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Lithium inhibits LPS-induced NO-synthase in cultured glial cells
162
;/02,04 I ~ I . , E U K I ~ I INCREASESTHE EXPRESSION OF ITS RECEPTORS (IL-XR)IN A RAT [~-PANCREATICCELL LINE (Rinm51~.
P07.05
LITHIUM INHIBITS LPS-INDU.CED NO-SYNTHASE IN CULTURED GLIAL CELLS M.K.A. Baunt, M. Bntger, Dept. of Psychiatry, U n l v ~ of F.relburg, F.R.G.
S.C-am*J.BristulP,L.I~trk *,D.Maltnowsky*,A.Sun~* and T.Barff'aP
*Nenrech~svy Dept./ImmunologyDepC,StockholmUr~v~sity. RinmSF cells represents an early stage of the [~-pancreatiecell maturation and they maintain some characteristics in common with neuronal cells. Using gene spedfic PCR primers we demonstrate that flmse ceils express both IL-1R subtypes mRNAs. Murine rIL-la and rat rIL-l~ (I0 ng/ml) treatment cause an upre~ulattoft of mRNA expremion for both IL-I R subtype. This effect is blocked by p r ~ z u m ~ n t with human rIL-lr& Binding studies ualn~ hrneI-Rel[~ h-ulicates that at least one type of IL-1R g e m product is premmt on untreated cells. IL-11~pretreatment (6 h) causes a two-fold increase of hrU'Sl-IL-ll~specific binding sites. Using indLrect inununo~uorascexce we detected an, IL-l-induced, increased presence of IL-1R lI at the cell surface level. A study on the IL-1R expreseicea is ~ premmed: Gluco~ (50raM) causes an upmgulatton of IL-tR I mRNA; D e ~ ( ~ lacks any effect;PMA (20 nM) induces upregulation of both IL-1R mRNAs; Pertuxiss Toxin pretreatment (100 ng/ml) partially blocks the IL-I[~induced expression of IL-IR I mRNA. We have sequenced the IL-IR II eDNA from this rat cell llne.The rat AA sequence of IL-IR 11shows respectively a 90% and 62% AA identity with the human and routine sequm~-es. The IL-1 R II from this rat cell line exhibits a six amino acid longer ( Q I K ] ~ ) cytosolic domain. Possible functional significance of this cytosollc extension will be discussed.
P01.02
N@'ic oxide l~as tmen ~ I as a member o~ a n e w class o~ ne~retranmnmm. R ~ e ~ dma smmoly m.,~mt a ro~ of NO in I m ~ e m l~/entiatiofl and Ion-tern dq~maion, little ~ Nm ~ devoted, Iwwov~, to NO ixoducod by ~ a l c~Is and oven l e ~ m oone expmsslon of NO..syrfd'msu in non-nsuronal ~ n ceils. The ~ of mmron~l c-
NOS m w d m
ot ~
~e~, o.r ~
NO~ has m e n ~ i n
nv-~a ~
slow rise of ~ ~ wee~ 1 and 2 h ual~dA ~ d
~a ~
~
etllumd amoo f ~ mzy-
W a ~ep ~ ~ ameJllum m 4 h I~ llqlb~ ~lffO~:
be~-
~n~ m~ma~ m m n ~ to m m unto 24 h m # ~ valm, morn man ~ m a m m l va~m o~ mlx~ ~ ~ aslroG'q,J~ pr_ndl'___r~._~ e ~ m ~
mere oJ~ums, wmzmmome t.PS.i~k,m~ ( c ~ I n ~ ~ . k e y to
:~,%s~-~ _ ~
mRNA
~ d ~oro t~S m
by mnte t h ~ 50 %. T h e ~ ~
, a r ~ @~xea effect of mhlum In ~
~
~
~,
reve~ m ~
in of but
mgl~ c~l
gone expremion.
