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Cytokine profile of myelin proteolipid protein specific T cell clones from SJL mice
193
W01.05
P05.05
NITRIC OXIDE AS 1MMUNESUPPRESSOR IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
ANTIBODIES TO HUMAN IIBAY SHOCK PRO'rEIN P A T I E N T S MZI['H N g U R O P S Y C H I A T R I C D I S O R D B R S
S.R. Ruul,% S. Van Der Linden, I. Huitinga and C.D. Dijkstra Dept. Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands
Sadiq, S.A., Asterloa, R., Strauss, D.H., Ogino, Y., Gorman, J.M., Latov, N. Columbia University, New York, N.Y., U.S.A
Blood-derived macrophages are considered to be important regulatory cells in the immune reactions taking place within the central nervous system (CNS) during experimental altergie encephalomyelitis (EAE). These cells can produce a number of immune regulatory products, among which is the free radical nitric oxide (NO). We isolated macrophages, by means of adherence to plastic, from cell suspensions of the CNS of Lewis rats in which EAE was induced. The spontaneous as well as the lipopolysaccharide (LPS)-induced NO production was measured in the supernatants of these cells. EAE was treated with inhibitors of NO-synthase: N°nitro-L-arginine was administered intraperitoneally,/W-monomethyl-L-arginine was administered into the lateral ventricle via a permanent cannule. We show that after the manifestation of clinical signs, isolated macrophages produced a high amount of NO, spontaneously as well as after stimulation with LPS. On eryostat sections of the CNS made at different timepoints after EAE induction NO-synthase was only expressed in newly infiltrating, blood-born macrophages, and not in (activated) microglial cells. Treatment of EAE with NOsynthase inhibitors, either intraperitoneally or intraventrieularly, resulted in a marked aggravation of the disease. Our results point at an important immunesuppressive role of NO during EAE. Furthermore, it seems that infiltrating, blood-born macrophages are the major source of NO in the CNS during EAE.
Introduction: A n t i b o d y reactivity to human heat shock protein 60 (hsp60) was determined in patients and normal controls. M a t e r i a l s and Methods: Serum at dilutions of I:I000, 1:4000, I:i0000, and CSF at IgG concentration of i0 ug/ml, from p a t i e n t s with schizophrenia, other p s y c h i a t r i c diseases, m u l t i p l e sclerosis, CNS i n f l a m m a t o r y disorders, other neurological disorders, and normal controls, was tested for binding to recombinant, a f f i n i t y - p u r i f i e d hsp60 by Western blot. Results: At d i l u t i o n s of I:I0000, 28 of 64 (44%) patients with schizophrenia reacted with hsp60, but none of the 59 patients with other p s y c h i a t r i c / n e u r o l o g i c diseases, or controls reacted. For CSF, r e a c t i v i t y to hsp60 was found in 3 of 7 patients with s c h i z o p h r e n i a but in only 1 of 50 patients with other disorders. Conclusion: Abnormal immune reactivity involving hsp60 is found in schizophrenia.
P01.14 THE ROLE OF REACTIVE OXYGEN SPECIES IN THE PATHOGENESIS OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
60
IN
P04. I0 Study of ILl0 levels in MS patients in different phases of lhe disease. Salmaggi As, Dufour A., Eoli M., Corsioi E., La Mantia L., Milanese C. and
S.R. Ruuls, J. Bauer, K. Sontrop, I Huitinga, B.A. 't Hart and C,D. Dijkstra Dept. Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands
Nespolo A.
Both blood-born, infiltrating macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions leading to neurological deficits in the demyelinating disease experimental allergic encephalomyelitis (EAE). These cells can produce several mediators of tissue damage, among which are the reactive oxygen species 0ROS). We measured the spontaneous and phorbol myristate acetate (PMA)-induced production of ROS by macrophages and microglial cells isolated from the central nervous system (CNS) of Lewis.rats, by means of a Percoll gradient, at different timepoints after the induction of EAE. Treatment with the ROS scavengers superoxide dismutase (SOD) and catalase was performed from day 7 until day 16 after the EAE induction. It was shown that only after the manifestation of clinical signs the isolated cells exhibited a high spontaneous as well as PMA-inducible production of ROS, In contrast, isolated peripheral blood mononuclear cells did not show an increased ROS production at any timepoint during EAE. The in vivo relevance of this high production of ROS by phagocytes within the CNS during EAE was furthermore demonstrated by treatment with ROS scavengers. Catalase, which scavenges H,O2, markedly suppressed the severity of the disease. However. SOD had no effect on the clinical signs. Our studies suggest a functional role tor ROS, in particular H202, produced within the CNS by macrophages and microglial cells, in the pathogenesis of EAE.
