- Email: [email protected]
Regulation of IL-6 mRNA levels in primary rat astrocytes by the calmodulin antagonist W7
169
P13.10 ANTIBODY ACTIVITIES TO SULFATIDE, Pit PROTEIN AND OLIGOSACCHARIDE CHAINS OF P0 PROTEIN IN POLYNEUROPATHIES T. Rosokawa, K. K i t a a n r a * , K. Nomura, a n d K. l l a ~ c h i . Department of Neurology, *Department of Physiology, Saitama Medical School, Saitama, Japan We h a v e e v a l u a t e d a n t i - S u l f a t i d e and PO p r o t e i n IgM antibody activities in s e r a f r o m one c a s e o f c h r o n i c p o l y n a u r o p a t h y w i t h IgM p a r a p r o t e i n e m i a (IgM-CP), 5 cases of chronic idiopathic p o l y n e u r o p a t h y ( C I P ) , 6 c a s e s o f acute idiopathic polyneuropathy (AIP) and 6 c a s e s o f controls. ELISA a n a l y s i s for anti-Sulfatide a n d P0 protein IgM a n t i b o d y r e v e a l e d h i g h t i t e r i n IgM-CP and significantly h i g h e r i n CIP t h a n i n AIP and c o n t r o l s . Furthermore, we c o u l d d e t e r m i n e the structure of oligoaaccharida c h a i n s o f P0 p r o t e i n , GP 4 and GP 5. These differences were only non-reducing terminal sugar o f t h e s e c h a i n s . GP 5 h a s a s u l f a t e d g l u c n r o n i o a c i d and GP 4 h a s a s i a l i c a c i d a t t h e t e r m i n a l . We e x a m i n e d t h e reactivitiea o f e l u t e d IgH f r o m one c a s e o f IgH-CP and 3 c a s e s o f CIP w i t h t h e s e c h a i n s . The IgM o f IgM-CP r e a c t e d w i t h GP 5 o n l y , and t h e IgM o f CIP r e a c t e d w i t h b o t h . These data suggested the presence of anti-Sulfatide and P0 p r o t e i n I g H a n t i b o d i e s i n t h e s e r a o f IgN-CP and C I P , and t h e e p i t o p e o f t h e s e r u m o f IgH-CP m i g h t be t h e s u l f a t e d g l u c u r o n i c a c i d i n GP 5.
P12.04 EXPRESSION OF THE SCLEROSIS LESIONS
CYTOKINE
RANTES
IN
MULTIPLE
J. Hyas, K. Sinding, J.R. Oksenbesg 1, G. ~ 2 a~d C.C.A. Bernasd Nenrvimmunology Laboratory, La Trobe University, Bundoo~ Victoria, 3083, Australia, ~'l~t of Nemolog~/, Udivea'sity of Cni~oxnia, San Fra~isco, California, 94143-0114, USA and '~L.A.R.C.H. ~ Research Unit, Royal Children's Hospital, Parkvtlle, Victoria, 3052, AusUniia. Multiple sclerosis (MS) is an inflanunatoey disease of the central nervous sysmm (C'NS), ~ by myelin destruction. The m~aa~iams of domyelination in MS are complex and lm3hably involve soluble immone mediators, partlcelm'lyc~s. Activated T cells, which pertici~ in the pathogeaexis of this disease, have beer shown to migrate from the pedphe~ lymphoid system to the CNS via the Hood ~ berrY. The re~ntly identified cym~ne Rantes, acts as a selective chemomeraotam to T cells and mediates their adherence to e~lethelial cells,thereby facilita~'g their enlry into the CNS. In the present study, we have used in-sRu hylwidisefion to locale Rantes and a semiquantilative polymemse chain reaction method to analyse the level of Rantes expressiou in MS brain lesions. The results show a highex expreuion of Rantes in MS lesions as compared to other nem'otogicni dtsoaden and healthy controls. These experiments implicate P,~tes as an ~ effectox in autoimmune demyelination and raise the possibility that its down-regnlation may have important therapeutic potential fox diseases like MS.
