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Variants in complement factor H affect complement activation in Henoch–Schonlein purpura nephritis
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Abstracts / Immunobiology 221 (2016) 1131–1225
kidney endothelial cells (hGEnCs) with and without a DM-like insult. Primary hGEnCs were grown in RPMI medium supplemented with either 6.5 mM (normal) or 25 mM (high) glucose. The cells were cultured for 8 days with media change every 48 h to allow exposure to a diabetic-like environment. During the final 48 h in culture, the cells were exposed to a second insult, i.e. TNF␣, IL1 or hypoxia. Gene expression levels were studied by qPCR using appropriate TaqMan probes. Untreated hGEnCs expressed moderately C2, C3, C4, C5, C7, C8 g main complement components, while C9 expression was very low. Moreover, the cells expressed moderately the auxiliary complement genes CFB, CFD, CFP and VTN, and at high levels CFH, CFI, CD46, CD55, CD59 and CLU. Neither unstimulated nor stimulated hGEnCs expressed C6, C8a, C8b, C4BPB, or CD35. Of the main complement components, only expression of C2 and C3 was regulated. C2 was increased 4× with TNF␣, while C3 was greatly upregulated by IL1 and TNF␣ (both approx. 500×). Neither hypoxia nor pre-treatment with high glucose had any effect. CFB was also highly regulated. Its expression increased 40× after TNF␣ and 20× after IL1 treatment, but remained unchanged after 48 h of hypoxia. The expression of CFP, CLU and VTN were decreased 3–8× after TNF␣, IL1 or hypoxia treatment. Human GEnCs express the majority of the main and auxiliary complement components, yet at different levels. Hypoxia as well as pro-inflammatory cytokines involved in DN pathology (TNF␣ and IL1) decreased the expression of CLU, CFP and VTN, while the cytokines caused also a considerable upregulation of C3 and CFB molecules. This is in line with the findings that both C3 and CFB are upregulated in glomeruli from DN patient cohorts. http://dx.doi.org/10.1016/j.imbio.2016.06.111 97 Variants in complement factor H affect complement activation in Henoch–Schonlein purpura nephritis Weiyi Guo ∗ , Li Zhu, Yalin Zhai, Hong Zhang Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China Objective: Henoch–Schonlein purpura nephritis (HSPN) and IgA nephropathy (IgAN) are widely considered as related diseases. Although extrarenal clinical signs are only present in HSPN, renal histological features are indistinguishable in HSPN and IgAN. Considerable evidences support the involvement of complement activation in both HSPN and IgAN. Our previous studies identified a genetic variant in complement factor H (CFH), rs6677604, as IgAN susceptible variant by GWAS, and further confirmed its linkage to CFHR3-1 and proved its influence to complement activation and thereby IgAN susceptibility. Based on the highly similarity of HSPN with IgAN, here we explored the role of rs6677604 in HSPN. Method: Totally, 2732 individuals were enrolled, including 652 patients with HSPN, 1178 patients with IgAN and 902 healthy volunteer. The genotype of rs6677604 were detected by TaqMan allele discrimination assays or extracted from GWAS data. Clinical and pathological information of enrolled patients were collected from medical records. Result: Compared to HSPN, the frequency of rs6677604-A allele was significantly lower in IgAN (HSPN vs. IgAN: 0.061 vs. 0.041, p = 0.009). However, no significant differences were observed between HSPN and controls, although rs6677604-A showed a little lower frequency in HSPN (HSPN vs. controls: 0.061 vs. 0.073, p = 0.187). Both in HSPN and IgAN, rs6677604-A was associated
with less intense of glomerular C3 deposits (rs6677604-A frequency in 0, 1+, 2+, 3+ ∼ 4+ of C3 deposits: HSPN: 10.2%, 6.9%, 5.0%, 3.9%, p = 0.002; IgAN: 5.2%, 6.6%, 3.4%, 3.0%, p = 0.009). As the frequency of rs6677604-A showed significant difference in HSPN and IgAN, we further compared the intensity of glomerular C3 deposits in these two diseases. In agreement with the higher frequency of rs6677604-A in HSPN, the glomerular C3 deposits of patients with HSPN were in less intensity than that in IgAN (0, 1+, 2+, 3+ ∼ 4+ of C3 deposits: HSPN: 16.6%, 24.4%, 35.6%, 23.5%; IgAN: 15.5%, 17.4%, 38.5%, 28.5%, p = 0.005). Conclusion: Our findings suggest that genetic variant in CFH (rs6677604) was involved in complement activation in both HSPN and IgAN. Moreover, rs6677604 might also contribute to the difference of complement activation intensity between HSPN and IgAN. http://dx.doi.org/10.1016/j.imbio.2016.06.112 98 Activation of complement and coagulation in xenotransplantation: Effect of growth hormone receptor knockout on porcine aortic endothelial cells Riccardo Sfriso 1,2,∗ , Nikolai Klymiuk 3 , Annegret Wuensch 3 , Joerg D. Seebach 4 , Eckhard Wolf 3 , Robert Rieben 1 1 Department of Clinical Research, University of Bern, Bern, Switzerland 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 3 Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany 4 Division of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland
