- Email: [email protected]
Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity
043- Clq
DEFICIENCY AND AUTOIMMUNITY: THE EFFECTS OF GENETIC BACKGROUND ON DISEASE EXPRESSION. Mitchell DA, Cook HT. Botto M, Walport MJ. Hammersmith Campus, ICSM, London, UK. Gene-targeted Clq-deficient mice, in the hybrid (129 x C57BLf6) genetic background, have been shown to develop spontaneously antinuclear antibodies and glomerulonephritis associated with multiple apoptotic bodies. The genetic background was believed to play an important role in disease expression since a small proportion of strain-matched control mice developed autoimmunity also. In order to investigate the influence of genetic background on disease expression in mutine Clq deficiency, the disrupted Clqa gene has been backcrossed for 7 generations onto the following inbred strains: C57BU6, 1291% and the Fasdeficient MRL/Mp.lprNpr and C57BW6.lpr/lpr strains. At 12 months of age, no serological or histological evidence of disease was found in Clq-deficient mice in the 129/Sv or C57BU6 strains, Analysis of Clq deficiency in the MRLMp.lprNpr strain showed no significant modification of disease onset or expression between Clq-l- and MRL&Ip.lprNpr wild-type mice at any of the 6, 9 or 12-week time points analysed. Similar findings were obtained from the analysis of Clq-deficient C57BL/6,lpr/lpr mice. However, significantly greater numbers of glomerular apoptotic cells were counted in the kidneys of Clq-deficient C57BW6.lpr/lpr than in wild-type C57BIJ6.IpwJpr mice. These findings highlight the critical significance of genetic background when analysing autoimmune phenotype in gene-targeted mice. In addition, this study fails to show an involvement of the classical pathway in regulating tolerance to nuclear antigens in IprApr mice, which has previously been reported in C4-deficient animals.
045-
Both I& and IgM anti-pig antibodies induce complement activation and cytotoxicity
Anja Roes, Maria Essers, DaniBlle J. van Gijlswijk-Janssen. Nicolai V. Bovin’, and Mohatned R. Daha, Deparrment of Nephrology, L&den University Medical Center, Leiden, the Netherlands, ’Shemyakin Institute ofRioorganic Chemistry Moscow, Russia Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of preformed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galal-3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galal-3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and complement-activating properties of IgG and IgM XAb. Both classes of antibodies were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galal-3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and IgM, pointing to Galal-3GaJ as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galal-3Gal antibodies was 22 times less potent than IgM in Induction of cytotoxicity. These results indicate a quantitative, but not a qualitative difference between IgM and IgG anti-pig antibodies concerning their :omplement-activating properties. Therefore, both isotypes of XAb ue of importance in the pathogenesis of hyperacute rejection, and he relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.
044- UVB-INDUCED
KERATINOCYTE APOPTOSIS IN C 1q DEFICIENT MICE MC Pickering, S Fischer, HT Cooke, MJ Walport. M Botto. Hammersmith Campus, ICSM, London, UK
Clq has been shown to specifically bind to apoptotic human keratinocytes in vitro. To test the hypothesis that Clq is involved in the clearance of apoptotic murine keratinocytes in Go we exposed wild type and CJqa-l- mice to UVB radiation and quantified the degree of subsequent epidermal keratinocyte apoptosis. The dorsal skin of 8-12 week, sex-matched wild type C57BLJ6 and Clqn-l- mice, back crossed on to C57BU6 for I generations. was shaved 24 hours prior to exposure to UVB radiation at doses of 130, 400 or 8OOmJ/cm’. Using morphological criteria, the number of apoptotic cells was determined in three separate sections of dorsal skin and the length of the epidermis measured using calibrated image analysis software. No apoptotic cells were seen in biopsies of normal or shaved skin. Following a UVB dose of 800mJ/cm*. no significant difference in keratinocyte apoptosis between wild type and Clq deficient mice was evident at 4, 8 16 and 24 hours. Following UVB doses of 4OOmJ/cm* or 13OmJ/cm*, no significant differences in apoptotic cell counts were evident up to 96hours after exposure, in the absence of epidermal inflammation. Immunostaining for Clq at baseline and following UVB exposure in wild type mice was consistently negative. Furthermore. a cohort of 8 week old C57BLJ6 wild type and Clq deficient mice were exposed three times a week to low dose UVB radiation for 6 months. Preliminary results indicate that neither group produced antinuclear antibodies up to the age of 1 year. Histological analysis of the kidneys is in progress. These initial findings suggest that murine Clq does not play a major physiological role in the clearance of apoptotic cells in the skin of mice. ---
046- OPSONIZED IMMUNE COMPLEXES STIMULATE THE PRODUCTION OF TNFa FROM MACROPHAGES. Lisa Piliero and Kathleen Sullivan. The Children’s Hospital of Philadelphia, Philadelphia, PA 19104
Immune complex deposition underlies much of the pathologic tissue damage in SLE. Identification of complement and immunoglobulin in affected tissues is an almost universal finding. Even in early complement component deficient patients with SLE, complement deposition is found. In those patients, complement activation is presumed to have bypassed the deficient component. End organs which are affected in SLE also are characterized by overexpression of tumor necrosis factor alpha (TNFa). We investigated whether immune complexes were capable of stimulating TNFa expression from macrophages. Immune complexes stimulated high levels of TNFa expression from macrophages at both 24 hours and 72 hours. Opsonization of the immune complexes resulted in a further increase in TNFcr expression. Co-incubation with y-interferon resulted in greater increases in TNFa expression when the opsonized immune complexes were used as the stimulus. This suggests that immune complex stimulation of TNFa production may be part of an auto-stimulatory feedback loop that perpetuates the inflammatory process in SLE.