P01.05
PI0.02
Centr~ Adn~,,,~'etion of Lipoconin R~uces Sevaity of Mild Exl~imeatni Allergic ~,~pllalomyeillis
O N T H E R O L E O F T C R V6 8.2 T - C E L L S I N L E W I S R A T EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)
L I-luitinf~L J. Bauorl, PJ.L.M. Strijbes2, N.J. Rothwell-L C.D. Dijkstrat. FJ.H. Tllders4 and F. Berkenbosch('D4. DelXs. of 1: Cell Biology and 4: Pharmacology, Vrije Universitelt, Amsterdam. The Netherlands, 2: Dept. of Pharmacology. The Wellcome Research Laboratories, KenL UK and 3: School of Biological Sciences, University of Manchester, Manchester, UK. Experimental autoimmune encephalomyelitis (EAE) w4xeseots an animal model for multiple sclerosis (MS). Cordceoternid treatment has proven to be benificlal in both MS and EAE. Suppressiou by corticostoroids ntight in part be mediated by the local reduction of inflammato~ reactions in the CNS. Corticosteroids induce expression of lipoconin-I (LC-I) which has been de~pon.¢a-ated to play a role in the control of inflammation. In the CNS of MS patients and EAE rats, LC-I is up resulated, which may be induced by elevated cortieostexone plasma levels during the disease. In this .study we show that central administration of an active recomhinant frasment (1-188 amino acids) of LC-I reduces clinical severity of ntild EAE in Lewis rats but not of sevevv EAB. lntmventficular injection of antibodies raised to the lipocortin 1-188 frogmant as well as anti-LC-I antibodies obtained from Zymod. did not aK~avate disease significantly. These results indicate that suRxeseive actions of corticesternids could in part be mediated by down reSulation of inflanuuation in the CNS via LC-I. Physiological relevance of •endogenous LC- l in the CNS during EAE however remains to be established.
P03.06 REGULATION OF IL-6 mRNA LEVELS IN PRIMARY RAT ASTROCYTES BY THE CALMODULIN ANTAGONIST W7 B.S. Hunevcutt and E. N. Benveniste• Depadment of Cell Biology. University of Alabama at Birmingham, Birmingham, Alabama 35294. INTRODUCTION: IL.6 expression in astrocytes is regulated by cytokines, neuropeptides, and neurotransmitters. We have previously shown that stimuh related to protein kinase C. such as PMA and calcium ionophore A23187, increase IL-6 expression, as do stimulators of the cAMP pathway (forskolin norepinephrifie (NE)). The purpose of this study was to determine the role of selmodu n tn regu at ng nduction of IL-6 mRNA. MATERIALS & METHODS: Rat astrocyles were preincubated with 0-60 p.M of W7, a calcium/cnimodulin inhibitor, for 30 rain prior to costimulation with PMA (80 nM) + A23187 (5 BM) NE (10 p.M) + A23187, or forskolin (20 I~M) + A23187. RNA was isolated and analyzed 1-4 h post stimulation for 11.-6 mRNA by ribonuclease protection assay• , ~ ,. RESULTS: W7, at concentrations above 25 ~tM. inauced IL-6 mHr,l,~ in astrocytes. The induction of IL-6 mRNA by W7 alone was both time and dose dependent. In contrast, a 30 min preincubation of rat astrccytes with 40-60 PM °f W7 inhibited the induction of IL-6 mRNA by PMA +..A2.3187: A transient inhibition of 50% with 40 BM of W/occurreo z-~ n post sttmu!a~on; however, preincubation with 60 p M of W7 resulted m a 50% mhibdton throughout the 4 h study period. This inhibition appeared to be specific for PMA + A23187 induction since stimulation of cells with forskolin + A23187 or NE + A23187 was not affected by W7. CONCLUSIONS: These results indicate that the W7 sensitive pathways appear to be composed of two mechanistically independent but antagonistic systems: one increasing steady state levels of IL.6 mRNA in the absence of known exogenous activators, and the other being an inhibitory system that blocks PMA/Ca ++ pathways. Whether this dual regulatory role of W7 is a consequence of specific Inhibition of calmodulindependent kinase/phosphatase systems remains to be determined
