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reoxygenation
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071- ACTIVATION OF COMPLEMENT BY APOPTOTIC ENDOTHELIAL CELLS FOLLOWING HYPOXIA/ REOXYGENATION. By C. Mold and CA. Morris, Dept. of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM, 87131. Reperfusion of ischemic tissue initiates an inflammatory reaction that induces tissue injury. This study investigated the mechanism of complement activation following 1reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfusion injury. HUVEC culmred in 1% oxygen followed by reoxygenation activated the classical pathway measured by C3 deposition. An increase in apoptotic cells was observed in these cultures by staining for annexin V binding and hypodiploid nuclei. To determine if these cells were responsible for the complement
activation, apoptotic cells were produced by growth factor deprivation. Apoptotic but not control HUVEC directly activated the classical pathway using purified components in the absence of antibody or other serum factors. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, the complement activation observed following reoxygenation of HUVEC was
eliminated by treatment of the cultures with a caspase inhibitor. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis. Apoptotic cells directly activate the classical pathway. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.
072- TISSUE EXPRESSION PATTERN OF THE MOUSE GPI-DAFAND ITS POTENTIAL ROLE IN A MIJRINE MODEL OF AUTOIMMUNE DISEASE Miwa T’, Maldonado M2, Sun
X’, Ohta R’, Okada H’, Harris C’, Morgan BP’, Eisenberg, RA2, and Song WC’ ‘Ctr Exp Therap and Dept Pharmacol, and biv. Rheumatol, Univ. Pennsylvania Sch Med, Philadelphia, PA 19104; ‘Dept Mol Biol Nagoya City Univ, Sch Med, Nagoya, Japan; ‘Dept Med Biochem, Univ of Wales Coil Med, Cardiff, Wales, UK Decay-accelerating factor (DAF) is a central membrane complement regulator. Two daf genes are present in the moose. One daf gene encodes a GPI-linked DAF (GPI-DAF) while the other makes predominantly a trammembrane form of DAF (TM-DAF). Using a panel of independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TMDAF, we have examined the relative expression levels of the two DAF proteins in the wild-type and GPI-daf knockout mice. We found that GPI-DAF but not TM-DAF is expressed on the mouse erythrocytes and leukocytes. Both T and B lymphocytes and splenic macrophages express GPI-DAF hut the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4 and CD8 T cells are positive. Both GPI-DAF and TM-DAF are expressed on mature sperm but GPI-DAF appears to be the predominant form in late-stage spetmatocytes. Recent in vitrostudies suggested that DAF is more active than another marine membrane complement regulator, Cny, in regulating the classical pathway of complement. To explore the role of GPI-DAF in antibody-mediated systemic autoimmune disease, we have crossed the GPI-DAF knockout mouse with the MRL/&r mouse, a model for SLE, for 4 generations. Wild-type, heterozygous and DAF knockout mice (>90% MRL&r) have been obtained and are being evaluated for development of the systemic autoimmune syndrome (dermatitis, nephritis and autoantibody production). This study should reveal what role DAF plays in this model of systemic autoimmunity, either as a complement regulator or in its capacity as a ligand for the newly discovered lymphocyte activation-associated antigen CD97.
073- MOLECULAR COMPLETE ClS WITH MULTIPLE M. EARLY ONSET.
BASIS OF A SELECTIVE AND DEFICIENCY ASSOCIATED AUTOIMMUNE DISEASES OF
A. Dragon-Durey, P. Quartier, V. FrCmeaux-Bacchi, J. Blouin, C. de Barace, A. Prieur, L. Weiss and W.H. Fridman. Service d’Immunologie and INSERM U430, HBpital Broussais. Paris, France. Unit6 d’Immunologie -h&matologiep&liatrique, H8pital Necker. Paris France. We have investigated the molecular basis of a selective and complete Cls deficiency in a little girl presenting with multiple autoimmune diseases including lupus-like syndrome, Hashimoto’s thyroiditis and autoimmune hepatitis initiated in early childhood. The complement profile of the patient was characterized by the lack of CH50 activity, Cl titnctional activity below 10%. undetectable levels of Cls antigen associated with normal levels of Clr and Clq antigens. Using exon specific amplification of genotnic DNA by Polymerase Chain Reaction
followed by direct sequence analysis, we identified in the genomic DNA a homozygous tmnsense mutation in the Cls gene’s excm XR at codon 534. It was caused by a nucleotide substitution from C EGA for Arginine) to T @-GA for stop cc&n). Both parents were found to be heterozygous for this mutation. We used the opportunity that the mutation created a new restriction site for the endonuclease Fok-I to detect the mutation in the genomic DNA of seven healthy family members. Four addi&nal heterozygotes for the mutation were identified in two generations. Our data characterizes for the first time the genetic defect of a selective and complete Cls deficiency in a Caucasien patient. This report underlines that. in addition to Clq deficiency, Cls deticiency is