Background: The phenotypic outcome of a mutation in the growth hormone receptor (GHR) gene – the Laron syndrome – is severe postnatal growth retardation and insulin-like growth factor-I deficiency despite high levels of serum GH. In the context of xenotransplantation the absence of GHR signaling might be beneficial because Laron pigs can be suitable donors due to their small organ sizes. In addition, people affected by the Laron syndrome have a reduced occurrence of cardiovascular diseases. This study therefore aimed at investigating whether the GHRKO phenotype may alter endothelial activation induced by pro-inflammatory stimuli in the context of delayed xenograft rejection. Methods: Immortalized porcine aortic endothelial cells lacking ␣Gal and GHR expression (PED GalTKO/GHRKO) were obtained after induction of a GHR gene mutation on the original PED GalTKO. The endothelial cell phenotype was assessed by immunofluorescence, assuring the expression of endothelial cell markers such as CD31, VE-cadherin and von Willebrand Factor (vWF). A cell ELISA was carried out in order to evaluate the effect of the GHR deletion on deposition of complement (C3b/c, C4b/c) and human antibodies (IgM, IgG) after incubation with pooled normal human serum. Effects on coagulation were also assessed performing whole blood clotting assays using endothelial cell-coated microcarriers. Results: A strong expression of CD31, vWF, VE-cadherin, and heparan sulfate proteoglycans on PED GalTKO/GHRKO was observed by immunofluorescence. Significantly lower IgM, IgG, C3b/c and C4b/c deposition was observed on PED GalTKO and PED GalTKO/GHRKO compared to wild type porcine endothelial cells. Furthermore, growth hormone resistant cells grown on microbeads significantly delayed the clotting time of freshly drawn, whole,
Abstracts / Immunobiology 221 (2016) 1131–1225
kidney endothelial cells (hGEnCs) with and without a DM-like insult. Primary hGEnCs were grown in RPMI medium supplemented with either 6.5 mM (normal) or 25 mM (high) glucose. The cells were cultured for 8 days with media change every 48 h to allow exposure to a diabetic-like environment. During the final 48 h in culture, the cells were exposed to a second insult, i.e. TNF␣, IL1 or hypoxia. Gene expression levels were studied by qPCR using appropriate TaqMan probes. Untreated hGEnCs expressed moderately C2, C3, C4, C5, C7, C8 g main complement components, while C9 expression was very low. Moreover, the cells expressed moderately the auxiliary complement genes CFB, CFD, CFP and VTN, and at high levels CFH, CFI, CD46, CD55, CD59 and CLU. Neither unstimulated nor stimulated hGEnCs expressed C6, C8a, C8b, C4BPB, or CD35. Of the main complement components, only expression of C2 and C3 was regulated. C2 was increased 4× with TNF␣, while C3 was greatly upregulated by IL1 and TNF␣ (both approx. 500×). Neither hypoxia nor pre-treatment with high glucose had any effect. CFB was also highly regulated. Its expression increased 40× after TNF␣ and 20× after IL1 treatment, but remained unchanged after 48 h of hypoxia. The expression of CFP, CLU and VTN were decreased 3–8× after TNF␣, IL1 or hypoxia treatment. Human GEnCs express the majority of the main and auxiliary complement components, yet at different levels. Hypoxia as well as pro-inflammatory cytokines involved in DN pathology (TNF␣ and IL1) decreased the expression of CLU, CFP and VTN, while the cytokines caused also a considerable upregulation of C3 and CFB molecules. This is in line with the findings that both C3 and CFB are upregulated in glomeruli from DN patient cohorts. http://dx.doi.org/10.1016/j.imbio.2016.06.111 97 Variants in complement factor H affect complement activation in Henoch–Schonlein purpura nephritis Weiyi Guo ∗ , Li Zhu, Yalin Zhai, Hong Zhang Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China Objective: Henoch–Schonlein purpura nephritis (HSPN) and IgA nephropathy (IgAN) are widely considered as related