H.Imrich. C.Kugler, N. Torres-Nagel, R. DOmes and T. Hiinig. lnsL f. Virologic nod Immunbiniogie, Universit.~RWtirzburg, Germany The predominance ofTCR V8 8.2 utilization in encephalitogenic T-cell lines isolated from rats and mice after induction of EAE is well established. The in vivo role of these cells for induction of the disease is, however, still controversial. Using a monoclonni antibody (mAb) specific for TCR VB 8.2 we dcmensWate an accumulation of TCR VII 8.2 cells in the brain of Lewis rats ahcr immunisation with MBP. In the pefipbery the vast majority of naive Tcells masks the increase of TCR VII 8.2 cells. However, after isolation of peripheral blood cells (PBL) and expansion of activated T-cells by culturing in IL-2 enriched medium, an increase of TCR VII 8.2 cells can be detected. Postnatal U'eaunent of rats with the mAb to TCR VII 8.2 eliminates VII 8.2 bearing cells and prevents the induction of severe EAE after MBP-immanisatioo in most of the animals. Moreover, treatment of adult Lewis rats with this anibody as late as on day 12 after MBP immunisation suppresses the development of neurological symptoms, Proliferation assays indicate that although the pool of MBP-specific T-cells may be reduced after postnatal depletion of TCR VII 8.2 cells, MBP spccifk: proliferation can be detected. Thus, VII 8.2 bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) V6 8.2-expressing T-cell subset provides an effective therapeutic saategy in this animal model.
P l l .06 ANALYSIS OF ANTIBODY RESPONSE TO PREDOMINANT LINEAR EPITOPES OF THEILER'S MURINE ENCEPHALOMYELITIS VIRUS A, lnoue*, Y-K Choe**, B. S. Kim** *Department of Medicine (Neurology) Shinshu University School o f M e d i c i n e **Department of Microbiology-Immunology and Pathology, N o r t h w e s t e r n U n i v e r s i t y Medical s c h o o l Intracerebral inoculation of Theiler's murine encephalomyelitis virus(TMEV) into susceptible mouse s t r a i n s r e s u l t s in a p r i m a r y d e m y e l i n a t i n g d i s e a s e . T h i s m o d e l s y s t e m is u s e d to s t u d y t h e h u m a n d i s e a s e m u h i p l e s c l e r o s i s (MS). D e t e r m i n a t i o n o f t h e e p i t o p e s o f TMEV t h a t a r e i n v o l v e d in t h e d i s e a s e p r o c e s s is a n i m p o r t a n t a n d critical s t e p in e x p l o r i n g t h e m e c h a n i s m o f p a t h o g e n e s i s o f this v i r u s . U s i n g s y n t h e t i c p e p t i d e s , w e h a v e a n a l y z e d a n t i b o d y r e s p o n s e s to TMEV in s u s c e p t i b l e SJL/J a n d r e s i s t a n t C 5 7 B L / 6 a n d BALB/c m i c e . T h e m a j o r a n t i b o d y e p i t o p e s d e f i n e d r e m a i n l a r g e l y t h e s a m e as t h o s e d e t e r m i n e d with f u s i o n p r o t e i n s b e f o r e , i . e . A-1A(VP113-27), A-1B(VP1 1 4 5 - 1 5 9 ) , A - 1 C b ( V P 1 2 6 2 - 2 7 6 ) , A-2A(VP22-16), A-2B(VP2165179) a n d A - 3 A ( V P 3 2 4 - 2 7 ) . S u s c e p t i b l e SJL/J m i c e s t r o n g l y a n d s e l e c t i v e l y r e c o g n i z e t h e A 1 C b e p i t o p e o f t h e VP1 as c o m p a r e d to r e s i s t a n t 8ALB/c o r C 5 7 B L / 6 m i c e . N e u t r a l i z a t i o n a s s a y o f in v i t r o v i r a l p l a q u e f o r m a t i o n b y antibodies specific for individual epitopes suggest that AICb is t h e m o s t p o t e n t n e u t r a l i z i n g e p i t o p e a m o n g t h e l i n e a r antibody epitopes.