W08.01
BINDING AND P R E S E N T A T I O N O F MYELIN BASIC PROTEIN PEPTIDE A c l - l l BY MHC CLASS II MOLECULES AaUAl~u. A nand M. Gautam and Sarah Weenink
THE IMMUNOLOGICAL "SELF" OF NEUitOENDOCIUNE PROTEIN FAMILIES: PHY~[OLO(~C-A~MPI.~Iq[4~WS
#Division of Clinical Sciences, The John Curtin School of Medical Research, Canberra, ACT, Australia. Myelin Baisc Protein (MBP) peptide Ac] - I 1 is presented by MHC class I[ molecules AaUA[3U.The present study demonstrates the minimal structural requirements for peptide presentation by MHC class II molecules. We show that substitution of all but four amino acids with alanines in Acl-11 resnlts in peptide which binds to AaUA[~U, stimulate specific T cells and induces experimental autoimmune encephalomyelis (EAE) in mice. Since peptide Ac3.5.6 ( A c ~ t ~ J ~ A A A A A ; native MBP residues arc shown as underlined bold letters) stimulats A c l - I 1 specific T cells, we have chosen this pcptide to study its conformation by introducing D-amino acids at various positions in this peptide. Our data show that D-amino acids can be tolerated only from C-terminus and that the pcptide binds in an extended conformation with a "kink" in the middle. These studies have also led us to identify a six residue peptide with only five native MBP residues which induces EAE. Overall, these results show essential requirements in recognition of MHC class Ii - bound peptide by the TcR. The data also provide insight into the mechanisms which may be involved in induction of autoimmunity.
V. C,~n~'~,Rcseare.hAssociateof BeigismNFSR institute of PathologyCHU-B23.Univ. of Liege-San Tilman, E-44J00Belgium Thymic epithelial and "nurse" cells (TEC/TNC) are the site of synthesis of precursors belongingto neurohypophysial (NHP), mchykinin and insulinfatuities. Oxytocin (O1") is the thymic peptide of the NHP family although, unlike hypothaiamic OT, it is nOt secreted along classic ~ (NE} pathways. Thymic OT is synthesized under the form of a 55-kD chimmic precursor which contains both a neurophysin (10 kD)-, as well as a MHC class I (45 kD)-selatcd domains. The MHC class-ld~ain could be used for membrane tasgening of the precursor in TEC/TNC, while the neurophysin one would bind OT for its pses=ntation to developing T cells. These fmdinsa s~ro~iy suggest that the intrathymic presentationof the OT self-a=digenis not restrictedby MHC alleles likethe periphcnd pmsunt~on of ago- or amo-anfigem, Inmli=-lik¢growth fa~orII (IGF-II) is the dominant thymic peptide of the insulin family which is synthesizedby TEC/TNC.Acco~d/ngm our model the ge,smmtion by TEC/TNC of IGF-II~leriveds d f - ~ s could induce the ccetral immmmlogicaltoleranceof the whole insulinfamily and, seco*Mmfly, of imalin-m~n~ng islet8 cells. We have also provided a seriesof experimental ~ t s supporting the role of thymic NE self-t~ptides as accessory signals in T cell positive selection. The novd type of cryptmainesignallingtinongh NHP-relatedsignals aud receptorswas demonstratedbetw~n TEC/TNC and pse-T cells. This type of signalling was shown to mediate direct nlitog~c effects of OT upon human and murinc thymocytes,as well as m induce the ~ l a t i o n of focal adhesion kiatases in murine pre-T cells. Altogether.our results dearly show that the dual l~ysiological role of the thymus in T cell pmifive and negative selection may be tnmsposerlat the levd of the thymicrepanotreof NE sdf-pepti~ p~mu'sm~.
W07.03
P01.03
INTERLEUKIN-3,
IL-3 RECEPTOR-8, AND JAK EXPRESSION IN RAT MICROGLIA K. A R o e l S ~ Dept. of P s ~ h i a ~ , ~ of IW~m~}, F.R.G.
