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Effect of anti-C1q antibodies from SLE patients on complement activation
278
Abstracts / Molecular Immunology 56 (2013) 240–316
suggest that current PD protocols might impair peritoneal function by modulating the activation and regulation of the complement system. http://dx.doi.org/10.1016/j.molimm.2013.05.110 P CD 17 Atypical haemolytic uraemic syndrome associated with a novel hybrid CFH/CFHR3 gene R. Challis 1,∗ , H. Anderson 1 , E. Wong 1 , G. Reis 1 , A. Awan 2 , M. Waldron 2 , L. Strain 1 , K. Marchbank 3 , T. Goodship 1 , D. Kavanagh 1 1
Newcastle University, Institute of Genetic Medicine, Newcastle upon Tyne, United Kingdom 2 Children’s University Hospital, Department of Nephrology, Dublin, Ireland 3 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom
Atypical haemolytic uraemic syndrome (aHUS) is a severe renal disease caused by deregulation of the alternative complement pathway. The RCA (Regulators of Complement Activation) gene cluster contains several genes that have been implicated in the pathogenesis of aHUS, including Complement Factor H (CFH). The gene for CFH is in close proximity to the genes encoding the 5 Complement Factor H related proteins (CFHRs). These are thought to have arisen from several large genomic duplications and thus have a very high degree of sequence identity to CFH. This homology predisposes this area to gene conversions and genomic rearrangements through non-allelic homologous recombination (NAHR) and microhomology-mediated end joining (MMEJ). In a sporadic case of aHUS, using multiplex ligation-dependent probe amplification, we describe a de novo deletion in the RCA cluster. Sequencing the PCR product across the deletion identified the breakpoint, with a 6.3 kb deletion between CFH intron 20 and the 3 UTR of CFH. Immediately adjacent to the breakpoint was a 7bp region of microhomology. This is predicted to result in a novel hybrid CFH/CFHR3 gene. Western blotting confirmed 2 distinct antifH species in the sera of the affected individual. Using specific monoclonal antibodies, the higher molecular weight band gave a signal in keeping with the predicted CFH/CFHR3 hybrid protein. In conclusion we describe a novel deletion which occurred through MMEJ in aHUS. This produced a CFH/CFHR3 hybrid protein predicted to have impaired cell surface complement regulation, predisposing to disease. This highlights the importance of undertaking copy number variation analysis when undertaking genetic screening of aHUS patients. http://dx.doi.org/10.1016/j.molimm.2013.05.111 P CD 18 Shiga toxin induces release of soluble ICAM-1 protein in primary human glomerular microvacular endothelial cells E. Volokhina 1,∗ , T. van der Velden 1 , D. Westra 1 , N. van de Kar 1 , L. van den Heuvel 1,2,3 1
UMC St. Radboud, Pediatric Nephrology, Nijmegen, Netherlands UMC St. Radboud, Department of Laboratory Medicine, Nijmegen, Netherlands 3 University Hospitals Leuven, Department of Pediatrics, Leuven, Netherlands 2
Question: Hemolytic uremic syndrome (HUS) is often preceded by infection with Shiga toxin (Stx) producing Escherichia coli (STEC). The exact mechanism of how Stx causes HUS is still poorly under-
stood. Adhesion molecules are known to play important role in endothelial inflammation. In this work we investigated whether increased release of ICAM-1 from endothelial cells might be important in HUS pathogenesis. Methods: HUVEC and human glomerular microvascular endothelial cells (GMVEC) from three donors were treated for 6, 12, 24 and 48 h with 0, 0.1, and 1.0 nM of Stx1 with or without prestimulation with TNF␣. The culture supernatant and cells were collected for ELISA and qPCR analyses to assess the expression of ICAM-1 on protein and mRNA levels. Results: Stx1 upregulated levels of soluble ICAM-1 in culture supernatant of GMVEC when cells were preincubated with TNF␣ in time- and dose-dependent manner for up to 20 times (p < 0.001). This upregulation on the protein level was consistent with enhanced ICAM-1 mRNA expression. No effect of Stx1 on HUVEC was observed. Incubation with TNF␣ only enhanced release of soluble ICAM-1 in both cell lines for up to 10 fold (p < 0.001). Conclusions: Our results indicate that increased production of ICAM-1 might be important in pathogenesis of STEC HUS and play role in glomerular endothelial damage, possibly via enhanced recruitment of leukocytes to the endothelial surface. http://dx.doi.org/10.1016/j.molimm.2013.05.112 P CD 19 Effect of anti-C1q antibodies from SLE patients on complement activation S. Thanei 1,∗ , D. Vanhecke 1 , C. Ploix 2 , M. Trendelenburg 1,3 1
