymphocytea was atudled. HIV infection did not induced IL-IO syntheais/release by these calla. Moreover, the constitutive lowlevel production of IL-tO by EBV-immortalized B cell lines was completely suppressed by HIV infection. Our date suggest that the cellular source of intrathecally synthesized IL-IO in HIV-infacted patients are likely uninfacted IL-IO producing cells (a.g., reactive microgltal cells).
P. GASQUE°, C. MAUGERb, P CHANb, MT. SCHOUFTb, M. DIEPdCI-I~, B.P MORGAN° & M, FONTAINEb a) UWCM,Med. Biochem,Heath park, Cardiff,CF44XN, UK.;b) INSERMU78, BP73, F76233, Bois Guillaumecedex, France.; c) Institutefor hygiene,hmsbruk, A6010, Austria. During these last years mm~erous studies have shown that cells from the CNS express munerous inunune fimetions. Astrocyte seems to be the major residem thununocoml~etent cell in the brain, lmvingthe the capacity to present antigen, to synthesise rations cytokines, and to secrete proinflanmmtoryproducts. Usingdiffereuthuman astrogliomacell lines, we lmve shown that astweyte can produce all components of the complement, and recently we have extended this investigationto the study of cotuplement receptor expression. Of the multiple receptors for C3 fragmants, ouly CD2I is expressed by human astrogliomacell lines (T98, T193, T109, Ul lSMG). We have not deteetod the expression of CD35 (CRI), CD11b/CDI8 (CR3) or CDI Ic./CDlg (CR4) on these cells using FACS, coufocal laser microscopy, westorn blot, mid RTPCR to detect nlRNA. For each r~:eptor we have used at least two Mahs -Pab, and in the case of CD21, we have used eight Mabs anti-huCR2. The glial CR2 seems to be identical to the lymphocyte CR2, 140 to 145 kDa m nou-reducmg conditions, and can biod polymeric C3d. These observations are ill agreement with the expression of an OKB7 bindiugmembrane protein detected ill vitro an human fetal astrocytos Expression of CR2 on astrocytos would explain the frequent infection of the CNS by EBV and perhaps implicatethis agent in braindiseases, includingMS
W05.05
W11.04
QUALITATIVE AND QUANTITATIVE DIFFERENCES IN HSP60 AND TCR ?6 EXPRESSION IN MURINE CHRt)NIC.RELAPSING EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE).
Interactions between EAE and Theiler's v i r u s i n f e c t i o n in the CNS o f P L / J mice. A. GATRILL, P. TONKS, D. WRAITH & A.A. NASH. Dept. Veterinary Pathology, Royal (Dick) School o f Vet. Studies, Edinburgh, U.K, and Dept. Pathology, University o f Cambridge, U.K. Animal models for multiple sclerosis include an autoimmune model, experimental allergic encephalomyelitis (EAE), and models of CNS virus infection such as Theiler's murine e n c e p h a l o m y e l i t i s v i r u s (TMEV) which provokes an i m m u n o p a t h o l o g i c a l d e m y e l i n a t i n g disease. T cell responses and immunopathology have been well studied in both these models. PL/J (H-2 u) mice are highly s u s c e p t i b l e to d e m y e l i n a t i n g p a t h o l o g y in both T M E V infection and EAE. We have examined the effect o f an u n d e r l y i n g p e r s i s t e n t CNS TMEV infection upon the outcome of EAE induced in PL/J mice by p r i m i n g with spinal cord homogenate. TMEV-infected a n i m a l s displayed the same time of onset, incidence and severity of EAE as that seen in u n i n f e c t e d animals. H o w e v e r , in c o n t r a s t to uninfected animals, which undergo relapsing and remitting clinical signs, infected mice generally failed to recover fully from the first round of paralysis. The results of immunohistological studies to determine the distribution of TMEV in the CNS o f these animals in relation to demyelinating lesions will be presented.
Y-L. Gao, C.F. Brosnan and C.S. Rainc. Dept. of Pathology, Albert Einstein Coil Medicine, Bronx, NY, USA Introduction: In both multiple sclerosis (MS) and EAE, the disease may take an acute exacerbating-remitting course or may develop a chronic course. We have proposed that heat shock proteins (hap) may function as additional targets of the immune response and perhaps contribute to the development of chronic disease. Methods: With immunocytochemistry, FACS analysis and Western blots, we assessed hsp6(I and "76 TCR expression in the chronic-relapsing mouse model of EAE. Results: Normal mouse spinal cord showed hsp60 located to mitochondria. In animals with acute EAE, lesions were clearly defined by increased expression of hsp00. Within the lesion, strong immunoreactivity for hsp60 was detected on perivascular inflammatory cells, and in oligodendrocytes and astrocytes. In chronic EAE, extensive immunoreactivity for hsp60 was found in submeningeal locations, and this correlated with increased expression in Western blots. "t6 T cells were rare in acute EAE lesions hut were more frequent in chronic EAE. Furthermore, '76 T cells showed a higher frequency of CD8 expression. Conclusion: The development of autoimmunc-directed inflammation in the CNS is associated with qualitative and quantitative differences in hs|~50 expression, and an increased frequency of '76 T cells. These may contribute to the development of the chronic disease. Supported by NS11920, ( NS08952, ( NS31919 ( a nd NMSS 10ql-H-8.