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Arthritis susceptibility in mice transgenic for human type II collagen
320
Methods: IIF test, DD, CIE, immunoblotting, phenol extraction, following proteinase K digestion for isolation of DNA and Dot-blot hybridization. For statistical analysis x2 and 4 Yule’s tests were used. Ruultaz The strong, positive association between the presence of autoantibodies against dsDNA, ssDNA, Sm and (Ul) RNP, and DNA sequences homologous to gag-pol region of HIV-1 was observed. Conoluskm: It is possible, that sequences homologous to gag-pol region of HIV-1 within DNA isolated from patients with CTD may became antigenic or can induce synthesis of antibodies, which recognize dsDNA or ssDNA, and may also induce production of autoantibodies, which recognize Sm and (Ul) RNP antigens but not Ro, La, ACA, antinucleolar antigens or M-2.
(1 -
Experimental autoimmune myocarditls: Hlktologlcal and immunohlstbchemlcal characteristics
M. Milenkovic', P. Milosavijevic *, M. Colic2, M. Dimitrijevic’. 1Department of Micmbiolcgy and Immunology Faculty of Pharmacy Belgrade, Vugaslavia, 2It&We
24 June 1997 - Posterpresentations
Pathogenesis of autoimmunity
of Medical Research, MMA, Belgrade, Yugoslavia
Introduction: Autoimmune myccarditis is a disease of unknown ethiology and not well understood pathogenetic mechanisms. An experimental disease model has been developed to induce autoimmune myocarditis in genetically susceptible DA rats with the aim to investigate immunohistological changes in the course of the disease. Materials and Methods: Male DA rats were received porcine cardiac myosin (5 m&g body weight) in complete Freunds adjuvant (FA). Control animals were immunised with complete FAalone. Rats were sacrificed 8th. Wth, 21st and 34th day after the disease induction. lmmunopathological changes of the heart tissue and phenotype of infiltrating cells were examined using immunoperoxidase and immunoalcaline phosphatase methods and monoclonal antibodies specific for TcR@ (R73). CD4 (W3/25), CD8 (0X8), CDlla (WTI), CDllb (0X42), CDlld (R-MC42), CD18 (WT3), CD64 (lAZ9), MHC class II (0X6), pan macrophage (M+) marker (EDl), marker of a subset of M4 (ED2), and marker of granulocytes and a subset of M4 (R-MC46). Results: Macroscopically, the hearts were remarkably enlarged, both ventricles were dilated and cardiac surface exhibited diffuse dlscoloured areas. Inflammatory infiltrates were composed of mononuclear cells, giant cells and rare granulocytes (Gr). On day 8, an increase in number of interstitial dendritic cells (0X6+) was observed, but infiltrates were not yet organised. On day 16, cell infiltrates were numerous. Whin them, predominant cells were dendrttic cells (0X8+), inflammatory Mg (EDl+), and T lymphocytes (R73+) of both CD4 and CD8 phenotypes. CD4 was also expressed on dendritic cells. Multinucleated giant cells were EDl+ED2- and appeared In the centres of inflammatory lesions. Mononuclear cells were mostly LFA-1 (CDlla/CD18)*, particularly those in the centres of infiltrates. Inflammatory Md (R-MC46+) coexpressed ICAM(CD54), a molecule that was also de&ted .on endothel/um. dn day 21, an increased number of residual W (ED1+R-MC42+) was observed at the periphery of infiltraties. Thirty four days aft& the disease induction. inflammatory infiltrates were reduced. Conclusions: Our results suggest that detailed immunohistochemicalanalysis of cell populations within inflammatory infiltrates might contribute to the better understandlng of imunopathogenesis in autoimmune myocarditis. It seems that interstitial dendritic cells are of the importance in initiation of the disease.