strongly associated with autoimmune disease
074- A CJA-RECmOR DEFlmlVE GUINEA PIG STRAIN IN EXHIBITS DECREASED BRONCHIAL REACTMTY
AN OVALBUMIN-IMIUCED ASTEMA MODEL M. Grove, ‘FL G. HoI. Meyer-Wiedenbach, Q. i!htmg, U. Raschke,N.Flemne,‘AUM~zuVilsendorWA.Klos,J. K&l, W. Bautsch; hutit& of Medical Miaotiology and *Dept. of Transplantaticn Surgery, Medical School, Harmova, Germany, ‘Fraoohofer hs!itue, Harmover, Germany ASthtUiSiiUdl~giCrep~Olltowards8Uinhaledpntigen, CbanlcterizedbyasIKmgbral&d~Gnandedema
fomlatial v&l s&qbEnt cell iluiktimi itIt0 the hmg pare&ma and alveoli, mainly lymphocytes and eosiriophils. Althcugh IgE-mediated ktamiue release is generally regarded as the major patho&siological pathway, other non-IgE mediated mebnismsalsoccutfn%utetothedisease.Amajorcandidstein thatreqeUistheC3aat4aphylatoxiqapotstdmm~aud adivatorofvariollscelItypes,iu&dingmacropbageSmastcells andeosillopllils,whi&isgenemtedbyitmmme almpkx-mediated activatim of the camplanent system. It actes via the C&mceptor, ~G-poteinooopledreeeptorthatbasbeenclonedfiomman, mouse,rstandguineapig.Bssedonthatsequeatce,weamplitied the C38-receptor coding sequence fKen the guinea pig strain CZBBiR- an inbred strain long Imown for a fimctioosl defect in the C3afC3a-w system. DNA sequence ennlysis revealed the prcsenceofapointmutati~with.inthecodingsequtncocreat@a ~stopcodonthatinacaivatestheCk-receptmThis.stmin was used to anslyu: the c4mttiLmtia1 of C3a to the early bmnch~dion iu an auimsl model of asthma. Immunized gliineapigsweretmatedwitht&eant&isl ‘_ pyl&m&aIld challwed with ovaIhumin by i&al&m. A significantly decmas&bNm~reactivity0falmos&30%was~~iuthe c3a-reeeptor defeuive stmiu czBB/R- when compared wirh strain C2BB/R+ harbcu@ a wiI&ype C3a-receptor. Fmther data will be pmsmted at themeeting.
071- ACTIVATION OF COMPLEMENT BY APOPTOTIC ENDOTHELIAL CELLS FOLLOWING HYPOXIA/ REOXYGENATION. By C. Mold and CA. Morris, Dept. of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM, 87131. Reperfusion of ischemic tissue initiates an inflammatory reaction that induces tissue injury. This study investigated the mechanism of complement activation following 1reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfusion injury. HUVEC culmred in 1% oxygen followed by reoxygenation activated the classical pathway measured by C3 deposition. An increase in apoptotic cells was observed in these cultures by staining for annexin V binding and hypodiploid nuclei. To determine if these cells were responsible for the complement
activation, apoptotic cells were produced by growth factor deprivation. Apoptotic but not control HUVEC directly activated the classical pathway using purified components in the absence of antibody or other serum factors. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, the complement activation observed following reoxygenation of HUVEC was
eliminated by treatment of the cultures with a caspase inhibitor. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis. Apoptotic cells directly activate the classical pathway. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.
072- TISSUE EXPRESSION PATTERN OF THE MOUSE GPI-DAFAND ITS POTENTIAL ROLE IN A MIJRINE MODEL OF AUTOIMMUNE DISEASE Miwa T’, Maldonado M2, Sun
X’, Ohta R’, Okada H’, Harris C’, Morgan BP’, Eisenberg, RA2, and Song WC’ ‘Ctr Exp Therap and Dept Pharmacol, and biv. Rheumatol, Univ. Pennsylvania Sch Med, Philadelphia, PA 19104; ‘Dept Mol Biol Nagoya City Univ, Sch Med, Nagoya, Japan; ‘Dept Med Biochem, Univ of Wales Coil Med, Cardiff, Wales, UK Decay-accelerating factor (DAF) is a central membrane complement regulator. Two daf genes are present in the moose. One daf gene encodes a GPI-linked DAF (GPI-DAF) while the other makes predominantly a trammembrane form of DAF (TM-DAF). Using a panel of independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TMDAF, we have examined the relative expression levels of the two DAF proteins in the wild-type and GPI-daf knockout mice. We found that GPI-DAF but not TM-DAF is expressed on the mouse erythrocytes and leukocytes. Both T and B lymphocytes and splenic macrophages express GPI-DAF hut the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4 and CD8 T cells are positive. Both GPI-DAF and TM-DAF are expressed on mature sperm but GPI-DAF appears to be the predominant form in late-stage spetmatocytes. Recent in vitrostudies suggested that DAF is more active than another marine membrane complement regulator, Cny, in regulating the classical pathway of complement. To explore the role of GPI-DAF in antibody-mediated systemic autoimmune disease, we have crossed the GPI-DAF knockout mouse with the MRL/&r mouse, a model for SLE, for 4 generations. Wild-type, heterozygous and DAF knockout mice (>90% MRL&r) have been obtained and are being evaluated for development of the systemic autoimmune syndrome (dermatitis, nephritis and autoantibody production). This study should reveal what role DAF plays in this model of systemic autoimmunity, either as a complement regulator or in its capacity as a ligand for the newly discovered lymphocyte activation-associated antigen CD97.
