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Cytokine production in autoimmune myasthenia gravis (MG)
190
P02.13
W15.01
IL-I0, IL-ll3 and TNF(xLEVELS IN SERUM AND CEREBRAL SPINAL FLUID (CSF) OF PATIENTS WITH GUILLAINBARI~ SYNDROME (GB$).
CYTOKINE-INDUCED
Pomm A.M.. Luga~esiA., Dell' Arciprete L., Gambi D. Imtimte o f ~ Neurology and Behavinml Sciences, University of Chieti, Italy
M K. Racke 1 , A. Bon0mo 2, D. E. Scott 3, 8. Cannella4,.A_ D. Levlne 5, C. S. Ralne4~ E. P1, Shevach 2, and M~ Rqcken2_ _ _ 1Neurolmmunology Branch, NIH, 2Laboratory of Immunology, NIH, 3FDA, Bethesda, MD, 4Albert Elnstein Col. of Med., Bronx, NY, andSMonsanto Co., St. Louis, MO
lntrodm'd(m: increased levels of mununoglobulins (Ig) and prom.flammatory cytokmes O"NFe.,IL-6) are detectable in the CSF of patients with GBS. IL-10, which inhibits cytokine synthesis and induces antibody production, may be involved in the mununopathogenesis of the disease. Aim of the study was to measure IL-10 levels in scram and CSF of patients with GBS and correlate them with levels of other eytokines and lg M~eelalt aed mt~otls: IL-10, IL-1[3 were assayed using commercial ELISA kits and TNFct using a bioassay in serum and CSF from 10 patiants with GBS and 10 patients with other non-inttammatory neurological diseases (NIND). Serum end CSF were drawn from patients with GBS within two weeks f~)rn the onset of elihieal symptoms. Result=: even though patients with GBS had detectable levels of serum and CSF IL-10 more fl~quently than pati(mts with NIND, we observed no significant differences in mean levels ofserurn and CSF IL-1010~weertthe two groups. IL-I [3was increased in serum from patients with GBS (51.1±21.1 pg/ml) compared to patients wath NIND (1.2~1.2 pg/ml) (1><0.05;two-roiled t-test). Serum and CSF levels of TNF(xwere similar between the two groups. No correlations were observed among ~ e m eytoldnes and between eytokines and Ig levels. Conduskm: IL-10 seems not to be involved in the acute clinical phase of GBS even though further studies at different times are warranted.
P12.08 STIMULATION OF IL-10 SECRETION MAY BE ONE MECHANISM OF THE THERAPEUTIC EFFECT OF RECOMBINANT HUMAN INTERFERON~3(rIFN Oser) IN MULTIPLE SCLEROSIS (MS). Pomdi A.M.~, Gambi D.t and Reder A.T.2 Institute of Clinical Neurology and Behavioral Sciences, University of Chieti, Italy; 2 Department of Neuro!ogy and Brain Research Institute, University of Chicago, IL, USA.
II'IMUNE D E V I A T I O N
AS
A THERAPY
FOR INFLAMMATORY AUTOli~UNE DISEASE
I n t r o d u c t i o n : To determine whether induction of Th2-1ike cells would modulate an inflammatory response in the prototyplc autolmmune disease, experimental atlerglc encephalomyelitis (EAE). M a t e r i a l s and Methods: IL-4 was administered to animals w i t h EAE produced by T h l - l i k e T cells specific for myelin basic protein (MBP). Cytoktne production of MBPspectfic T cells In the periphery and cytoktne gene exp~-,~stonln the CNS was also examined. Results: IL-4 treatment resulted In amelioration of c]lnical dlsease, the Induction of MBP-sDeciflc Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines In the CN$. Conclusion: Modulation of an I m m u n e response from one dominated by excessive activity of Th1-Hke T ceils to one dominated by the protective cytok~nes produced by Th2-11ke T,ceils m a y have applicability to the therapy of certain h u m a n autoimmune disorders,
N09.04