The T.~MI~ ir~leukin~ is a mitogen for microglla. We have shown re cer,W tms purmm, Wo;,~,-~,~ r e m~rog~ emress tms cym~ne mems~yes 1 ~ et al., 19~41.The imment . d ~ ~mln~ by non-com-
pemm RT-PCRrewm uwegum~n of mRNA by ~:qX~m~mride (LPS), S..1, T N F . ~ ~ o~m s u m t m ~ , and mh~Son by Ommmma-
sone. In se~0fi lnt ~ of the IL-3 receptor, the rnudne sec~enee of the 8..sUbun~(NC2A; Wlm0 et M., 19~) wm u x d to dev~e a nundmr of oligonuctso~M [~mem. WMho~s l~dr of prlmem, a 1.25 kb c0NA Iragment was ampgl~d, clormcl and UeClUmCe~. B a x d on thi~ ~ClUence, new wimers we,e ~lectad m~l ua~l In ceml~mtlon wi~ the ~ (6gBCO4gRL) to ~ mere ~ ~ in the 5"-fllrtt@~ region. Cltm'actecis~ f m t t u s w~ be ~ in o0ml~flsen w ~ mudne and htxnan sequemm. S~n~ ~ e-~tum laem tn~ns~ tyrmm tdrme ffK) a c ~ Im~ I ~ a ~ e s recql~m'-mmnc,~ed TKs JAK1,2 or I"YK2 (M011nt et al., 19~). # wire p(mMIm to ~ by RT-PCR s~'~ch~ of 2.7 and 2.1 kb of JAK 1 and 2 cONAs, ~ , from totul r~ m ~ RNA. Seque~Sme~mmdmr ~ ~ ~mamm of comew~on mtween sPecmso~rfe@@W~n~ ~ TI~ W@-w~mty o~ m~mmmmlR~l~g IL-3 an0 t.P8 ~n)u~W way anamWlm wll Im pmmnmd. Bern" !nMol'm Imo molemW rNmmlmm of cymkme m ~mm,m~n In m l m ~ can
G e l ~ ( : k e - ~ et ~1., !9~4, J . N ~ . 50, 203-214. Wang et M., 19e2, J.Biol.Ghem. 267, 979-983. MOlkwet a1.,1993, NaCre 366, 129-135.
TREATMENTOF EAE WITH BONE MARROWTRANSPLANTATION M.v~ C ~ and D.W.vanBekkum Biomed~celPrimateRescmchCenterTNO, Rijswij'k,The Netherlands We investigatedthe effect of bone marrowtmnsplm~afion(BMT) on the com'seof relapsing EAE--indnced by immunization with spinal cord homogenate and complemadjuvant--in tim Blip rat and tim Binzzi mouse. Animals west Izestedwi~ mini body ~ (TBI) mgl syngmalc or pscudoautologons (S)BMT. after octtmt of paresis. This treatment indnced complete remission in all aninm~ only 30% developed a spontlmeees relapse vs.IO0% in untreated animals.The incidanceu d onset o( relgpeeswm rehited to tbe degree of 1 ~ abiaina indncedby tha coml~iamn'g. Purthanxx~ me incidence of Immsis afterreimmmi,~n was comimm~ reduced in SBM-tremplaated rats (not ascribable to post BMT i m ~ y ) . It was shown immmmtdslochmnicagy that.donor-daiv~l(F1) inonan~ celk imkm:ed the ~ afterndmmunizafion. Aliogemcic BM'r from resistant d ¢ ~ . eithm"complete MHC mis-maghed or partially matched, resulted in significantly mdnced ~ rates comlmred with SBMT. However, remisalon couM only be iagnced befme extcesive gliceis and demyalimUim Imd develol~xlin tbe CNS. Oer data indices tl~ earn afterallogeaeic BMT can be attributed to 1. a ~abelinic4tlOngt-vemm~Hcet-Rca~Iionand 2. the newlydeveloped,EAE-tesistsntiramune-bemopoieticsystmaof tbe doso¢ strain. In conclusion, extensive lympho-ablafion and autOlOgOmBMT might be an effectivetreatmentof severeMS, whm mmbined with ~ tolmzing Ueatment as used in mmsplmtagonmedlgine.AliogeaalcBMT is a goed alternative,but only if clinicalGvHDis properlyixevented.33eammntshould be appliedbefore extensive irreversibledamageto the CNS has developed.