P.5.11.60
Arthrltls susceptibility in mice transgenlc for human type II collagen
Vivianne Malmstrbm, Kathryn SE. Cheah ‘, Rikard Holmdahl. De@of Cell & Molecular Biolog)! Section br Medical lnflemmation Research, Lund University.Sweden, ’ Biochemistry Dept, Hong Kong Univetsi& Hong Kong Intmductlon: Type II collagen (Cll) is a cartilage-specific protein which can induce severe arthritis in mice after intradennal immunization. This model (CIA) is used for studying rheumatoid arthritis (RA) especially the interactions between the immune system and cartilage components. In humans RA is associated to HLA-DR4 and in mice CIA is associated to H-2q. Interestingly, these MHC molecules are similar and they bind the same immunodominant CII peptide. Materlals and Methods: H-2q mice with transgenic expression of human CII were established and the susceptibility for arthritis after immunization with human CII was investigated. Results: Cartilage-specific expression of human CII in mice lead to a high degree of tolerance, but still 10% of the mice developed severe arthritis. Also transgenic mice mounted measurable B- and T-cell responses to human CII. Conclusions: Autoimmune reactions to human CII can take place and we now have a system that will help us understand the initiating events in ClA/RA and dissect immune tolerance.
(p.5.11.611 Selective
afflnlty of blndlng of GAD peptldes by I-Ag7 and I-Ed: Susceptlblllty to autolmmune dlabetes In the former and resistance to it In the latter
G.K. Papadopoulos. Laboratory of lmmunol~, L7eparttnentof Internal Medicine, Univemity of loannina Medical School, GR-451 10 loannina, Greece Introduction: The diabetes-susceptible non-obese diabetic (NOD) mice, contain in their genome at least 15 loci linked to the disease. The strongest linkage concerns kktml, that has been mapped to the unique MHC class II allele of NOD, I-Ag’. The aim of this research was to study the physicochemical basis of the antigen-binding properties of I-A’J’ and the transgenically protective I-Ed allele. Methods and Mat~~lals: Mdecular simulations of the structures of I-Ag7 and I-Ed were canied out using the known structure of the human MHC class II allele DRl as a starting point. Reaula: The modelled structure of I-Ag7is very much likethat of other known MHC dass II proteins. All polar residues that point towards the antigen-binding groove and hold antigenic peptide non-specifically are present with one semiconservative substitution (BGlTrp+Tyr). The presence of a His-Ser dipeptide in 856-57, instead of the Pro-Asp shown by most mouse MHC class II proteins gives extra positive charge to the amino-terminus of the j1J1helix. There is thus considerable repuslion between this side and the apposed a8OArg, a residue conserved in all species. This setting is probabiy responsible for the weak affinity to all antigenic peptides shown by I-AQ’. By contrast, I-Ed and all other mouse MHC molecules show an attractive force at this end of the molecule between ,z6OArgand fl57Asp, that is also essential in holding the antigenic peptide by forming with it two hydrogen bonds. In IAg7 we can distinguish the fiie anchor positions (pl, p4,p6, p7, p9), of which the most prominent are p4, p6 and p9. The latter favours the anchoring of negatively charged residues, aspartate and glutamate. Based on the physicochemical properties of the various pockets as seen by the modelling, we have determined the antigsnic epitopes of the autoantigenic peptides p34, ~35, ~36 and ~17, from glutamic add decarboxylase, for I-AU’. Each of these peptides also contains at least one motif of l-Ed, which by all indications fit quite well in the groove. Conclusion: Our results are consistent with the determinant capture model of protection and susceptibility to autoimmunity by various MHC class II alleles.
