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Interaction between adhesion molecules on murine brain microvascular endothelial cells and splenocytes
207 releasing properties of TRH on lymphoid cells. Interleukin (IL)-2 was significantly increased in TRH-treated but not mock or interferon (IFN)-gamma-treated Molt 4 T cells and human PBLs. The effect was abrogated by the pretreatment of the cells with anti-IFNy. The mRNA for IL-2 in TRH-treated cells rose significantly between 4 and 6 h and then declined. These cells also showed a significant increase in IFNy after treatment with TRH but not media. TRH plus IFNy showed a significant increase in the IFN levels induced by TRH or IFN alone. This suggests a synergistic or additive effect of TRH with IFN, which is abrogated by antisera to IFNy. The TRH-induced IFN had antiviral activity which ranged from 50 to 100 U//xl. T R H + IFNy showed up to 250 U//zl, also abrogated by pretreatment with antisera to IFNy. Mock and TRH treated Molt 4 cells released 1.5 + 0.4 and 2.5 + 0.5/zIU/ml of ir-TSH by RIA, respectively. PBLs released 0.7 and 1.3 /xIU/ml of ir-TSH after mock and TRH treatment, respectively. The addition of IFNy increased the TRH-induced ir-TSH levels by 2-3-fold in both cell types and was abrogated by antisera to IFNy. These data suggest that TRH induces the release of IFNy, IL-2 and ir-TSH. Mechanisms regulating lymphocyte interactions with brain endothelium and smooth muscle/pericyte cells
Michael N. Hart, Zsuzsanna Fabry, Mari Waldschmidt and Dana Hendrickson
Department of Pathology (Neuropathology), University of Iowa College of Medicine, Iowa City, IA, USA Using cultured lines of BALB/c brain microvessel endothelium (En) and smooth muscle/pericyte cells (SM/P), we determined ICAM-1 and ICAM-2 expression by flow cytometry. 30-40% of En cells expressed ICAM-1 which was upregulated by IFN-y, TNF-a, and ILl but not TGF-/3, in a dose-dependent manner with maximal expression at 24 h. ICAM-2 was expressed by 15-20% of En cells but could not be upregulated by cytokines. Both ICAM-1 and -2 were expressed at very low levels on SM/P. Adhesion of splenocytes to En was compared in the BALB/c and SJL/systems. At 37°C, 10.7% + 1.0 of BALB/c splenic cells were adherent to syngeneic En at 1 h compared to only 4.3% + 0.6 in the SJL/syngeneic system. Adhesion was enhanced following En activation by IFN-y, TNF-a and ILl-a, and this enhancement was abrogated by anti-CDlla/CD18 (LFA-1) in the BALB/c system but not in the SJL/j system. AntiICAM-1 did not block adhesion in either system. Adhesion was predictably diminished in the BALB/c system by adding Ca 2÷, Mg 2+ chelating agents (EDTA, EGTA) or at 4°C. However, in the SJL/j system, both 4°C and chelation paradoxically increased adhesion. In
an allogeneic system in which SJL/j splenocytes were placed on BALB/c En, the percentage of adherent ceils was similar to that of BALB/c syngeneic adhesion, whereas percentage of BALB/c splenocytes adherent to SJL/j En was similar to that of SJL/j syngeneic adhesion, indicating that BALB/c and SJL/j splenocytes exhibit similar properties of adhesiveness to En. Collectively, these results bespeak fundamental differences between BALB/c and SJL/j brain En in terms of their relationships with hematopoietic cells. The V-region disease hypothesis
E. Heber-Katz, X.-M. Zhang and T. Esch
The Wistar Institute, Philadelphia, PA, USA The shared usage of V/38 and Va2 by T cells in EAE, EAU and EAN in both rat and mouse led us to propose that these T cell receptors recognize a second ligand other than antigen + MHC. Recent results from our laboratory have indicated that a soluble beta chain from the TcR of a T cell which recognizes MBP in the context of the Lewis class II I-A molecule can bind to MBP in the absence of MHC. Such a result may indicate that MBP could be the second ligand and may bind to the V-region itself. On the other hand, it may be that MBP could be binding through the CDR3 region of the TcR beta chain and that the alpha chain may be the MHC restricting element. Binding studies in the presence of the TcR alpha chain are being carried out. Interaction between adhesion molecules on murine brain microvascular endothelial cells and splenocytes
Dana Hendrickson, Zsuzsanna Fabry and Michael N. Hart
University of Iowa, Iowa City, IA, USA Endothelial cells that form the blood-brain barrier interact with hematopoietic cells to regulate immune reactions in the brain. Endothelial cells express adhesion molecules which bind hematopoietic cells prior to their infiltration into the central nervous system. Previously we have shown that murine microvascular endothelial cells express the intercellular adhesion molecule-1 (ICAM-1) and Lgp55 (suggested mouse homologue of human ICAM-2), and that ICAM-1 expression is increased after activation with the recombinant murine cytokines y-interferon, tumor necrosis factor, and interleukin-la. We have also shown that ICAM-1 and Lgp55 expression is polarized on the luminal surface of endothelial cells by confocal laser microscopy. Here we show by confocal laser microscopy that ICAM-1 and Lgp55 adhesion molecules on endothelial cells localize to endothelial cellsplenocyte adhesion sites after endothelial cell incubation with splenocytes. The lymphocyte ligand for
208 ICAM-1 and Lgp55, lymphocyte function-related antigen (LFA-1), also localizes to endothelial cell-splenocyte adhesion sites. In addition, ICAM-1 localizes to endothelial cell-endothelial cell contact points after incubation at 37°C in contrast to a diffuse surface distribution at 4°C. The interaction between microvascular endothelial cells and hematopoietic cells via adhesion molecules may be important for the development of inflammatory reactions in the central nervous system.