University Hospital Basel, Department of Biomedicine, Basel, Switzerland 2 F.Hoffmann – La Roche, Immunosafety, Basel, Switzerland 3 University Hospital Basel, Internal Medicine, Basel, Switzerland Introduction: Antibodies against C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE) and they strongly correlate with the occurrence of severe lupus nephritis. Although a pathogenic role of anti-C1q has been suggested, the potential interference with the complement system remains to be elucidated. Objectives: The aim of this study was to determine the complement-activating potential of anti-C1q. Patients and methods: We developed an ELISA-based assay to investigate the ability of anti-C1q to activate the complement cascade using plates coated with C1q, anti-C1q positive or negative SLE patient sera as a source of anti-C1q and either purified complement components or normal human serum or complement-deficient sera diluted in specific buffers as a source of complement. The activation of the classical or the alternative pathway was assessed by detecting C4b or C3 deposition respectively. With this assay a total of 28 SLE patient sera and 25 healthy donor sera were analyzed. Results: We found no evidence for a direct activation of C1 leading to C4b deposition by anti-C1q themselves. There was also no interference of anti-C1q with the activation of the classical pathway when a classical complement activation ELISA was performed using IgM-coated plates. However, anti-C1q bound to immobilized C1q resulted in the activation of the classical pathway as reflected by C4b deposition in the presence of purified C1 and C4. Already low dilutions of anti-C1q positive SLE patient sera, such as 1:1000, were sufficient to trigger the activation of the classical pathway. The extent of C4b deposition correlated with the titers of anti-C1q in SLE patients but not in healthy controls. In contrast, we could not observe activation via the alternative pathway of complement when normal human serum was used as a source for complement components.
Abstracts / Molecular Immunology 56 (2013) 240–316
Conclusion: Our data indicate that SLE patient-derived anti-C1q are able to activate the classical pathway of complement in an in vitro assay by secondary binding of C1 molecules leading to C4b deposition.
279
P CD 21 Serum C3 levels are associated with the clinical manifestations and disease activity of microscopic polyangiitis D. Liu 1,2,3 , Y. Chen 3 , X. Min 1 , N. Wang 1 , Y. Jia 2 , K. Li 1,∗
http://dx.doi.org/10.1016/j.molimm.2013.05.113 P CD 20 CD59 is a blood group antigen M. Anliker 1,∗ , I. von Zabern 1 , B. Höchsmann 1 , C. DohnaSchwake 2 , H. Kyrieleis 3 , H. Schrezenmeier 1 , C. Weinstock 1 1
Inst. f. Klin. Transfusionsmedizin und Immungenetik Ulm, DRK BSD Baden-Württ.-Hessen, University of Ulm, Ulm, Germany 2 University Hospital of Essen, Essen, Germany 3 Hospital Bethanien, Moers, Germany Question: Homologous restriction factor (HRF), Membrane inhibitor of reactive lysis (MIRL), membrane attack complex inhibitory factor (MACIF) and CD59 are a few of the many designations for a glycoprotein of approximately 20 kDa that binds to the terminal complement complex C5b-9 and thereby limits the polymerization of C9. Although CD59 is known since a long time as part of the erythrocyte membrane, it so far has not been defined as a blood group antigen. Here, we give evidence for the presence of an anti-CD59 antibody in a patient affected by a homozygous CD59 deficiency. Methods: Erythrocyte membrane CD59 and CD55 were determined by flow cytometry using FITC labelled anti-human-CD59 and PE labelled anti-human-CD55 (Becton Dickinson Pharmingen). His tagged and