Conflicting results have emerged from previous studies on serum cytokine levels
Istituto Nazionale Neuroiogico "C. Besta" - MILANO
in the various phases of clinical activity of Multiple Sclerosis (MS). The recently characterized ILl0 molecule has a number of potentially immunosopressive functions, such as inhibition of amigen-presenting ability of monneytes via downregulation of HLA-class II molecules, inhibition of IL2 and 7Ib'N production by Th 1, and inhibition of monoknie production. We investigated serum levels of TNFct, ILl0 and soluble IL2R in 26 MS patients (9 in progression, 9 in acute relapse, 8 with stable disease) and 10 healthy controls, by commercially available EUSA kits. While no significant differences were detected In TNF0t and IL2R levels among MS patients (and their subgroups) and controls. ILl0 levels were lower in MS, especially in progressive ones (p < O.001 by Mann-Whimey U test) vs. controls. Further studies are needed about the possible relevance of ILl0 monitoring in relationship with clinical disease activity.
P15.07
~05.04
CYTOKINE PROFILE OF MYELIN PROTEOLIPID PROTEIN SPECIFIC T CELL CLONES FROM SJL MICE.
T LYMPHOCYTE RESPONSE TO 70-kD HEAT SHOCK PROTEINS IN" MULTIPLE SCLEROSIS. IN TUBERCULOSIS AND 1N HEALTHY INDIVIDUALS.
L. De Rvck (1,2), C. Vandevyver (2) p. Brouckaert (3) and J. Raus (1,2). (1) Limhurgs Universitair Centrum and (2) Dr. L. Willemsinstituut, Diepenbeek, and (3) Laboratory of Molecular Biology. Gent, Belgium. Introduction. The factors that contribute to the encephalitogenicity of T cell clones in experimental allergic encephalomyelitis (EAE) are not well understood. While some of our proteolipid protein (PLP)-specific T cell clones, selected with the encephalitogenic peptide 139-151, induce EAE, others do not. These clones cannot always been distinguished on the basis of surface markers or T cell receptor gent usage. To determine the basis for this functional heterogeneity, we have analyzed a panel of PLP peptide 139-151specific, encephalitogenic and nonencephalitogenic T cell clones for the production of various cyto&cines. Methods. Culture sapematants of PLP-pepride activated T cell clones where analyzed with respect to cytokine secretion patterns both by Elisa and hionssay measurement of IL-2. IL-4, IL-10, IFN-7 and TNF. Furthermore, cytokine mRNA levels of PLP*activated clones where analyzed by semiquantitative PCR. Results. The data indicate that various factms, including cytokioes, contribute to the et~ephalitogedicity of PLP-sl~cific T cell clones in vivo. Acknowledgments. This work was supported by an "NFWO-Levenstijn"Grant.
M. Salv¢lli. G. Ristori. C. Buttinelli, P. Fiori, M. Falcone, C. La Barbera, A Pismfi, W. Brinon j , and C. Pozzilli. I Cattedra di Clinica Neurologica, Dip. Sci. Neurol. Universith "La Sapienza", Rome, ITALY and IDept. of Medicine, University of Sydney, AUS'IqT,.AI_,IA. Introduction: We investigated the T cell response to 70kD heat shock proteins (HSPT0) in patients with multiple sclerosis (MS), in petients with tubea'culosisand in healthy individuals. Materials and methods:The relative frequency of the recognition of highly conserved sequences compared to variable ones was assessed by mapping experiments on antigen specific T lymphocyte lines. Analysis of the frequencies of PPD-specific T cell lines which proliferated in response to M. tuberculosis HSP70 was also performed. Results: The results on antigen-specific T lines showed that the response to conserved cpitopes is a frequent event in each of the three conditions studied, often leading to the cross-recognition of microbial and human sequences. In MS, we observed a significandy higher frequency of PPD-specific T lines responding to mycobacteTial HSP70 compared to the othe~ two groups. Conclusions: These findings support recent theories on "natural" or "benign" autoimmunity. More specifically to MS, we extended previous results from our group confirming, on a larger series, the increased T cell renetivity to M. tuberculosis HSP70 in this disease and showing that T cells with this specificity have an auto,aggressivepotential.