073- MOLECULAR COMPLETE ClS WITH MULTIPLE M. EARLY ONSET.
BASIS OF A SELECTIVE AND DEFICIENCY ASSOCIATED AUTOIMMUNE DISEASES OF
A. Dragon-Durey, P. Quartier, V. FrCmeaux-Bacchi, J. Blouin, C. de Barace, A. Prieur, L. Weiss and W.H. Fridman. Service d’Immunologie and INSERM U430, HBpital Broussais. Paris, France. Unit6 d’Immunologie -h&matologiep&liatrique, H8pital Necker. Paris France. We have investigated the molecular basis of a selective and complete Cls deficiency in a little girl presenting with multiple autoimmune diseases including lupus-like syndrome, Hashimoto’s thyroiditis and autoimmune hepatitis initiated in early childhood. The complement profile of the patient was characterized by the lack of CH50 activity, Cl titnctional activity below 10%. undetectable levels of Cls antigen associated with normal levels of Clr and Clq antigens. Using exon specific amplification of genotnic DNA by Polymerase Chain Reaction
followed by direct sequence analysis, we identified in the genomic DNA a homozygous tmnsense mutation in the Cls gene’s excm XR at codon 534. It was caused by a nucleotide substitution from C EGA for Arginine) to T @-GA for stop cc&n). Both parents were found to be heterozygous for this mutation. We used the opportunity that the mutation created a new restriction site for the endonuclease Fok-I to detect the mutation in the genomic DNA of seven healthy family members. Four addi&nal heterozygotes for the mutation were identified in two generations. Our data characterizes for the first time the genetic defect of a selective and complete Cls deficiency in a Caucasien patient. This report underlines that. in addition to Clq deficiency, Cls deticiency is
strongly associated with autoimmune disease
074- A CJA-RECmOR DEFlmlVE GUINEA PIG STRAIN IN EXHIBITS DECREASED BRONCHIAL REACTMTY
AN OVALBUMIN-IMIUCED ASTEMA MODEL M. Grove, ‘FL G. HoI. Meyer-Wiedenbach, Q. i!htmg, U. Raschke,N.Flemne,‘AUM~zuVilsendorWA.Klos,J. K&l, W. Bautsch; hutit& of Medical Miaotiology and *Dept. of Transplantaticn Surgery, Medical School, Harmova, Germany, ‘Fraoohofer hs!itue, Harmover, Germany ASthtUiSiiUdl~giCrep~Olltowards8Uinhaledpntigen, CbanlcterizedbyasIKmgbral&d~Gnandedema
fomlatial v&l s&qbEnt cell iluiktimi itIt0 the hmg pare&ma and alveoli, mainly lymphocytes and eosiriophils. Althcugh IgE-mediated ktamiue release is generally regarded as the major patho&siological pathway, other non-IgE mediated mebnismsalsoccutfn%utetothedisease.Amajorcandidstein thatreqeUistheC3aat4aphylatoxiqapotstdmm~aud adivatorofvariollscelItypes,iu&dingmacropbageSmastcells andeosillopllils,whi&isgenemtedbyitmmme almpkx-mediated activatim of the camplanent system. It actes via the C&mceptor, ~G-poteinooopledreeeptorthatbasbeenclonedfiomman, mouse,rstandguineapig.Bssedonthatsequeatce,weamplitied the C38-receptor coding sequence fKen the guinea pig strain CZBBiR- an inbred strain long Imown for a fimctioosl defect in the C3afC3a-w system. DNA sequence ennlysis revealed the prcsenceofapointmutati~with.inthecodingsequtncocreat@a ~stopcodonthatinacaivatestheCk-receptmThis.stmin was used to anslyu: the c4mttiLmtia1 of C3a to the early bmnch~dion iu an auimsl model of asthma. Immunized gliineapigsweretmatedwitht&eant&isl ‘_ pyl&m&aIld challwed with ovaIhumin by i&al&m. A significantly decmas&bNm~reactivity0falmos&30%was~~iuthe c3a-reeeptor defeuive stmiu czBB/R- when compared wirh strain C2BB/R+ harbcu@ a wiI&ype C3a-receptor. Fmther data will be pmsmted at themeeting.