Microglial activation and the susceptibility to CNS inflammation William F. Hickey
Dartmouth Medical School, Hanover, NH, USA All areas of the CNS are not equally susceptible to inflammation associated with experimental allergic encephalomyelitis (EAE). One typically resistant area is the superior colliculus (SC) of the dorsal midbrain. This area can be rendered fully susceptible to inflammation within 24 h following optic nerve transection. Investigations into the changes occurring in the SC which permit this novel E A E susceptibility have directed attention to the microglial cells. Immunohistochemical studies have shown that M H C class I and II molecules are rapidly noted, and CD45, CD4, and C D l l b / c are likewise upregulated. Furthermore, in situ hybridization demonstrates that c-los, c-jun and c-fms are activated early in the area of microglial activation, although the specific cells doing so cannot be identified with certainty. Interestingly, there also appears to be significantly enhanced neuronal elaboration of nitric oxide in the deafferented colliculus. Studies are ongoing to determine the extent of the molecular changes occurring in the SC following optic nerve transection. The potential role of nitric oxide in the acquisition of susceptibility to E A E inflammation in an otherwise EAE-resistant area is also being examined.
Susceptibility to experimental allergic encephalomyelitis is linked to defined behavioural responses I. Huitinga a, C.D. Dijkstra a, G. Kraal b, S. Ried b and A.R. Cools b
a Department of Cell Biology, Medical Faculty, Vrije Unic,ersiteit, Amsterdam, and b Psychoneuropharmacological Research-Unit, University of Nijmegen, Netherlands Susceptibility to the induction of experimental allergic encephalomyelitis (EAE) is strain-correlated, e.g. Lewis rats are highly susceptible, whereas P V G rats do not develop disease after sensitization. Wistar rats are intermediate susceptible. In the present study, we
demonstrate how a bimodal variation in behavioural and pharmacological responses within an outbred Wistar rat population is correlated with different onset and severity of clinical signs of EAE. The different behavioural responses to the dopamine agonist apomorphine reflect neurochemical and functional differences within the central nervous system, i.e. the ventral striatum [1]. It has been found that corticoid receptors within the hippocampus, which directly controls the ventral striatum, show different density and corticosteroid binding characteristics in the two pharmacologically selected rat types [2]. Interestingly, these corticold receptors in the hippocampus are also involved in neuroendocrine regulation under resting as well as stress conditions. Since the importance of stress hormones in susceptibility to experimental autoimmune diseases has been well established [3], we induced E A E in both pharmacologically selected rat types. We observed that the apomorphine susceptible rats developed no or significantly retarded and much less severe clinical signs of E A E as compared to the apomorphine unsusceptible rats. 1 Cools, A.R. et al. (1990) Brain Res. Bull. 24, 49. 2 Win Sutano et at. (1989) Neurosci. Res. Commun. 5, 19. 3 Sternberg, E. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2374.