;/02,04 I ~ I . , E U K I ~ I INCREASESTHE EXPRESSION OF ITS RECEPTORS (IL-XR)IN A RAT [~-PANCREATICCELL LINE (Rinm51~.
P07.05
LITHIUM INHIBITS LPS-INDU.CED NO-SYNTHASE IN CULTURED GLIAL CELLS M.K.A. Baunt, M. Bntger, Dept. of Psychiatry, U n l v ~ of F.relburg, F.R.G.
S.C-am*J.BristulP,L.I~trk *,D.Maltnowsky*,A.Sun~* and T.Barff'aP
*Nenrech~svy Dept./ImmunologyDepC,StockholmUr~v~sity. RinmSF cells represents an early stage of the [~-pancreatiecell maturation and they maintain some characteristics in common with neuronal cells. Using gene spedfic PCR primers we demonstrate that flmse ceils express both IL-1R subtypes mRNAs. Murine rIL-la and rat rIL-l~ (I0 ng/ml) treatment cause an upre~ulattoft of mRNA expremion for both IL-I R subtype. This effect is blocked by p r ~ z u m ~ n t with human rIL-lr& Binding studies ualn~ hrneI-Rel[~ h-ulicates that at least one type of IL-1R g e m product is premmt on untreated cells. IL-11~pretreatment (6 h) causes a two-fold increase of hrU'Sl-IL-ll~specific binding sites. Using indLrect inununo~uorascexce we detected an, IL-l-induced, increased presence of IL-1R lI at the cell surface level. A study on the IL-1R expreseicea is ~ premmed: Gluco~ (50raM) causes an upmgulatton of IL-tR I mRNA; D e ~ ( ~ lacks any effect;PMA (20 nM) induces upregulation of both IL-1R mRNAs; Pertuxiss Toxin pretreatment (100 ng/ml) partially blocks the IL-I[~induced expression of IL-IR I mRNA. We have sequenced the IL-IR II eDNA from this rat cell llne.The rat AA sequence of IL-IR 11shows respectively a 90% and 62% AA identity with the human and routine sequm~-es. The IL-1 R II from this rat cell line exhibits a six amino acid longer ( Q I K ] ~ ) cytosolic domain. Possible functional significance of this cytosollc extension will be discussed.
P01.02
N@'ic oxide l~as tmen ~ I as a member o~ a n e w class o~ ne~retranmnmm. R ~ e ~ dma smmoly m.,~mt a ro~ of NO in I m ~ e m l~/entiatiofl and Ion-tern dq~maion, little ~ Nm ~ devoted, Iwwov~, to NO ixoducod by ~ a l c~Is and oven l e ~ m oone expmsslon of NO..syrfd'msu in non-nsuronal ~ n ceils. The ~ of mmron~l c-
NOS m w d m
ot ~
~e~, o.r ~
NO~ has m e n ~ i n
nv-~a ~
slow rise of ~ ~ wee~ 1 and 2 h ual~dA ~ d
~a ~
~
etllumd amoo f ~ mzy-
W a ~ep ~ ~ ameJllum m 4 h I~ llqlb~ ~lffO~:
be~-
~n~ m~ma~ m m n ~ to m m unto 24 h m # ~ valm, morn man ~ m a m m l va~m o~ mlx~ ~ ~ aslroG'q,J~ pr_ndl'___r~._~ e ~ m ~
mere oJ~ums, wmzmmome t.PS.i~k,m~ ( c ~ I n ~ ~ . k e y to
:~,%s~-~ _ ~
mRNA
~ d ~oro t~S m
by mnte t h ~ 50 %. T h e ~ ~
, a r ~ @~xea effect of mhlum In ~
~
~
~,
reve~ m ~
in of but
mgl~ c~l
gone expremion.