P13.10 ANTIBODY ACTIVITIES TO SULFATIDE, Pit PROTEIN AND OLIGOSACCHARIDE CHAINS OF P0 PROTEIN IN POLYNEUROPATHIES T. Rosokawa, K. K i t a a n r a * , K. Nomura, a n d K. l l a ~ c h i . Department of Neurology, *Department of Physiology, Saitama Medical School, Saitama, Japan We h a v e e v a l u a t e d a n t i - S u l f a t i d e and PO p r o t e i n IgM antibody activities in s e r a f r o m one c a s e o f c h r o n i c p o l y n a u r o p a t h y w i t h IgM p a r a p r o t e i n e m i a (IgM-CP), 5 cases of chronic idiopathic p o l y n e u r o p a t h y ( C I P ) , 6 c a s e s o f acute idiopathic polyneuropathy (AIP) and 6 c a s e s o f controls. ELISA a n a l y s i s for anti-Sulfatide a n d P0 protein IgM a n t i b o d y r e v e a l e d h i g h t i t e r i n IgM-CP and significantly h i g h e r i n CIP t h a n i n AIP and c o n t r o l s . Furthermore, we c o u l d d e t e r m i n e the structure of oligoaaccharida c h a i n s o f P0 p r o t e i n , GP 4 and GP 5. These differences were only non-reducing terminal sugar o f t h e s e c h a i n s . GP 5 h a s a s u l f a t e d g l u c n r o n i o a c i d and GP 4 h a s a s i a l i c a c i d a t t h e t e r m i n a l . We e x a m i n e d t h e reactivitiea o f e l u t e d IgH f r o m one c a s e o f IgH-CP and 3 c a s e s o f CIP w i t h t h e s e c h a i n s . The IgM o f IgM-CP r e a c t e d w i t h GP 5 o n l y , and t h e IgM o f CIP r e a c t e d w i t h b o t h . These data suggested the presence of anti-Sulfatide and P0 p r o t e i n I g H a n t i b o d i e s i n t h e s e r a o f IgN-CP and C I P , and t h e e p i t o p e o f t h e s e r u m o f IgH-CP m i g h t be t h e s u l f a t e d g l u c u r o n i c a c i d i n GP 5.
P12.04 EXPRESSION OF THE SCLEROSIS LESIONS
CYTOKINE
RANTES
IN
MULTIPLE
J. Hyas, K. Sinding, J.R. Oksenbesg 1, G. ~ 2 a~d C.C.A. Bernasd Nenrvimmunology Laboratory, La Trobe University, Bundoo~ Victoria, 3083, Australia, ~'l~t of Nemolog~/, Udivea'sity of Cni~oxnia, San Fra~isco, California, 94143-0114, USA and '~L.A.R.C.H. ~ Research Unit, Royal Children's Hospital, Parkvtlle, Victoria, 3052, AusUniia. Multiple sclerosis (MS) is an inflanunatoey disease of the central nervous sysmm (C'NS), ~ by myelin destruction. The m~aa~iams of domyelination in MS are complex and lm3hably involve soluble immone mediators, partlcelm'lyc~s. Activated T cells, which pertici~ in the pathogeaexis of this disease, have beer shown to migrate from the pedphe~ lymphoid system to the CNS via the Hood ~ berrY. The re~ntly identified cym~ne Rantes, acts as a selective chemomeraotam to T cells and mediates their adherence to e~lethelial cells,thereby facilita~'g their enlry into the CNS. In the present study, we have used in-sRu hylwidisefion to locale Rantes and a semiquantilative polymemse chain reaction method to analyse the level of Rantes expressiou in MS brain lesions. The results show a highex expreuion of Rantes in MS lesions as compared to other nem'otogicni dtsoaden and healthy controls. These experiments implicate P,~tes as an ~ effectox in autoimmune demyelination and raise the possibility that its down-regnlation may have important therapeutic potential fox diseases like MS.