Adhesion molecules of the blood-brain barrier H. Lassmann
Res. Unit for Exp. Neuropathology, Austrian Academy of Sciences, Vienna, Austria Transendothelial migration of leucocytes in the course of immune surveillance and inflammation involves a complex interaction of cellular adhesion molecules. A cascade of different molecular interactions are involved in the process of primary binding of cells to the endothelial surface and in migration through the endothelial layer. Up to now, our knowledge on the in vivo expression and functional role of adhesion molecules at the b l o o d - b r a i n barrier is limited. Under normal conditions, only a minority of brain vessels reveal endothelial expression of ICAM1, LFA3, CD44 and CD9. Reactivity is mainly found on microvessels (capillaries and venules), but rarely on arteries and arterioles. ICAM1 and LFA3 are also expressed on the astrocytic end feet of the glia limitans. Their respective ligands are found on leukocytic infiltrates and microglia (LFA1) or on lymphocytes only (CD2). Within inflammatory lesions, an upregulation of endothelial ICAM1 and LFA3 expression is found, whereas CD44 remains similar to controls. In addition, adhesion molecules, typical for high endothelial venules, appear on cerebral endothelial cells in the course of inflammation. Animals with experimental autoimmune en-
Michael N. Hart, Zsuzsanna Fabry, Mari Waldschmidt and Dana Hendrickson
Department of Pathology (Neuropathology), University of Iowa College of Medicine, Iowa City, IA, USA Using cultured lines of BALB/c brain microvessel endothelium (En) and smooth muscle/pericyte cells (SM/P), we determined ICAM-1 and ICAM-2 expression by flow cytometry. 30-40% of En cells expressed ICAM-1 which was upregulated by IFN-y, TNF-a, and ILl but not TGF-/3, in a dose-dependent manner with maximal expression at 24 h. ICAM-2 was expressed by 15-20% of En cells but could not be upregulated by cytokines. Both ICAM-1 and -2 were expressed at very low levels on SM/P. Adhesion of splenocytes to En was compared in the BALB/c and SJL/systems. At 37°C, 10.7% + 1.0 of BALB/c splenic cells were adherent to syngeneic En at 1 h compared to only 4.3% + 0.6 in the SJL/syngeneic system. Adhesion was enhanced following En activation by IFN-y, TNF-a and ILl-a, and this enhancement was abrogated by anti-CDlla/CD18 (LFA-1) in the BALB/c system but not in the SJL/j system. AntiICAM-1 did not block adhesion in either system. Adhesion was predictably diminished in the BALB/c system by adding Ca 2÷, Mg 2+ chelating agents (EDTA, EGTA) or at 4°C. However, in the SJL/j system, both 4°C and chelation paradoxically increased adhesion. In
an allogeneic system in which SJL/j splenocytes were placed on BALB/c En, the percentage of adherent ceils was similar to that of BALB/c syngeneic adhesion, whereas percentage of BALB/c splenocytes adherent to SJL/j En was similar to that of SJL/j syngeneic adhesion, indicating that BALB/c and SJL/j splenocytes exhibit similar properties of adhesiveness to En. Collectively, these results bespeak fundamental differences between BALB/c and SJL/j brain En in terms of their relationships with hematopoietic cells. The V-region disease hypothesis
E. Heber-Katz, X.-M. Zhang and T. Esch
The Wistar Institute, Philadelphia, PA, USA The shared usage of V/38 and Va2 by T cells in EAE, EAU and EAN in both rat and mouse led us to propose that these T cell receptors recognize a second ligand other than antigen + MHC. Recent results from our laboratory have indicated that a soluble beta chain from the TcR of a T cell which recognizes MBP in the context of the Lewis class II I-A molecule can bind to MBP in the absence of MHC. Such a result may indicate that MBP could be the second ligand and may bind to the V-region itself. On the other hand, it may be that MBP could be binding through the CDR3 region of the TcR beta chain and that the alpha chain may be the MHC restricting element. Binding studies in the presence of the TcR alpha chain are being carried out. Interaction between adhesion molecules on murine brain microvascular endothelial cells and splenocytes
Dana Hendrickson, Zsuzsanna Fabry and Michael N. Hart
University of Iowa, Iowa City, IA, USA Endothelial cells that form the blood-brain barrier interact with hematopoietic cells to regulate immune reactions in the brain. Endothelial cells express adhesion molecules which bind hematopoietic cells prior to their infiltration into the central nervous system. Previously we have shown that murine microvascular endothelial cells express the intercellular adhesion molecule-1 (ICAM-1) and Lgp55 (suggested mouse homologue of human ICAM-2), and that ICAM-1 expression is increased after activation with the recombinant murine cytokines y-interferon, tumor necrosis factor, and interleukin-la. We have also shown that ICAM-1 and Lgp55 expression is polarized on the luminal surface of endothelial cells by confocal laser microscopy. Here we show by confocal laser microscopy that ICAM-1 and Lgp55 adhesion molecules on endothelial cells localize to endothelial cellsplenocyte adhesion sites after endothelial cell incubation with splenocytes. The lymphocyte ligand for
208 ICAM-1 and Lgp55, lymphocyte function-related antigen (LFA-1), also localizes to endothelial cell-splenocyte adhesion sites. In addition, ICAM-1 localizes to endothelial cell-endothelial cell contact points after incubation at 37°C in contrast to a diffuse surface distribution at 4°C. The interaction between microvascular endothelial cells and hematopoietic cells via adhesion molecules may be important for the development of inflammatory reactions in the central nervous system.