non-glycosylated recombinant soluble CD59 protein was obtained in lyophilized form (Creative Bio Mart). Results: Six cases of an isolated deficiency of CD59 with two molecular bases have been published so far. An additional case of an isolated homozygous CD59 defect in a child of Turkish origin caused by the deletion c.146delA has been reported. The erythrocytes of the affected child lacked CD59. The plasma contained an antibody against a high frequency erythrocyte antigen, likely acquired by transfusions. To identify the specificity of the antibody, soluble recombinant CD59 was added to the plasma of the CD59 deficient patient. As controls, monoclonal anti-CD59 and monoclonal anti-CD55 were used instead of plasma. Flow cytometric analysis demonstrated inhibition of the patient antibody and of commercial monoclonal anti-CD59 by the recombinant soluble CD59 protein while the reactivity of anti-CD55 remained unaffected. The CD59 deficiency was inherited from the parents who both were heterozygous for the same allele. Conclusions: We give evidence that a CD59 deficient patient had formed an anti-CD59 antibody, easily detectable by blocking the antibody reactivity by recombinant soluble CD59. The erythrocyte membrane glycoprotein CD59 is well characterized on a molecular level, but this is the first demonstration of a human antibody directed against it. Therefore, CD59 is a blood group antigen and represents a candidate for the definition of a new blood group system. http://dx.doi.org/10.1016/j.molimm.2013.05.114
1
The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Core Research Laboratory, Xi’an, China 2 Peking University People’s Hospital, Department of Rheumatology and Immunology, Beijing, China 3 The Fifth Hospital of Xi’an, Department of Rheumatology, Xi’an, China
Introduction: Microscopic polyangiitis (MPA) is the most commonanti-neutrophil cytoplasmic antibodies (ANCA) associated small-vessel vasculitis with specificity of the ANCA to myeloperoxidase (MPO) of neutrophils. MPA mainly affects kidneys and lungs. The kidneys are the most commonly affected organ; and kidney vasculitis can result in renal failure. The interplay of ANCA, neutrophil and complement activation has been implicated in the pathogenesis of MPA. The objective: To establish the relationship of complement activation and the disease activity of MPA and to identify potential biomarkers for this disease. Patients and methods: Blood samples were collected from 45 MPO-ANCA positive patients who had clinical diagnosis of MPA in People’s Hospital, Peking University, after informed consent. The samples, according to serum C3 levels, were divided into two groups: normal C3 group: ≥0.8 g/L (n = 18) and low C3 group: C3 < 0.8 g/L (n = 27). Inflammatory markers (i.e. ESR, CRP), renal functional markers (i.e. BUN, creatinine, 24-h urinary protein level), and lung diseases were evaluated in the two groups. Birmingham Vasculitis Activity Score-version 3 (BVAS[V3]) was calculated. Data was analyzed using t test, chi-square test, linear correlation analysis and binary logistic regression, respectively. Results: (1) Compared with normal C3 group, low C3 group had significantly lowered C4 (0.17 ± 0.08 g/L vs 0.25 ± 0.08 g/L) but significantly increased CRP (101 ± 57 mg/L vs 33 ± 33 mg/L), 24-h urinary protein (1.4 ± 1.3 g/d vs 0.6 ± 0.5 g/d), BUN (17.08 ± 9.02 mmol/L vs 8.62 ± 5.28 mmol/L) and BVAS(V3) (21.4 ± 3.6 vs 15.4 ± 3.8). (2) BVAS (V3) has a significant linear correlation with serum complement C3 level (R2 = 0.43, P < 0.01). (3) Morbidity of severe lung injury (i.e. interstitial lung disease, pneumorrhagia) in low C3 group was higher than that in normal C3 group (92.6% vs 33.3%, X2 = 17.69, P < 0.01). (4) C3, CRP and ESR levels were closely related to the occurrence of severe lung injury, and the logistic regression equation was: P(y) = 1/(1 + e−2.1+7.54×C3−0.054×CRP−0.027×ESR ), in which the overall correct percentage was 93.3%. Conclusion: Serum complement C3 levels were associated with the disease activity and clinical manifestations of MPA. Therefore, serum C3 level may represent as a biomarker for disease activity and an indicator for organ injury of MPA. http://dx.doi.org/10.1016/j.molimm.2013.05.115