P01.05
PI0.02
Centr~ Adn~,,,~'etion of Lipoconin R~uces Sevaity of Mild Exl~imeatni Allergic ~,~pllalomyeillis
O N T H E R O L E O F T C R V6 8.2 T - C E L L S I N L E W I S R A T EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)
L I-luitinf~L J. Bauorl, PJ.L.M. Strijbes2, N.J. Rothwell-L C.D. Dijkstrat. FJ.H. Tllders4 and F. Berkenbosch('D4. DelXs. of 1: Cell Biology and 4: Pharmacology, Vrije Universitelt, Amsterdam. The Netherlands, 2: Dept. of Pharmacology. The Wellcome Research Laboratories, KenL UK and 3: School of Biological Sciences, University of Manchester, Manchester, UK. Experimental autoimmune encephalomyelitis (EAE) w4xeseots an animal model for multiple sclerosis (MS). Cordceoternid treatment has proven to be benificlal in both MS and EAE. Suppressiou by corticostoroids ntight in part be mediated by the local reduction of inflammato~ reactions in the CNS. Corticosteroids induce expression of lipoconin-I (LC-I) which has been de~pon.¢a-ated to play a role in the control of inflammation. In the CNS of MS patients and EAE rats, LC-I is up resulated, which may be induced by elevated cortieostexone plasma levels during the disease. In this .study we show that central administration of an active recomhinant frasment (1-188 amino acids) of LC-I reduces clinical severity of ntild EAE in Lewis rats but not of sevevv EAB. lntmventficular injection of antibodies raised to the lipocortin 1-188 frogmant as well as anti-LC-I antibodies obtained from Zymod. did not aK~avate disease significantly. These results indicate that suRxeseive actions of corticesternids could in part be mediated by down reSulation of inflanuuation in the CNS via LC-I. Physiological relevance of •endogenous LC- l in the CNS during EAE however remains to be established.
P03.06 REGULATION OF IL-6 mRNA LEVELS IN PRIMARY RAT ASTROCYTES BY THE CALMODULIN ANTAGONIST W7 B.S. Hunevcutt and E. N. Benveniste• Depadment of Cell Biology. University of Alabama at Birmingham, Birmingham, Alabama 35294. INTRODUCTION: IL.6 expression in astrocytes is regulated by cytokines, neuropeptides, and neurotransmitters. We have previously shown that stimuh related to protein kinase C. such as PMA and calcium ionophore A23187, increase IL-6 expression, as do stimulators of the cAMP pathway (forskolin norepinephrifie (NE)). The purpose of this study was to determine the role of selmodu n tn regu at ng nduction of IL-6 mRNA. MATERIALS & METHODS: Rat astrocyles were preincubated with 0-60 p.M of W7, a calcium/cnimodulin inhibitor, for 30 rain prior to costimulation with PMA (80 nM) + A23187 (5 BM) NE (10 p.M) + A23187, or forskolin (20 I~M) + A23187. RNA was isolated and analyzed 1-4 h post stimulation for 11.-6 mRNA by ribonuclease protection assay• , ~ ,. RESULTS: W7, at concentrations above 25 ~tM. inauced IL-6 mHr,l,~ in astrocytes. The induction of IL-6 mRNA by W7 alone was both time and dose dependent. In contrast, a 30 min preincubation of rat astrccytes with 40-60 PM °f W7 inhibited the induction of IL-6 mRNA by PMA +..A2.3187: A transient inhibition of 50% with 40 BM of W/occurreo z-~ n post sttmu!a~on; however, preincubation with 60 p M of W7 resulted m a 50% mhibdton throughout the 4 h study period. This inhibition appeared to be specific for PMA + A23187 induction since stimulation of cells with forskolin + A23187 or NE + A23187 was not affected by W7. CONCLUSIONS: These results indicate that the W7 sensitive pathways appear to be composed of two mechanistically independent but antagonistic systems: one increasing steady state levels of IL.6 mRNA in the absence of known exogenous activators, and the other being an inhibitory system that blocks PMA/Ca ++ pathways. Whether this dual regulatory role of W7 is a consequence of specific Inhibition of calmodulindependent kinase/phosphatase systems remains to be determined