W01.05
P05.05
NITRIC OXIDE AS 1MMUNESUPPRESSOR IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
ANTIBODIES TO HUMAN IIBAY SHOCK PRO'rEIN P A T I E N T S MZI['H N g U R O P S Y C H I A T R I C D I S O R D B R S
S.R. Ruul,% S. Van Der Linden, I. Huitinga and C.D. Dijkstra Dept. Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands
Sadiq, S.A., Asterloa, R., Strauss, D.H., Ogino, Y., Gorman, J.M., Latov, N. Columbia University, New York, N.Y., U.S.A
Blood-derived macrophages are considered to be important regulatory cells in the immune reactions taking place within the central nervous system (CNS) during experimental altergie encephalomyelitis (EAE). These cells can produce a number of immune regulatory products, among which is the free radical nitric oxide (NO). We isolated macrophages, by means of adherence to plastic, from cell suspensions of the CNS of Lewis rats in which EAE was induced. The spontaneous as well as the lipopolysaccharide (LPS)-induced NO production was measured in the supernatants of these cells. EAE was treated with inhibitors of NO-synthase: N°nitro-L-arginine was administered intraperitoneally,/W-monomethyl-L-arginine was administered into the lateral ventricle via a permanent cannule. We show that after the manifestation of clinical signs, isolated macrophages produced a high amount of NO, spontaneously as well as after stimulation with LPS. On eryostat sections of the CNS made at different timepoints after EAE induction NO-synthase was only expressed in newly infiltrating, blood-born macrophages, and not in (activated) microglial cells. Treatment of EAE with NOsynthase inhibitors, either intraperitoneally or intraventrieularly, resulted in a marked aggravation of the disease. Our results point at an important immunesuppressive role of NO during EAE. Furthermore, it seems that infiltrating, blood-born macrophages are the major source of NO in the CNS during EAE.
Introduction: A n t i b o d y reactivity to human heat shock protein 60 (hsp60) was determined in patients and normal controls. M a t e r i a l s and Methods: Serum at dilutions of I:I000, 1:4000, I:i0000, and CSF at IgG concentration of i0 ug/ml, from p a t i e n t s with schizophrenia, other p s y c h i a t r i c diseases, m u l t i p l e sclerosis, CNS i n f l a m m a t o r y disorders, other neurological disorders, and normal controls, was tested for binding to recombinant, a f f i n i t y - p u r i f i e d hsp60 by Western blot. Results: At d i l u t i o n s of I:I0000, 28 of 64 (44%) patients with schizophrenia reacted with hsp60, but none of the 59 patients with other p s y c h i a t r i c / n e u r o l o g i c diseases, or controls reacted. For CSF, r e a c t i v i t y to hsp60 was found in 3 of 7 patients with s c h i z o p h r e n i a but in only 1 of 50 patients with other disorders. Conclusion: Abnormal immune reactivity involving hsp60 is found in schizophrenia.
P01.14 THE ROLE OF REACTIVE OXYGEN SPECIES IN THE PATHOGENESIS OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
60
IN
P04. I0 Study of ILl0 levels in MS patients in different phases of lhe disease. Salmaggi As, Dufour A., Eoli M., Corsioi E., La Mantia L., Milanese C. and
S.R. Ruuls, J. Bauer, K. Sontrop, I Huitinga, B.A. 't Hart and C,D. Dijkstra Dept. Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands
Nespolo A.
Both blood-born, infiltrating macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions leading to neurological deficits in the demyelinating disease experimental allergic encephalomyelitis (EAE). These cells can produce several mediators of tissue damage, among which are the reactive oxygen species 0ROS). We measured the spontaneous and phorbol myristate acetate (PMA)-induced production of ROS by macrophages and microglial cells isolated from the central nervous system (CNS) of Lewis.rats, by means of a Percoll gradient, at different timepoints after the induction of EAE. Treatment with the ROS scavengers superoxide dismutase (SOD) and catalase was performed from day 7 until day 16 after the EAE induction. It was shown that only after the manifestation of clinical signs the isolated cells exhibited a high spontaneous as well as PMA-inducible production of ROS, In contrast, isolated peripheral blood mononuclear cells did not show an increased ROS production at any timepoint during EAE. The in vivo relevance of this high production of ROS by phagocytes within the CNS during EAE was furthermore demonstrated by treatment with ROS scavengers. Catalase, which scavenges H,O2, markedly suppressed the severity of the disease. However. SOD had no effect on the clinical signs. Our studies suggest a functional role tor ROS, in particular H202, produced within the CNS by macrophages and microglial cells, in the pathogenesis of EAE.