CYTOKINE PRODUCTION IN AUTOIMMUNE MYASTHENIA GRAVIS (MG), S. Raoheb and R. P. Lisak Division of Neuroimmunology, Department of Neurology, Wayne State University, Detroit, MI, USA Introduction: In MG, production of anti-acetylcholine receptor (AChR) antibodies by B-ceits b T:cell dependent. T/B-oell interactions are mediated in pad through ~ Wtekine signals: O~t 0 1 : ~ e iS to measure in vitro ptOCt~lg~ ~ cy~klmm by T-t~Ig ffom~lJlle~lB "~qhldlG; M e ~ e d ~ & 4 ~ m u l ~ P ~ p h ~ = b~od ~ ce~
lntrodm:tkm: MS is amehomted by rlFNI3ser, treatment with IF'N?worsens the disease. The mechanism of action oflFNs Jn M S isstillunclear. Promflammat~y cytokin~ (IL- 113,TNFct) are involvedin demyelination end MS exaced~tions (IFN7). IL-10reduc~ seeretion'oftbe~e p ~ cy~kines. Aim ofthe study was to investisate the effects of tFNs on IL-I0 secretion by monocytes (Mo) from MS patients. Mathwbls mtd m~thedz: using a commercial ELISA kit we measured IL-IOleve~in s ~ t m a t s of M o flora lO M S patienlsand 10 healthy centrols (NL) in~mbetsdfor t8 hours with rlFNflse r (50U/ml and 1000U/ml),IFN? (t00O/ml) and r IFN[~ser+IFN? Correlation betwsen the levels oflL-10 and proinflammatoty cytokines (IL-ll3, TNFot, IL-6) was also inves~gated. Rmnlts: high dose rlFNI3ser (1000U/ml) induced IL-10 secretion by MS Mo (54~19.5 pg/rnl vs spontaneous seeredon=8.23~:2.4pg/ml; p<0.04; parred t-test) and NL Mo (384±11.8 pg/ml ~ spontaneous seeretion=5.6~:2:3pg/ml; p<0.02): IFN? and low dose rlFNI3se r (50U/ml) did not stimulate IL-I0 secretion, IFN7 had no effect on rlFN[3ser-induced IL-IOproduction.Levels oflL-lOsecreted after incubation with rlFNI3ser and IFNy eorrelsted with IL-6 levels in MS (p<0.003 and p<0.05, respectively) but not in NL. C o m : i u ~ : rlFN~ser may be therapeutic in MS throt~ the induction oflL-lO secfeffon.
corresponding to segments of the ct subun# of ~ ACItR. Ce~ wore cultured with medie alone to datAm~ine cot~titutive ~ I~oduction and cultured wiS~tatantm texoid (TT) ,~, a positivo c o b o l Levob for interferon-y (IFN-y), intedeukin-2 (IL-2), IL-4, and 1L-6 in tim culture supematants were measured by EUSA. ~ culture8 weeesat up to measure proliferation by =H-thymidine incorpore~ten in an 8 day culture. Ro~: Pept~ldstlm-ulat~ PBMC proliferated a6d $~..~e[~l h i ~ levels of IL-6, some IL-4, and little to no IL-2 and IFN-7. By coffd~, TTstimulated ee~ secreted high levels of 11.-2and tFN-7, moderate levele of IL-6, and no IL-4. Them was some constitutive IL-6 production by unsttrnulatedPBM~:
P17.12
W13.01
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l
q
~
]
J&JL T.4~I~J J, M I G R A T I O N IS D]g]BENDF, NT ON ANTIGEN STIMULATION, I L - 2 , 131ME ~ ADHESION M O L E C U L E EXPRE&~ION Gareth Pryce, John G r e e n w o o d and David Male. Institutes o f Ophthalmology and Psychiatry, London, I n t r o d u c t i o n : W e have studied the mechanisms determining T-celt migration eeross cerebral-derived endothelium o f the rat with the longterm aim o f developing a possible drug-delivery vector. M a t e r i a l s a n d M e t h o d s : T h e effect o f T-cell line activation and adhesion molec~d~ blockade on ttans~Ldo~eliai migration were asessed by time-lapse video-microsonpy. Adhesion molecule and 11-2 receptor expressioa was determined by flow cytometry. Results: Peak levels o f T-cell migration was achieved after antigen stimulation followed by 4 days culture in I L - 2 containing media. Migration was found to be L F A - 1 / I C A M - 1 dependent. Conclusion: W e propose that T-cell migration across CNS-derived endothelia requires at least two separate signals. Upregulation o f the IL-2 receptor by antigen stimulation precedes IL-2 induced proliferation and increase in adhesion molecule expression and migratory capacity.