diseases. Although extrarenal clinical signs are only present in HSPN, renal histological features are indistinguishable in HSPN and IgAN. Considerable evidences support the involvement of complement activation in both HSPN and IgAN. Our previous studies identified a genetic variant in complement factor H (CFH), rs6677604, as IgAN susceptible variant by GWAS, and further confirmed its linkage to CFHR3-1 and proved its influence to complement activation and thereby IgAN susceptibility. Based on the highly similarity of HSPN with IgAN, here we explored the role of rs6677604 in HSPN. Method: Totally, 2732 individuals were enrolled, including 652 patients with HSPN, 1178 patients with IgAN and 902 healthy volunteer. The genotype of rs6677604 were detected by TaqMan allele discrimination assays or extracted from GWAS data. Clinical and pathological information of enrolled patients were collected from medical records. Result: Compared to HSPN, the frequency of rs6677604-A allele was significantly lower in IgAN (HSPN vs. IgAN: 0.061 vs. 0.041, p = 0.009). However, no significant differences were observed between HSPN and controls, although rs6677604-A showed a little lower frequency in HSPN (HSPN vs. controls: 0.061 vs. 0.073, p = 0.187). Both in HSPN and IgAN, rs6677604-A was associated
with less intense of glomerular C3 deposits (rs6677604-A frequency in 0, 1+, 2+, 3+ ∼ 4+ of C3 deposits: HSPN: 10.2%, 6.9%, 5.0%, 3.9%, p = 0.002; IgAN: 5.2%, 6.6%, 3.4%, 3.0%, p = 0.009). As the frequency of rs6677604-A showed significant difference in HSPN and IgAN, we further compared the intensity of glomerular C3 deposits in these two diseases. In agreement with the higher frequency of rs6677604-A in HSPN, the glomerular C3 deposits of patients with HSPN were in less intensity than that in IgAN (0, 1+, 2+, 3+ ∼ 4+ of C3 deposits: HSPN: 16.6%, 24.4%, 35.6%, 23.5%; IgAN: 15.5%, 17.4%, 38.5%, 28.5%, p = 0.005). Conclusion: Our findings suggest that genetic variant in CFH (rs6677604) was involved in complement activation in both HSPN and IgAN. Moreover, rs6677604 might also contribute to the difference of complement activation intensity between HSPN and IgAN. http://dx.doi.org/10.1016/j.imbio.2016.06.112 98 Activation of complement and coagulation in xenotransplantation: Effect of growth hormone receptor knockout on porcine aortic endothelial cells Riccardo Sfriso 1,2,∗ , Nikolai Klymiuk 3 , Annegret Wuensch 3 , Joerg D. Seebach 4 , Eckhard Wolf 3 , Robert Rieben 1 1 Department of Clinical Research, University of Bern, Bern, Switzerland 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 3 Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany 4 Division of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland
Background: The phenotypic outcome of a mutation in the growth hormone receptor (GHR) gene – the Laron syndrome – is severe postnatal growth retardation and insulin-like growth factor-I deficiency despite high levels of serum GH. In the context of xenotransplantation the absence of GHR signaling might be beneficial because Laron pigs can be suitable donors due to their small organ sizes. In addition, people affected by the Laron syndrome have a reduced occurrence of cardiovascular diseases. This study therefore aimed at investigating whether the GHRKO phenotype may alter endothelial activation induced by pro-inflammatory stimuli in the context of delayed xenograft rejection. Methods: Immortalized porcine aortic endothelial cells lacking ␣Gal and GHR expression (PED GalTKO/GHRKO) were obtained after induction of a GHR gene mutation on the original PED GalTKO. The endothelial cell phenotype was assessed by immunofluorescence, assuring the expression of endothelial cell markers such as CD31, VE-cadherin and von Willebrand Factor (vWF). A cell ELISA was carried out in order to evaluate the effect of the GHR deletion on deposition of complement (C3b/c, C4b/c) and human antibodies (IgM, IgG) after incubation with pooled normal human serum. Effects on coagulation were also assessed performing whole blood clotting assays using endothelial cell-coated microcarriers. Results: A strong expression of CD31, vWF, VE-cadherin, and heparan sulfate proteoglycans on PED GalTKO/GHRKO was observed by immunofluorescence. Significantly lower IgM, IgG, C3b/c and C4b/c deposition was observed on PED GalTKO and PED GalTKO/GHRKO compared to wild type porcine endothelial cells. Furthermore, growth hormone resistant cells grown on microbeads significantly delayed the clotting time of freshly drawn, whole,