W08.01
BINDING AND P R E S E N T A T I O N O F MYELIN BASIC PROTEIN PEPTIDE A c l - l l BY MHC CLASS II MOLECULES AaUAl~u. A nand M. Gautam and Sarah Weenink
THE IMMUNOLOGICAL "SELF" OF NEUitOENDOCIUNE PROTEIN FAMILIES: PHY~[OLO(~C-A~MPI.~Iq[4~WS
#Division of Clinical Sciences, The John Curtin School of Medical Research, Canberra, ACT, Australia. Myelin Baisc Protein (MBP) peptide Ac] - I 1 is presented by MHC class I[ molecules AaUA[3U.The present study demonstrates the minimal structural requirements for peptide presentation by MHC class II molecules. We show that substitution of all but four amino acids with alanines in Acl-11 resnlts in peptide which binds to AaUA[~U, stimulate specific T cells and induces experimental autoimmune encephalomyelis (EAE) in mice. Since peptide Ac3.5.6 ( A c ~ t ~ J ~ A A A A A ; native MBP residues arc shown as underlined bold letters) stimulats A c l - I 1 specific T cells, we have chosen this pcptide to study its conformation by introducing D-amino acids at various positions in this peptide. Our data show that D-amino acids can be tolerated only from C-terminus and that the pcptide binds in an extended conformation with a "kink" in the middle. These studies have also led us to identify a six residue peptide with only five native MBP residues which induces EAE. Overall, these results show essential requirements in recognition of MHC class Ii - bound peptide by the TcR. The data also provide insight into the mechanisms which may be involved in induction of autoimmunity.
V. C,~n~'~,Rcseare.hAssociateof BeigismNFSR institute of PathologyCHU-B23.Univ. of Liege-San Tilman, E-44J00Belgium Thymic epithelial and "nurse" cells (TEC/TNC) are the site of synthesis of precursors belongingto neurohypophysial (NHP), mchykinin and insulinfatuities. Oxytocin (O1") is the thymic peptide of the NHP family although, unlike hypothaiamic OT, it is nOt secreted along classic ~ (NE} pathways. Thymic OT is synthesized under the form of a 55-kD chimmic precursor which contains both a neurophysin (10 kD)-, as well as a MHC class I (45 kD)-selatcd domains. The MHC class-ld~ain could be used for membrane tasgening of the precursor in TEC/TNC, while the neurophysin one would bind OT for its pses=ntation to developing T cells. These fmdinsa s~ro~iy suggest that the intrathymic presentationof the OT self-a=digenis not restrictedby MHC alleles likethe periphcnd pmsunt~on of ago- or amo-anfigem, Inmli=-lik¢growth fa~orII (IGF-II) is the dominant thymic peptide of the insulin family which is synthesizedby TEC/TNC.Acco~d/ngm our model the ge,smmtion by TEC/TNC of IGF-II~leriveds d f - ~ s could induce the ccetral immmmlogicaltoleranceof the whole insulinfamily and, seco*Mmfly, of imalin-m~n~ng islet8 cells. We have also provided a seriesof experimental ~ t s supporting the role of thymic NE self-t~ptides as accessory signals in T cell positive selection. The novd type of cryptmainesignallingtinongh NHP-relatedsignals aud receptorswas demonstratedbetw~n TEC/TNC and pse-T cells. This type of signalling was shown to mediate direct nlitog~c effects of OT upon human and murinc thymocytes,as well as m induce the ~ l a t i o n of focal adhesion kiatases in murine pre-T cells. Altogether.our results dearly show that the dual l~ysiological role of the thymus in T cell pmifive and negative selection may be tnmsposerlat the levd of the thymicrepanotreof NE sdf-pepti~ p~mu'sm~.
W07.03
P01.03
INTERLEUKIN-3,
IL-3 RECEPTOR-8, AND JAK EXPRESSION IN RAT MICROGLIA K. A R o e l S ~ Dept. of P s ~ h i a ~ , ~ of IW~m~}, F.R.G.