Abstracts / Molecular Immunology 56 (2013) 240–316
suggest that current PD protocols might impair peritoneal function by modulating the activation and regulation of the complement system. http://dx.doi.org/10.1016/j.molimm.2013.05.110 P CD 17 Atypical haemolytic uraemic syndrome associated with a novel hybrid CFH/CFHR3 gene R. Challis 1,∗ , H. Anderson 1 , E. Wong 1 , G. Reis 1 , A. Awan 2 , M. Waldron 2 , L. Strain 1 , K. Marchbank 3 , T. Goodship 1 , D. Kavanagh 1 1
Newcastle University, Institute of Genetic Medicine, Newcastle upon Tyne, United Kingdom 2 Children’s University Hospital, Department of Nephrology, Dublin, Ireland 3 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom
Atypical haemolytic uraemic syndrome (aHUS) is a severe renal disease caused by deregulation of the alternative complement pathway. The RCA (Regulators of Complement Activation) gene cluster contains several genes that have been implicated in the pathogenesis of aHUS, including Complement Factor H (CFH). The gene for CFH is in close proximity to the genes encoding the 5 Complement Factor H related proteins (CFHRs). These are thought to have arisen from several large genomic duplications and thus have a very high degree of sequence identity to CFH. This homology predisposes this area to gene conversions and genomic rearrangements through non-allelic homologous recombination (NAHR) and microhomology-mediated end joining (MMEJ). In a sporadic case of aHUS, using multiplex ligation-dependent probe amplification, we describe a de novo deletion in the RCA cluster. Sequencing the PCR product across the deletion identified the breakpoint, with a 6.3 kb deletion between CFH intron 20 and the 3 UTR of CFH. Immediately adjacent to the breakpoint was a 7bp region of microhomology. This is predicted to result in a novel hybrid CFH/CFHR3 gene. Western blotting confirmed 2 distinct antifH species in the sera of the affected individual. Using specific monoclonal antibodies, the higher molecular weight band gave a signal in keeping with the predicted CFH/CFHR3 hybrid protein. In conclusion we describe a novel deletion which occurred through MMEJ in aHUS. This produced a CFH/CFHR3 hybrid protein predicted to have impaired cell surface complement regulation, predisposing to disease. This highlights the importance of undertaking copy number variation analysis when undertaking genetic screening of aHUS patients. http://dx.doi.org/10.1016/j.molimm.2013.05.111 P CD 18 Shiga toxin induces release of soluble ICAM-1 protein in primary human glomerular microvacular endothelial cells E. Volokhina 1,∗ , T. van der Velden 1 , D. Westra 1 , N. van de Kar 1 , L. van den Heuvel 1,2,3 1
UMC St. Radboud, Pediatric Nephrology, Nijmegen, Netherlands UMC St. Radboud, Department of Laboratory Medicine, Nijmegen, Netherlands 3 University Hospitals Leuven, Department of Pediatrics, Leuven, Netherlands 2
Question: Hemolytic uremic syndrome (HUS) is often preceded by infection with Shiga toxin (Stx) producing Escherichia coli (STEC). The exact mechanism of how Stx causes HUS is still poorly under-
stood. Adhesion molecules are known to play important role in endothelial inflammation. In this work we investigated whether increased release of ICAM-1 from endothelial cells might be important in HUS pathogenesis. Methods: HUVEC and human glomerular microvascular endothelial cells (GMVEC) from three donors were treated for 6, 12, 24 and 48 h with 0, 0.1, and 1.0 nM of Stx1 with or without prestimulation with TNF␣. The culture supernatant and cells were collected for ELISA and qPCR analyses to assess the expression of ICAM-1 on protein and mRNA levels. Results: Stx1 upregulated levels of soluble ICAM-1 in culture supernatant of GMVEC when cells were preincubated with TNF␣ in time- and dose-dependent manner for up to 20 times (p < 0.001). This upregulation on the protein level was consistent with enhanced ICAM-1 mRNA expression. No effect of Stx1 on HUVEC was observed. Incubation with TNF␣ only enhanced release of soluble ICAM-1 in both cell lines for up to 10 fold (p < 0.001). Conclusions: Our results indicate that increased production of ICAM-1 might be important in pathogenesis of STEC HUS and play role in glomerular endothelial damage, possibly via enhanced recruitment of leukocytes to the endothelial surface. http://dx.doi.org/10.1016/j.molimm.2013.05.112 P CD 19 Effect of anti-C1q antibodies from SLE patients on complement activation S. Thanei 1,∗ , D. Vanhecke 1 , C. Ploix 2 , M. Trendelenburg 1,3 1