DEFICIENCY AND AUTOIMMUNITY: THE EFFECTS OF GENETIC BACKGROUND ON DISEASE EXPRESSION. Mitchell DA, Cook HT. Botto M, Walport MJ. Hammersmith Campus, ICSM, London, UK. Gene-targeted Clq-deficient mice, in the hybrid (129 x C57BLf6) genetic background, have been shown to develop spontaneously antinuclear antibodies and glomerulonephritis associated with multiple apoptotic bodies. The genetic background was believed to play an important role in disease expression since a small proportion of strain-matched control mice developed autoimmunity also. In order to investigate the influence of genetic background on disease expression in mutine Clq deficiency, the disrupted Clqa gene has been backcrossed for 7 generations onto the following inbred strains: C57BU6, 1291% and the Fasdeficient MRL/Mp.lprNpr and C57BW6.lpr/lpr strains. At 12 months of age, no serological or histological evidence of disease was found in Clq-deficient mice in the 129/Sv or C57BU6 strains, Analysis of Clq deficiency in the MRLMp.lprNpr strain showed no significant modification of disease onset or expression between Clq-l- and MRL&Ip.lprNpr wild-type mice at any of the 6, 9 or 12-week time points analysed. Similar findings were obtained from the analysis of Clq-deficient C57BL/6,lpr/lpr mice. However, significantly greater numbers of glomerular apoptotic cells were counted in the kidneys of Clq-deficient C57BW6.lpr/lpr than in wild-type C57BIJ6.IpwJpr mice. These findings highlight the critical significance of genetic background when analysing autoimmune phenotype in gene-targeted mice. In addition, this study fails to show an involvement of the classical pathway in regulating tolerance to nuclear antigens in IprApr mice, which has previously been reported in C4-deficient animals.
045-
Both I& and IgM anti-pig antibodies induce complement activation and cytotoxicity
Anja Roes, Maria Essers, DaniBlle J. van Gijlswijk-Janssen. Nicolai V. Bovin’, and Mohatned R. Daha, Deparrment of Nephrology, L&den University Medical Center, Leiden, the Netherlands, ’Shemyakin Institute ofRioorganic Chemistry Moscow, Russia Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of preformed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galal-3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galal-3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and complement-activating properties of IgG and IgM XAb. Both classes of antibodies were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galal-3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and IgM, pointing to Galal-3GaJ as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galal-3Gal antibodies was 22 times less potent than IgM in Induction of cytotoxicity. These results indicate a quantitative, but not a qualitative difference between IgM and IgG anti-pig antibodies concerning their :omplement-activating properties. Therefore, both isotypes of XAb ue of importance in the pathogenesis of hyperacute rejection, and he relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.
044- UVB-INDUCED
KERATINOCYTE APOPTOSIS IN C 1q DEFICIENT MICE MC Pickering, S Fischer, HT Cooke, MJ Walport. M Botto. Hammersmith Campus, ICSM, London, UK
Clq has been shown to specifically bind to apoptotic human keratinocytes in vitro. To test the hypothesis that Clq is involved in the clearance of apoptotic murine keratinocytes in Go we exposed wild type and CJqa-l- mice to UVB radiation and quantified the degree of subsequent epidermal keratinocyte apoptosis. The dorsal skin of 8-12 week, sex-matched wild type C57BLJ6 and Clqn-l- mice, back crossed on to C57BU6 for I generations. was shaved 24 hours prior to exposure to UVB radiation at doses of 130, 400 or 8OOmJ/cm’. Using morphological criteria, the number of apoptotic cells was determined in three separate sections of dorsal skin and the length of the epidermis measured using calibrated image analysis software. No apoptotic cells were seen in biopsies of normal or shaved skin. Following a UVB dose of 800mJ/cm*. no significant difference in keratinocyte apoptosis between wild type and Clq deficient mice was evident at 4, 8 16 and 24 hours. Following UVB doses of 4OOmJ/cm* or 13OmJ/cm*, no significant differences in apoptotic cell counts were evident up to 96hours after exposure, in the absence of epidermal inflammation. Immunostaining for Clq at baseline and following UVB exposure in wild type mice was consistently negative. Furthermore. a cohort of 8 week old C57BLJ6 wild type and Clq deficient mice were exposed three times a week to low dose UVB radiation for 6 months. Preliminary results indicate that neither group produced antinuclear antibodies up to the age of 1 year. Histological analysis of the kidneys is in progress. These initial findings suggest that murine Clq does not play a major physiological role in the clearance of apoptotic cells in the skin of mice. ---
046- OPSONIZED IMMUNE COMPLEXES STIMULATE THE PRODUCTION OF TNFa FROM MACROPHAGES. Lisa Piliero and Kathleen Sullivan. The Children’s Hospital of Philadelphia, Philadelphia, PA 19104
Immune complex deposition underlies much of the pathologic tissue damage in SLE. Identification of complement and immunoglobulin in affected tissues is an almost universal finding. Even in early complement component deficient patients with SLE, complement deposition is found. In those patients, complement activation is presumed to have bypassed the deficient component. End organs which are affected in SLE also are characterized by overexpression of tumor necrosis factor alpha (TNFa). We investigated whether immune complexes were capable of stimulating TNFa expression from macrophages. Immune complexes stimulated high levels of TNFa expression from macrophages at both 24 hours and 72 hours. Opsonization of the immune complexes resulted in a further increase in TNFcr expression. Co-incubation with y-interferon resulted in greater increases in TNFa expression when the opsonized immune complexes were used as the stimulus. This suggests that immune complex stimulation of TNFa production may be part of an auto-stimulatory feedback loop that perpetuates the inflammatory process in SLE.