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ConcluSion: Our data indicatethatAChR-responsive T-celisin patients
with MG produce pdmadly IL-6, a TH2 cytokine which is important for Bcell growth and differentiation.
]MMUNOLOGIC~L CRO$ SPJEACI"IVITYBETWEEN C.jrEjuAt/L£FOPOLYSACCH~QPJ[DE ANDGANGLIOSlDEGM1 IN PATIENTSWITHGU1LLAINBAling SYm~OME J H Reas ~, NA Gregsonz, G Rajagopatan I and RAC Hushes l Department of Neurology (1) and Anatomy (2), UMDS, Guy's Hospital
Campylobacterjejuni (C. jejuni) enteritis is a common antecedent infection preceding Guillain Barr~ syndrome (GBS). In a prospective case controlled study 22 out of 102 patients with GBS had evidence of recent C. jejuni infection compared with 1 out of 193 controls (P=0.00001). 12 out of those 22 patients h a d anttlx~as against ganglioside GMI (GM1). We have demonstrated immunological crossreactivity between gangiioside GMI and the purified lipopol]aeu~hRride (LPS) fraction from different C jejuni serotypcs isolated from patients with GBS. These LPS fractions had ~ g amounts of GM1 hapten as determined by cholera toxin biading. Affinity purified anti-GM1 antibodies froirt a panel Of GBS pattent~-te~ted with different .Penner serotypes. Furthermore the serum of one of the patients from whom the LPS was derived was not strongly reactive with this LPS and did not contain anti-OM1 antibodies. These results indicate that the GMI hapten can occur in the LPS from different C. jejuni serotypes and is not restricted to Penner 19 strains as has been suggestedx. The presence of this carbohydrate sequence in the LPS does not necessarily induce anti-GM1 antibodies. 1.
Yuki N, Taki T, Inagaki F et aL J Exp Med 1993; 178:1771-1775
P02.13
W15.01
IL-I0, IL-ll3 and TNF(xLEVELS IN SERUM AND CEREBRAL SPINAL FLUID (CSF) OF PATIENTS WITH GUILLAINBARI~ SYNDROME (GB$).
CYTOKINE-INDUCED
Pomm A.M.. Luga~esiA., Dell' Arciprete L., Gambi D. Imtimte o f ~ Neurology and Behavinml Sciences, University of Chieti, Italy
M K. Racke 1 , A. Bon0mo 2, D. E. Scott 3, 8. Cannella4,.A_ D. Levlne 5, C. S. Ralne4~ E. P1, Shevach 2, and M~ Rqcken2_ _ _ 1Neurolmmunology Branch, NIH, 2Laboratory of Immunology, NIH, 3FDA, Bethesda, MD, 4Albert Elnstein Col. of Med., Bronx, NY, andSMonsanto Co., St. Louis, MO
lntrodm'd(m: increased levels of mununoglobulins (Ig) and prom.flammatory cytokmes O"NFe.,IL-6) are detectable in the CSF of patients with GBS. IL-10, which inhibits cytokine synthesis and induces antibody production, may be involved in the mununopathogenesis of the disease. Aim of the study was to measure IL-10 levels in scram and CSF of patients with GBS and correlate them with levels of other eytokines and lg M~eelalt aed mt~otls: IL-10, IL-1[3 were assayed using commercial ELISA kits and TNFct using a bioassay in serum and CSF from 10 patiants with GBS and 10 patients with other non-inttammatory neurological diseases (NIND). Serum end CSF were drawn from patients with GBS within two weeks f~)rn the onset of elihieal symptoms. Result=: even though patients with GBS had detectable levels of serum and CSF IL-10 more fl~quently than pati(mts with NIND, we observed no significant differences in mean levels ofserurn and CSF IL-1010~weertthe two groups. IL-I [3was increased in serum from patients with GBS (51.1±21.1 pg/ml) compared to patients wath NIND (1.2~1.2 pg/ml) (1><0.05;two-roiled t-test). Serum and CSF levels of TNF(xwere similar between the two groups. No correlations were observed among ~ e m eytoldnes and between eytokines and Ig levels. Conduskm: IL-10 seems not to be involved in the acute clinical phase of GBS even though further studies at different times are warranted.