P.5.11.61
Altered apoptosls and lymphocyte subpopulatlons In SLE patients
G. Lakos, E. Gytmesi, P. Tatjan’. I. Sonkoly ‘, E. Kiss, I. Kovacs, S. Sipka, Gy. Szegedi. U&e&y Medical School of Deixecen, Hunga% ’ Hefenyi Geza CountyHospital, Hungary Introduction: It has been shown, that autoimmune phenomena might be caused by a failure in the elimination of autoreactive lymphocytes. Defective regulation of programmed cell death (apoptosis) may play a role in the development of autoimmune diseases. Three independent mutations involving the Apo-Was receptor/CD95 or its ligand have led to lupuslike diseases in different strains of mice. To evaluate the putative imbalance between the production and destruction of various types of immunocompetent cells, we examined the distribution of lymphocyte subpopulations, the expression of Fas receptor and lymphocyte apoptosis in systemic lupus erythematosus (SLE) patients. Matsrlals and Methods: CD3,CD4, CD8, CD19 and CD56 expression was evaluated by immunfluorescence analysis on peripheral blood lymphocytes (PBL) of 30 SLE patients and 21 healthy controls. In vitro apoptosis of cultured PBLs from 10 patients and 10 controls was determined at 0, 24, and 72 hours of culture by propidium iodide staining and flow cytometty. PBLs were cultured either in RPM1medium alone, or with pokeweed-mitogen at a final concentration of 20 &ml. CD95 (Fas) expression was measured by two colour immunfluorescence analysis at 0, 24 and 72 hours of culture on CD4, CD8 and CD19 positive cells, as well as apoptosis was investigated in these cell populations by combining immunophenotyping and propidium iodide staining in 6 patients. Results: The CD4/CD8 ratio was decreased in SLE patients: 1.38 +/0.81, compared to the controls: 1.76 +/- 0.63. (p < 0.05) 12 out of the 30 patients but no one out of the controls had CD4ICD8 ratio -=I= 1. Moreover, patients had significantly less CD19 positive cells, than the controls (p = 0.001). In vitro apoptosis was accelerated in patients at 24 and 72 hours of culture, but the difference did not reach the significant level. CD95 expression was found to be moderately elevated on unfractionated PBLs of patients, mainly on stimulated cells. However, CD95 expression on CD4 posit&e cells was significantly higher at any time point, both on stimulated and unstlmulated cells. This finding correlated with the higher apoptosis rate of CD4 positive cells. There was no significant difference in Fas expression and apoptosis concerning the CD8 and CD19 positive cells. Conclusion: There is an alteration in the distribution of lymphocyte subpopulations in SLE patients, which may reflect an imbalance between the production and elimination of cells. On the basis of our results, dysregulation of programmed cell death may contribute to the detected alterations. Elevated CD95 expression
Methods: IIF test, DD, CIE, immunoblotting, phenol extraction, following proteinase K digestion for isolation of DNA and Dot-blot hybridization. For statistical analysis x2 and 4 Yule’s tests were used. Ruultaz The strong, positive association between the presence of autoantibodies against dsDNA, ssDNA, Sm and (Ul) RNP, and DNA sequences homologous to gag-pol region of HIV-1 was observed. Conoluskm: It is possible, that sequences homologous to gag-pol region of HIV-1 within DNA isolated from patients with CTD may became antigenic or can induce synthesis of antibodies, which recognize dsDNA or ssDNA, and may also induce production of autoantibodies, which recognize Sm and (Ul) RNP antigens but not Ro, La, ACA, antinucleolar antigens or M-2.
(1 -
Experimental autoimmune myocarditls: Hlktologlcal and immunohlstbchemlcal characteristics
M. Milenkovic', P. Milosavijevic *, M. Colic2, M. Dimitrijevic’. 1Department of Micmbiolcgy and Immunology Faculty of Pharmacy Belgrade, Vugaslavia, 2It&We