Conflicting results have emerged from previous studies on serum cytokine levels
Istituto Nazionale Neuroiogico "C. Besta" - MILANO
in the various phases of clinical activity of Multiple Sclerosis (MS). The recently characterized ILl0 molecule has a number of potentially immunosopressive functions, such as inhibition of amigen-presenting ability of monneytes via downregulation of HLA-class II molecules, inhibition of IL2 and 7Ib'N production by Th 1, and inhibition of monoknie production. We investigated serum levels of TNFct, ILl0 and soluble IL2R in 26 MS patients (9 in progression, 9 in acute relapse, 8 with stable disease) and 10 healthy controls, by commercially available EUSA kits. While no significant differences were detected In TNF0t and IL2R levels among MS patients (and their subgroups) and controls. ILl0 levels were lower in MS, especially in progressive ones (p < O.001 by Mann-Whimey U test) vs. controls. Further studies are needed about the possible relevance of ILl0 monitoring in relationship with clinical disease activity.
P15.07
~05.04
CYTOKINE PROFILE OF MYELIN PROTEOLIPID PROTEIN SPECIFIC T CELL CLONES FROM SJL MICE.
T LYMPHOCYTE RESPONSE TO 70-kD HEAT SHOCK PROTEINS IN" MULTIPLE SCLEROSIS. IN TUBERCULOSIS AND 1N HEALTHY INDIVIDUALS.
L. De Rvck (1,2), C. Vandevyver (2) p. Brouckaert (3) and J. Raus (1,2). (1) Limhurgs Universitair Centrum and (2) Dr. L. Willemsinstituut, Diepenbeek, and (3) Laboratory of Molecular Biology. Gent, Belgium. Introduction. The factors that contribute to the encephalitogenicity of T cell clones in experimental allergic encephalomyelitis (EAE) are not well understood. While some of our proteolipid protein (PLP)-specific T cell clones, selected with the encephalitogenic peptide 139-151, induce EAE, others do not. These clones cannot always been distinguished on the basis of surface markers or T cell receptor gent usage. To determine the basis for this functional heterogeneity, we have analyzed a panel of PLP peptide 139-151specific, encephalitogenic and nonencephalitogenic T cell clones for the production of various cyto&cines. Methods. Culture sapematants of PLP-pepride activated T cell clones where analyzed with respect to cytokine secretion patterns both by Elisa and hionssay measurement of IL-2. IL-4, IL-10, IFN-7 and TNF. Furthermore, cytokine mRNA levels of PLP*activated clones where analyzed by semiquantitative PCR. Results. The data indicate that various factms, including cytokioes, contribute to the et~ephalitogedicity of PLP-sl~cific T cell clones in vivo. Acknowledgments. This work was supported by an "NFWO-Levenstijn"Grant.
M. Salv¢lli. G. Ristori. C. Buttinelli, P. Fiori, M. Falcone, C. La Barbera, A Pismfi, W. Brinon j , and C. Pozzilli. I Cattedra di Clinica Neurologica, Dip. Sci. Neurol. Universith "La Sapienza", Rome, ITALY and IDept. of Medicine, University of Sydney, AUS'IqT,.AI_,IA. Introduction: We investigated the T cell response to 70kD heat shock proteins (HSPT0) in patients with multiple sclerosis (MS), in petients with tubea'culosisand in healthy individuals. Materials and methods:The relative frequency of the recognition of highly conserved sequences compared to variable ones was assessed by mapping experiments on antigen specific T lymphocyte lines. Analysis of the frequencies of PPD-specific T cell lines which proliferated in response to M. tuberculosis HSP70 was also performed. Results: The results on antigen-specific T lines showed that the response to conserved cpitopes is a frequent event in each of the three conditions studied, often leading to the cross-recognition of microbial and human sequences. In MS, we observed a significandy higher frequency of PPD-specific T lines responding to mycobacteTial HSP70 compared to the othe~ two groups. Conclusions: These findings support recent theories on "natural" or "benign" autoimmunity. More specifically to MS, we extended previous results from our group confirming, on a larger series, the increased T cell renetivity to M. tuberculosis HSP70 in this disease and showing that T cells with this specificity have an auto,aggressivepotential.