P12.08 STIMULATION OF IL-10 SECRETION MAY BE ONE MECHANISM OF THE THERAPEUTIC EFFECT OF RECOMBINANT HUMAN INTERFERON~3(rIFN Oser) IN MULTIPLE SCLEROSIS (MS). Pomdi A.M.~, Gambi D.t and Reder A.T.2 Institute of Clinical Neurology and Behavioral Sciences, University of Chieti, Italy; 2 Department of Neuro!ogy and Brain Research Institute, University of Chicago, IL, USA.
II'IMUNE D E V I A T I O N
AS
A THERAPY
FOR INFLAMMATORY AUTOli~UNE DISEASE
I n t r o d u c t i o n : To determine whether induction of Th2-1ike cells would modulate an inflammatory response in the prototyplc autolmmune disease, experimental atlerglc encephalomyelitis (EAE). M a t e r i a l s and Methods: IL-4 was administered to animals w i t h EAE produced by T h l - l i k e T cells specific for myelin basic protein (MBP). Cytoktne production of MBPspectfic T cells In the periphery and cytoktne gene exp~-,~stonln the CNS was also examined. Results: IL-4 treatment resulted In amelioration of c]lnical dlsease, the Induction of MBP-sDeciflc Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines In the CN$. Conclusion: Modulation of an I m m u n e response from one dominated by excessive activity of Th1-Hke T ceils to one dominated by the protective cytok~nes produced by Th2-11ke T,ceils m a y have applicability to the therapy of certain h u m a n autoimmune disorders,
N09.04
CYTOKINE PRODUCTION IN AUTOIMMUNE MYASTHENIA GRAVIS (MG), S. Raoheb and R. P. Lisak Division of Neuroimmunology, Department of Neurology, Wayne State University, Detroit, MI, USA Introduction: In MG, production of anti-acetylcholine receptor (AChR) antibodies by B-ceits b T:cell dependent. T/B-oell interactions are mediated in pad through ~ Wtekine signals: O~t 0 1 : ~ e iS to measure in vitro ptOCt~lg~ ~ cy~klmm by T-t~Ig ffom~lJlle~lB "~qhldlG; M e ~ e d ~ & 4 ~ m u l ~ P ~ p h ~ = b~od ~ ce~
lntrodm:tkm: MS is amehomted by rlFNI3ser, treatment with IF'N?worsens the disease. The mechanism of action oflFNs Jn M S isstillunclear. Promflammat~y cytokin~ (IL- 113,TNFct) are involvedin demyelination end MS exaced~tions (IFN7). IL-10reduc~ seeretion'oftbe~e p ~ cy~kines. Aim ofthe study was to investisate the effects of tFNs on IL-I0 secretion by monocytes (Mo) from MS patients. Mathwbls mtd m~thedz: using a commercial ELISA kit we measured IL-IOleve~in s ~ t m a t s of M o flora lO M S patienlsand 10 healthy centrols (NL) in~mbetsdfor t8 hours with rlFNflse r (50U/ml and 1000U/ml),IFN? (t00O/ml) and r IFN[~ser+IFN? Correlation betwsen the levels oflL-10 and proinflammatoty cytokines (IL-ll3, TNFot, IL-6) was also inves~gated. Rmnlts: high dose rlFNI3ser (1000U/ml) induced IL-10 secretion by MS Mo (54~19.5 pg/rnl vs spontaneous seeredon=8.23~:2.4pg/ml; p<0.04; parred t-test) and NL Mo (384±11.8 pg/ml ~ spontaneous seeretion=5.6~:2:3pg/ml; p<0.02): IFN? and low dose rlFNI3se r (50U/ml) did not stimulate IL-I0 secretion, IFN7 had no effect on rlFN[3ser-induced IL-IOproduction.Levels oflL-lOsecreted after incubation with rlFNI3ser and IFNy eorrelsted with IL-6 levels in MS (p<0.003 and p<0.05, respectively) but not in NL. C o m : i u ~ : rlFN~ser may be therapeutic in MS throt~ the induction oflL-lO secfeffon.