Microglial activation and the susceptibility to CNS inflammation William F. Hickey
Dartmouth Medical School, Hanover, NH, USA All areas of the CNS are not equally susceptible to inflammation associated with experimental allergic encephalomyelitis (EAE). One typically resistant area is the superior colliculus (SC) of the dorsal midbrain. This area can be rendered fully susceptible to inflammation within 24 h following optic nerve transection. Investigations into the changes occurring in the SC which permit this novel E A E susceptibility have directed attention to the microglial cells. Immunohistochemical studies have shown that M H C class I and II molecules are rapidly noted, and CD45, CD4, and C D l l b / c are likewise upregulated. Furthermore, in situ hybridization demonstrates that c-los, c-jun and c-fms are activated early in the area of microglial activation, although the specific cells doing so cannot be identified with certainty. Interestingly, there also appears to be significantly enhanced neuronal elaboration of nitric oxide in the deafferented colliculus. Studies are ongoing to determine the extent of the molecular changes occurring in the SC following optic nerve transection. The potential role of nitric oxide in the acquisition of susceptibility to E A E inflammation in an otherwise EAE-resistant area is also being examined.
Susceptibility to experimental allergic encephalomyelitis is linked to defined behavioural responses I. Huitinga a, C.D. Dijkstra a, G. Kraal b, S. Ried b and A.R. Cools b
a Department of Cell Biology, Medical Faculty, Vrije Unic,ersiteit, Amsterdam, and b Psychoneuropharmacological Research-Unit, University of Nijmegen, Netherlands Susceptibility to the induction of experimental allergic encephalomyelitis (EAE) is strain-correlated, e.g. Lewis rats are highly susceptible, whereas P V G rats do not develop disease after sensitization. Wistar rats are intermediate susceptible. In the present study, we
demonstrate how a bimodal variation in behavioural and pharmacological responses within an outbred Wistar rat population is correlated with different onset and severity of clinical signs of EAE. The different behavioural responses to the dopamine agonist apomorphine reflect neurochemical and functional differences within the central nervous system, i.e. the ventral striatum [1]. It has been found that corticoid receptors within the hippocampus, which directly controls the ventral striatum, show different density and corticosteroid binding characteristics in the two pharmacologically selected rat types [2]. Interestingly, these corticold receptors in the hippocampus are also involved in neuroendocrine regulation under resting as well as stress conditions. Since the importance of stress hormones in susceptibility to experimental autoimmune diseases has been well established [3], we induced E A E in both pharmacologically selected rat types. We observed that the apomorphine susceptible rats developed no or significantly retarded and much less severe clinical signs of E A E as compared to the apomorphine unsusceptible rats. 1 Cools, A.R. et al. (1990) Brain Res. Bull. 24, 49. 2 Win Sutano et at. (1989) Neurosci. Res. Commun. 5, 19. 3 Sternberg, E. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2374.
Adhesion molecules of the blood-brain barrier H. Lassmann
Res. Unit for Exp. Neuropathology, Austrian Academy of Sciences, Vienna, Austria Transendothelial migration of leucocytes in the course of immune surveillance and inflammation involves a complex interaction of cellular adhesion molecules. A cascade of different molecular interactions are involved in the process of primary binding of cells to the endothelial surface and in migration through the endothelial layer. Up to now, our knowledge on the in vivo expression and functional role of adhesion molecules at the b l o o d - b r a i n barrier is limited. Under normal conditions, only a minority of brain vessels reveal endothelial expression of ICAM1, LFA3, CD44 and CD9. Reactivity is mainly found on microvessels (capillaries and venules), but rarely on arteries and arterioles. ICAM1 and LFA3 are also expressed on the astrocytic end feet of the glia limitans. Their respective ligands are found on leukocytic infiltrates and microglia (LFA1) or on lymphocytes only (CD2). Within inflammatory lesions, an upregulation of endothelial ICAM1 and LFA3 expression is found, whereas CD44 remains similar to controls. In addition, adhesion molecules, typical for high endothelial venules, appear on cerebral endothelial cells in the course of inflammation. Animals with experimental autoimmune en-