The T.~MI~ ir~leukin~ is a mitogen for microglla. We have shown re cer,W tms purmm, Wo;,~,-~,~ r e m~rog~ emress tms cym~ne mems~yes 1 ~ et al., 19~41.The imment . d ~ ~mln~ by non-com-
pemm RT-PCRrewm uwegum~n of mRNA by ~:qX~m~mride (LPS), S..1, T N F . ~ ~ o~m s u m t m ~ , and mh~Son by Ommmma-
sone. In se~0fi lnt ~ of the IL-3 receptor, the rnudne sec~enee of the 8..sUbun~(NC2A; Wlm0 et M., 19~) wm u x d to dev~e a nundmr of oligonuctso~M [~mem. WMho~s l~dr of prlmem, a 1.25 kb c0NA Iragment was ampgl~d, clormcl and UeClUmCe~. B a x d on thi~ ~ClUence, new wimers we,e ~lectad m~l ua~l In ceml~mtlon wi~ the ~ (6gBCO4gRL) to ~ mere ~ ~ in the 5"-fllrtt@~ region. Cltm'actecis~ f m t t u s w~ be ~ in o0ml~flsen w ~ mudne and htxnan sequemm. S~n~ ~ e-~tum laem tn~ns~ tyrmm tdrme ffK) a c ~ Im~ I ~ a ~ e s recql~m'-mmnc,~ed TKs JAK1,2 or I"YK2 (M011nt et al., 19~). # wire p(mMIm to ~ by RT-PCR s~'~ch~ of 2.7 and 2.1 kb of JAK 1 and 2 cONAs, ~ , from totul r~ m ~ RNA. Seque~Sme~mmdmr ~ ~ ~mamm of comew~on mtween sPecmso~rfe@@W~n~ ~ TI~ W@-w~mty o~ m~mmmmlR~l~g IL-3 an0 t.P8 ~n)u~W way anamWlm wll Im pmmnmd. Bern" !nMol'm Imo molemW rNmmlmm of cymkme m ~mm,m~n In m l m ~ can
G e l ~ ( : k e - ~ et ~1., !9~4, J . N ~ . 50, 203-214. Wang et M., 19e2, J.Biol.Ghem. 267, 979-983. MOlkwet a1.,1993, NaCre 366, 129-135.
TREATMENTOF EAE WITH BONE MARROWTRANSPLANTATION M.v~ C ~ and D.W.vanBekkum Biomed~celPrimateRescmchCenterTNO, Rijswij'k,The Netherlands We investigatedthe effect of bone marrowtmnsplm~afion(BMT) on the com'seof relapsing EAE--indnced by immunization with spinal cord homogenate and complemadjuvant--in tim Blip rat and tim Binzzi mouse. Animals west Izestedwi~ mini body ~ (TBI) mgl syngmalc or pscudoautologons (S)BMT. after octtmt of paresis. This treatment indnced complete remission in all aninm~ only 30% developed a spontlmeees relapse vs.IO0% in untreated animals.The incidanceu d onset o( relgpeeswm rehited to tbe degree of 1 ~ abiaina indncedby tha coml~iamn'g. Purthanxx~ me incidence of Immsis afterreimmmi,~n was comimm~ reduced in SBM-tremplaated rats (not ascribable to post BMT i m ~ y ) . It was shown immmmtdslochmnicagy that.donor-daiv~l(F1) inonan~ celk imkm:ed the ~ afterndmmunizafion. Aliogemcic BM'r from resistant d ¢ ~ . eithm"complete MHC mis-maghed or partially matched, resulted in significantly mdnced ~ rates comlmred with SBMT. However, remisalon couM only be iagnced befme extcesive gliceis and demyalimUim Imd develol~xlin tbe CNS. Oer data indices tl~ earn afterallogeaeic BMT can be attributed to 1. a ~abelinic4tlOngt-vemm~Hcet-Rca~Iionand 2. the newlydeveloped,EAE-tesistsntiramune-bemopoieticsystmaof tbe doso¢ strain. In conclusion, extensive lympho-ablafion and autOlOgOmBMT might be an effectivetreatmentof severeMS, whm mmbined with ~ tolmzing Ueatment as used in mmsplmtagonmedlgine.AliogeaalcBMT is a goed alternative,but only if clinicalGvHDis properlyixevented.33eammntshould be appliedbefore extensive irreversibledamageto the CNS has developed.