University Hospital Basel, Department of Biomedicine, Basel, Switzerland 2 F.Hoffmann – La Roche, Immunosafety, Basel, Switzerland 3 University Hospital Basel, Internal Medicine, Basel, Switzerland Introduction: Antibodies against C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE) and they strongly correlate with the occurrence of severe lupus nephritis. Although a pathogenic role of anti-C1q has been suggested, the potential interference with the complement system remains to be elucidated. Objectives: The aim of this study was to determine the complement-activating potential of anti-C1q. Patients and methods: We developed an ELISA-based assay to investigate the ability of anti-C1q to activate the complement cascade using plates coated with C1q, anti-C1q positive or negative SLE patient sera as a source of anti-C1q and either purified complement components or normal human serum or complement-deficient sera diluted in specific buffers as a source of complement. The activation of the classical or the alternative pathway was assessed by detecting C4b or C3 deposition respectively. With this assay a total of 28 SLE patient sera and 25 healthy donor sera were analyzed. Results: We found no evidence for a direct activation of C1 leading to C4b deposition by anti-C1q themselves. There was also no interference of anti-C1q with the activation of the classical pathway when a classical complement activation ELISA was performed using IgM-coated plates. However, anti-C1q bound to immobilized C1q resulted in the activation of the classical pathway as reflected by C4b deposition in the presence of purified C1 and C4. Already low dilutions of anti-C1q positive SLE patient sera, such as 1:1000, were sufficient to trigger the activation of the classical pathway. The extent of C4b deposition correlated with the titers of anti-C1q in SLE patients but not in healthy controls. In contrast, we could not observe activation via the alternative pathway of complement when normal human serum was used as a source for complement components.
Abstracts / Molecular Immunology 56 (2013) 240–316
Conclusion: Our data indicate that SLE patient-derived anti-C1q are able to activate the classical pathway of complement in an in vitro assay by secondary binding of C1 molecules leading to C4b deposition.
279
P CD 21 Serum C3 levels are associated with the clinical manifestations and disease activity of microscopic polyangiitis D. Liu 1,2,3 , Y. Chen 3 , X. Min 1 , N. Wang 1 , Y. Jia 2 , K. Li 1,∗
http://dx.doi.org/10.1016/j.molimm.2013.05.113 P CD 20 CD59 is a blood group antigen M. Anliker 1,∗ , I. von Zabern 1 , B. Höchsmann 1 , C. DohnaSchwake 2 , H. Kyrieleis 3 , H. Schrezenmeier 1 , C. Weinstock 1 1
Inst. f. Klin. Transfusionsmedizin und Immungenetik Ulm, DRK BSD Baden-Württ.-Hessen, University of Ulm, Ulm, Germany 2 University Hospital of Essen, Essen, Germany 3 Hospital Bethanien, Moers, Germany Question: Homologous restriction factor (HRF), Membrane inhibitor of reactive lysis (MIRL), membrane attack complex inhibitory factor (MACIF) and CD59 are a few of the many designations for a glycoprotein of approximately 20 kDa that binds to the terminal complement complex C5b-9 and thereby limits the polymerization of C9. Although CD59 is known since a long time as part of the erythrocyte membrane, it so far has not been defined as a blood group antigen. Here, we give evidence for the presence of an anti-CD59 antibody in a patient affected by a homozygous CD59 deficiency. Methods: Erythrocyte membrane CD59 and CD55 were determined by flow cytometry using FITC labelled anti-human-CD59 and PE labelled anti-human-CD55 (Becton Dickinson Pharmingen). His tagged and non-glycosylated recombinant soluble CD59 protein was obtained in