24 June 1997 - Posterpresentations
Pathogenesis of autoimmunity
of Medical Research, MMA, Belgrade, Yugoslavia
Introduction: Autoimmune myccarditis is a disease of unknown ethiology and not well understood pathogenetic mechanisms. An experimental disease model has been developed to induce autoimmune myocarditis in genetically susceptible DA rats with the aim to investigate immunohistological changes in the course of the disease. Materials and Methods: Male DA rats were received porcine cardiac myosin (5 m&g body weight) in complete Freunds adjuvant (FA). Control animals were immunised with complete FAalone. Rats were sacrificed 8th. Wth, 21st and 34th day after the disease induction. lmmunopathological changes of the heart tissue and phenotype of infiltrating cells were examined using immunoperoxidase and immunoalcaline phosphatase methods and monoclonal antibodies specific for TcR@ (R73). CD4 (W3/25), CD8 (0X8), CDlla (WTI), CDllb (0X42), CDlld (R-MC42), CD18 (WT3), CD64 (lAZ9), MHC class II (0X6), pan macrophage (M+) marker (EDl), marker of a subset of M4 (ED2), and marker of granulocytes and a subset of M4 (R-MC46). Results: Macroscopically, the hearts were remarkably enlarged, both ventricles were dilated and cardiac surface exhibited diffuse dlscoloured areas. Inflammatory infiltrates were composed of mononuclear cells, giant cells and rare granulocytes (Gr). On day 8, an increase in number of interstitial dendritic cells (0X6+) was observed, but infiltrates were not yet organised. On day 16, cell infiltrates were numerous. Whin them, predominant cells were dendrttic cells (0X8+), inflammatory Mg (EDl+), and T lymphocytes (R73+) of both CD4 and CD8 phenotypes. CD4 was also expressed on dendritic cells. Multinucleated giant cells were EDl+ED2- and appeared In the centres of inflammatory lesions. Mononuclear cells were mostly LFA-1 (CDlla/CD18)*, particularly those in the centres of infiltrates. Inflammatory Md (R-MC46+) coexpressed ICAM(CD54), a molecule that was also de&ted .on endothel/um. dn day 21, an increased number of residual W (ED1+R-MC42+) was observed at the periphery of infiltraties. Thirty four days aft& the disease induction. inflammatory infiltrates were reduced. Conclusions: Our results suggest that detailed immunohistochemicalanalysis of cell populations within inflammatory infiltrates might contribute to the better understandlng of imunopathogenesis in autoimmune myocarditis. It seems that interstitial dendritic cells are of the importance in initiation of the disease.
P.5.11.60
Arthrltls susceptibility in mice transgenlc for human type II collagen
Vivianne Malmstrbm, Kathryn SE. Cheah ‘, Rikard Holmdahl. De@of Cell & Molecular Biolog)! Section br Medical lnflemmation Research, Lund University.Sweden, ’ Biochemistry Dept, Hong Kong Univetsi& Hong Kong Intmductlon: Type II collagen (Cll) is a cartilage-specific protein which can induce severe arthritis in mice after intradennal immunization. This model (CIA) is used for studying rheumatoid arthritis (RA) especially the interactions between the immune system and cartilage components. In humans RA is associated to HLA-DR4 and in mice CIA is associated to H-2q. Interestingly, these MHC molecules are similar and they bind the same immunodominant CII peptide. Materlals and Methods: H-2q mice with transgenic expression of human CII were established and the susceptibility for arthritis after immunization with human CII was investigated. Results: Cartilage-specific expression of human CII in mice lead to a high degree of tolerance, but still 10% of the mice developed severe arthritis. Also transgenic mice mounted measurable B- and T-cell responses to human CII. Conclusions: Autoimmune reactions to human CII can take place and we now have a system that will help us understand the initiating events in ClA/RA and dissect immune tolerance.