H.Imrich. C.Kugler, N. Torres-Nagel, R. DOmes and T. Hiinig. lnsL f. Virologic nod Immunbiniogie, Universit.~RWtirzburg, Germany The predominance ofTCR V8 8.2 utilization in encephalitogenic T-cell lines isolated from rats and mice after induction of EAE is well established. The in vivo role of these cells for induction of the disease is, however, still controversial. Using a monoclonni antibody (mAb) specific for TCR VB 8.2 we dcmensWate an accumulation of TCR VII 8.2 cells in the brain of Lewis rats ahcr immunisation with MBP. In the pefipbery the vast majority of naive Tcells masks the increase of TCR VII 8.2 cells. However, after isolation of peripheral blood cells (PBL) and expansion of activated T-cells by culturing in IL-2 enriched medium, an increase of TCR VII 8.2 cells can be detected. Postnatal U'eaunent of rats with the mAb to TCR VII 8.2 eliminates VII 8.2 bearing cells and prevents the induction of severe EAE after MBP-immanisatioo in most of the animals. Moreover, treatment of adult Lewis rats with this anibody as late as on day 12 after MBP immunisation suppresses the development of neurological symptoms, Proliferation assays indicate that although the pool of MBP-specific T-cells may be reduced after postnatal depletion of TCR VII 8.2 cells, MBP spccifk: proliferation can be detected. Thus, VII 8.2 bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) V6 8.2-expressing T-cell subset provides an effective therapeutic saategy in this animal model.
P l l .06 ANALYSIS OF ANTIBODY RESPONSE TO PREDOMINANT LINEAR EPITOPES OF THEILER'S MURINE ENCEPHALOMYELITIS VIRUS A, lnoue*, Y-K Choe**, B. S. Kim** *Department of Medicine (Neurology) Shinshu University School o f M e d i c i n e **Department of Microbiology-Immunology and Pathology, N o r t h w e s t e r n U n i v e r s i t y Medical s c h o o l Intracerebral inoculation of Theiler's murine encephalomyelitis virus(TMEV) into susceptible mouse s t r a i n s r e s u l t s in a p r i m a r y d e m y e l i n a t i n g d i s e a s e . T h i s m o d e l s y s t e m is u s e d to s t u d y t h e h u m a n d i s e a s e m u h i p l e s c l e r o s i s (MS). D e t e r m i n a t i o n o f t h e e p i t o p e s o f TMEV t h a t a r e i n v o l v e d in t h e d i s e a s e p r o c e s s is a n i m p o r t a n t a n d critical s t e p in e x p l o r i n g t h e m e c h a n i s m o f p a t h o g e n e s i s o f this v i r u s . U s i n g s y n t h e t i c p e p t i d e s , w e h a v e a n a l y z e d a n t i b o d y r e s p o n s e s to TMEV in s u s c e p t i b l e SJL/J a n d r e s i s t a n t C 5 7 B L / 6 a n d BALB/c m i c e . T h e m a j o r a n t i b o d y e p i t o p e s d e f i n e d r e m a i n l a r g e l y t h e s a m e as t h o s e d e t e r m i n e d with f u s i o n p r o t e i n s b e f o r e , i . e . A-1A(VP113-27), A-1B(VP1 1 4 5 - 1 5 9 ) , A - 1 C b ( V P 1 2 6 2 - 2 7 6 ) , A-2A(VP22-16), A-2B(VP2165179) a n d A - 3 A ( V P 3 2 4 - 2 7 ) . S u s c e p t i b l e SJL/J m i c e s t r o n g l y a n d s e l e c t i v e l y r e c o g n i z e t h e A 1 C b e p i t o p e o f t h e VP1 as c o m p a r e d to r e s i s t a n t 8ALB/c o r C 5 7 B L / 6 m i c e . N e u t r a l i z a t i o n a s s a y o f in v i t r o v i r a l p l a q u e f o r m a t i o n b y antibodies specific for individual epitopes suggest that AICb is t h e m o s t p o t e n t n e u t r a l i z i n g e p i t o p e a m o n g t h e l i n e a r antibody epitopes.