lyophilized form (Creative Bio Mart). Results: Six cases of an isolated deficiency of CD59 with two molecular bases have been published so far. An additional case of an isolated homozygous CD59 defect in a child of Turkish origin caused by the deletion c.146delA has been reported. The erythrocytes of the affected child lacked CD59. The plasma contained an antibody against a high frequency erythrocyte antigen, likely acquired by transfusions. To identify the specificity of the antibody, soluble recombinant CD59 was added to the plasma of the CD59 deficient patient. As controls, monoclonal anti-CD59 and monoclonal anti-CD55 were used instead of plasma. Flow cytometric analysis demonstrated inhibition of the patient antibody and of commercial monoclonal anti-CD59 by the recombinant soluble CD59 protein while the reactivity of anti-CD55 remained unaffected. The CD59 deficiency was inherited from the parents who both were heterozygous for the same allele. Conclusions: We give evidence that a CD59 deficient patient had formed an anti-CD59 antibody, easily detectable by blocking the antibody reactivity by recombinant soluble CD59. The erythrocyte membrane glycoprotein CD59 is well characterized on a molecular level, but this is the first demonstration of a human antibody directed against it. Therefore, CD59 is a blood group antigen and represents a candidate for the definition of a new blood group system. http://dx.doi.org/10.1016/j.molimm.2013.05.114
1
The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Core Research Laboratory, Xi’an, China 2 Peking University People’s Hospital, Department of Rheumatology and Immunology, Beijing, China 3 The Fifth Hospital of Xi’an, Department of Rheumatology, Xi’an, China
Introduction: Microscopic polyangiitis (MPA) is the most commonanti-neutrophil cytoplasmic antibodies (ANCA) associated small-vessel vasculitis with specificity of the ANCA to myeloperoxidase (MPO) of neutrophils. MPA mainly affects kidneys and lungs. The kidneys are the most commonly affected organ; and kidney vasculitis can result in renal failure. The interplay of ANCA, neutrophil and complement activation has been implicated in the pathogenesis of MPA. The objective: To establish the relationship of complement activation and the disease activity of MPA and to identify potential biomarkers for this disease. Patients and methods: Blood samples were collected from 45 MPO-ANCA positive patients who had clinical diagnosis of MPA in People’s Hospital, Peking University, after informed consent. The samples, according to serum C3 levels, were divided into two groups: normal C3 group: ≥0.8 g/L (n = 18) and low C3 group: C3 < 0.8 g/L (n = 27). Inflammatory markers (i.e. ESR, CRP), renal functional markers (i.e. BUN, creatinine, 24-h urinary protein level), and lung diseases were evaluated in the two groups. Birmingham Vasculitis Activity Score-version 3 (BVAS[V3]) was calculated. Data was analyzed using t test, chi-square test, linear correlation analysis and binary logistic regression, respectively. Results: (1) Compared with normal C3 group, low C3 group had significantly lowered C4 (0.17 ± 0.08 g/L vs 0.25 ± 0.08 g/L) but significantly increased CRP (101 ± 57 mg/L vs 33 ± 33 mg/L), 24-h urinary protein (1.4 ± 1.3 g/d vs 0.6 ± 0.5 g/d), BUN (17.08 ± 9.02 mmol/L vs 8.62 ± 5.28 mmol/L) and BVAS(V3) (21.4 ± 3.6 vs 15.4 ± 3.8). (2) BVAS (V3) has a significant linear correlation with serum complement C3 level (R2 = 0.43, P < 0.01). (3) Morbidity of severe lung injury (i.e. interstitial lung disease, pneumorrhagia) in low C3 group was higher than that in normal C3 group (92.6% vs 33.3%, X2 = 17.69, P < 0.01). (4) C3, CRP and ESR levels were closely related to the occurrence of severe lung injury, and the logistic regression equation was: P(y) = 1/(1 + e−2.1+7.54×C3−0.054×CRP−0.027×ESR ), in which the overall correct percentage was 93.3%. Conclusion: Serum complement C3 levels were associated with the disease activity and clinical manifestations of MPA. Therefore, serum C3 level may represent as a biomarker for disease activity and an indicator for organ injury of MPA. http://dx.doi.org/10.1016/j.molimm.2013.05.115