corresponding to segments of the ct subun# of ~ ACItR. Ce~ wore cultured with medie alone to datAm~ine cot~titutive ~ I~oduction and cultured wiS~tatantm texoid (TT) ,~, a positivo c o b o l Levob for interferon-y (IFN-y), intedeukin-2 (IL-2), IL-4, and 1L-6 in tim culture supematants were measured by EUSA. ~ culture8 weeesat up to measure proliferation by =H-thymidine incorpore~ten in an 8 day culture. Ro~: Pept~ldstlm-ulat~ PBMC proliferated a6d $~..~e[~l h i ~ levels of IL-6, some IL-4, and little to no IL-2 and IFN-7. By coffd~, TTstimulated ee~ secreted high levels of 11.-2and tFN-7, moderate levele of IL-6, and no IL-4. Them was some constitutive IL-6 production by unsttrnulatedPBM~:
P17.12
W13.01
~
l
q
~
]
J&JL T.4~I~J J, M I G R A T I O N IS D]g]BENDF, NT ON ANTIGEN STIMULATION, I L - 2 , 131ME ~ ADHESION M O L E C U L E EXPRE&~ION Gareth Pryce, John G r e e n w o o d and David Male. Institutes o f Ophthalmology and Psychiatry, London, I n t r o d u c t i o n : W e have studied the mechanisms determining T-celt migration eeross cerebral-derived endothelium o f the rat with the longterm aim o f developing a possible drug-delivery vector. M a t e r i a l s a n d M e t h o d s : T h e effect o f T-cell line activation and adhesion molec~d~ blockade on ttans~Ldo~eliai migration were asessed by time-lapse video-microsonpy. Adhesion molecule and 11-2 receptor expressioa was determined by flow cytometry. Results: Peak levels o f T-cell migration was achieved after antigen stimulation followed by 4 days culture in I L - 2 containing media. Migration was found to be L F A - 1 / I C A M - 1 dependent. Conclusion: W e propose that T-cell migration across CNS-derived endothelia requires at least two separate signals. Upregulation o f the IL-2 receptor by antigen stimulation precedes IL-2 induced proliferation and increase in adhesion molecule expression and migratory capacity.
.
.
.
.
.
.
.
.
.
.
.
.
.
ConcluSion: Our data indicatethatAChR-responsive T-celisin patients
with MG produce pdmadly IL-6, a TH2 cytokine which is important for Bcell growth and differentiation.
]MMUNOLOGIC~L CRO$ SPJEACI"IVITYBETWEEN C.jrEjuAt/L£FOPOLYSACCH~QPJ[DE ANDGANGLIOSlDEGM1 IN PATIENTSWITHGU1LLAINBAling SYm~OME J H Reas ~, NA Gregsonz, G Rajagopatan I and RAC Hushes l Department of Neurology (1) and Anatomy (2), UMDS, Guy's Hospital
Campylobacterjejuni (C. jejuni) enteritis is a common antecedent infection preceding Guillain Barr~ syndrome (GBS). In a prospective case controlled study 22 out of 102 patients with GBS had evidence of recent C. jejuni infection compared with 1 out of 193 controls (P=0.00001). 12 out of those 22 patients h a d anttlx~as against ganglioside GMI (GM1). We have demonstrated immunological crossreactivity between gangiioside GMI and the purified lipopol]aeu~hRride (LPS) fraction from different C jejuni serotypcs isolated from patients with GBS. These LPS fractions had ~ g amounts of GM1 hapten as determined by cholera toxin biading. Affinity purified anti-GM1 antibodies froirt a panel Of GBS pattent~-te~ted with different .Penner serotypes. Furthermore the serum of one of the patients from whom the LPS was derived was not strongly reactive with this LPS and did not contain anti-OM1 antibodies. These results indicate that the GMI hapten can occur in the LPS from different C. jejuni serotypes and is not restricted to Penner 19 strains as has been suggestedx. The presence of this carbohydrate sequence in the LPS does not necessarily induce anti-GM1 antibodies. 1.
Yuki N, Taki T, Inagaki F et aL J Exp Med 1993; 178:1771-1775