(p.5.11.611 Selective
afflnlty of blndlng of GAD peptldes by I-Ag7 and I-Ed: Susceptlblllty to autolmmune dlabetes In the former and resistance to it In the latter
G.K. Papadopoulos. Laboratory of lmmunol~, L7eparttnentof Internal Medicine, Univemity of loannina Medical School, GR-451 10 loannina, Greece Introduction: The diabetes-susceptible non-obese diabetic (NOD) mice, contain in their genome at least 15 loci linked to the disease. The strongest linkage concerns kktml, that has been mapped to the unique MHC class II allele of NOD, I-Ag’. The aim of this research was to study the physicochemical basis of the antigen-binding properties of I-A’J’ and the transgenically protective I-Ed allele. Methods and Mat~~lals: Mdecular simulations of the structures of I-Ag7 and I-Ed were canied out using the known structure of the human MHC class II allele DRl as a starting point. Reaula: The modelled structure of I-Ag7is very much likethat of other known MHC dass II proteins. All polar residues that point towards the antigen-binding groove and hold antigenic peptide non-specifically are present with one semiconservative substitution (BGlTrp+Tyr). The presence of a His-Ser dipeptide in 856-57, instead of the Pro-Asp shown by most mouse MHC class II proteins gives extra positive charge to the amino-terminus of the j1J1helix. There is thus considerable repuslion between this side and the apposed a8OArg, a residue conserved in all species. This setting is probabiy responsible for the weak affinity to all antigenic peptides shown by I-AQ’. By contrast, I-Ed and all other mouse MHC molecules show an attractive force at this end of the molecule between ,z6OArgand fl57Asp, that is also essential in holding the antigenic peptide by forming with it two hydrogen bonds. In IAg7 we can distinguish the fiie anchor positions (pl, p4,p6, p7, p9), of which the most prominent are p4, p6 and p9. The latter favours the anchoring of negatively charged residues, aspartate and glutamate. Based on the physicochemical properties of the various pockets as seen by the modelling, we have determined the antigsnic epitopes of the autoantigenic peptides p34, ~35, ~36 and ~17, from glutamic add decarboxylase, for I-AU’. Each of these peptides also contains at least one motif of l-Ed, which by all indications fit quite well in the groove. Conclusion: Our results are consistent with the determinant capture model of protection and susceptibility to autoimmunity by various MHC class II alleles.
P.5.11.61
Altered apoptosls and lymphocyte subpopulatlons In SLE patients
G. Lakos, E. Gytmesi, P. Tatjan’. I. Sonkoly ‘, E. Kiss, I. Kovacs, S. Sipka, Gy. Szegedi. U&e&y Medical School of Deixecen, Hunga% ’ Hefenyi Geza CountyHospital, Hungary Introduction: It has been shown, that autoimmune phenomena might be caused by a failure in the elimination of autoreactive lymphocytes. Defective regulation of programmed cell death (apoptosis) may play a role in the development of autoimmune diseases. Three independent mutations involving the Apo-Was receptor/CD95 or its ligand have led to lupuslike diseases in different strains of mice. To evaluate the putative imbalance between the production and destruction of various types of immunocompetent cells, we examined the distribution of lymphocyte subpopulations, the expression of Fas receptor and lymphocyte apoptosis in systemic lupus erythematosus (SLE) patients. Matsrlals and Methods: CD3,CD4, CD8, CD19 and CD56 expression was evaluated by immunfluorescence analysis on peripheral blood lymphocytes (PBL) of 30 SLE patients and 21 healthy controls. In vitro apoptosis of cultured PBLs from 10 patients and 10 controls was determined at 0, 24, and 72 hours of culture by propidium iodide staining and flow cytometty. PBLs were cultured either in RPM1medium alone, or with pokeweed-mitogen at a final concentration of 20 &ml. CD95 (Fas) expression was measured by two colour immunfluorescence analysis at 0, 24 and 72 hours of culture on CD4, CD8 and CD19 positive cells, as well as apoptosis was investigated in these cell populations by combining immunophenotyping and propidium iodide staining in 6 patients. Results: The CD4/CD8 ratio was decreased in SLE patients: 1.38 +/0.81, compared to the controls: 1.76 +/- 0.63. (p < 0.05) 12 out of the 30 patients but no one out of the controls had CD4ICD8 ratio -=I= 1. Moreover, patients had significantly less CD19 positive cells, than the controls (p = 0.001). In vitro apoptosis was accelerated in patients at 24 and 72 hours of culture, but the difference did not reach the significant level. CD95 expression was found to be moderately elevated on unfractionated PBLs of patients, mainly on stimulated cells. However, CD95 expression on CD4 posit&e cells was significantly higher at any time point, both on stimulated and unstlmulated cells. This finding correlated with the higher apoptosis rate of CD4 positive cells. There was no significant difference in Fas expression and apoptosis concerning the CD8 and CD19 positive cells. Conclusion: There is an alteration in the distribution of lymphocyte subpopulations in SLE patients, which may reflect an imbalance between the production and elimination of cells. On the basis of our results, dysregulation of programmed cell death may contribute to the detected alterations. Elevated CD95 expression