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Collectin-11 deficiency reduces renal inflammation and ameliorates tubulointerstitial fibrosis following renal ischemia reperfusion injury
Abstracts / Immunobiology 221 (2016) 1131–1225
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Activation of complement by pigment epithelium derived protein in synovial fluid of rheumatoid arthritis patients
Collectin-11 deficiency reduces renal inflammation and ameliorates tubulointerstitial fibrosis following renal ischemia reperfusion injury
Leonie M. Vogt 1,∗ , Simone Talens 1 , Carsten Scavenius 2 , Jan J. Enghild 2 , Tore Saxne 3 , Anna M. Blom 1 1
Lund University, Department of Translational Medicine, Malmö, Sweden 2 Aarhus University, Department of Molecular Biology and Genetics, Aarhus, Denmark 3 Lund University, Department of Clinical Sciences, Lund, Sweden Introduction: Rheumatoid arthritis (RA) is a disease in which complement activation leading to inflammation plays a detrimental role. Evidence for complement activation in synovial joints in arthritis comes both from clinical studies and animal models. However, it is unclear which molecules trigger complement activation under these conditions, and the aim of this study was to identify these activators. Methods: C4d, the final cleavage products resulting from activation of C4, is found in high amounts in the diseased joint, and it may bind covalently to complement-activating molecules. Therefore, we used a novel, highly specific antibody against a cleavage neoepitope in C4d for affinity purification of C4d in complexes with molecules, which were specifically bound to C4d, from pooled synovial fluid of four RA patients. As a control, pre-immune IgG from the same rabbit was coupled to a second column. Using mass spectrometry analysis, molecules eluted from both columns were identified. Those present only in the eluates from the anti-C4d column were subsequently studied for their ability to activate the classical complement pathway. Results: Besides immunoglobulins and several components of the classical pathway, one of the C4d binding molecules identified was pigment epithelium derived protein (PEDF). PEDF is a broadly expressed multifunctional member of the serine proteinase inhibitor (serpin) family. It plays critical roles in many physiological and pathophysiological processes, including neuroprotection, angiogenesis, fibrogenesis and inflammation. We expressed PEDF in eukaryotic cells and purified it using affinity chromatography. Upon subsequent testing with normal human serum, PEDF immobilized in a microtiter plate was found to induce C4b and C3b deposition. C1q-depleted serum, heat inactivated serum, and serum containing 20 mM EDTA all completely abrogated the complement activating potential of PEDF, indicating that PEDF operates through the classical pathway. Further, using specific antibodies we detected complexes between PEDF and C4d in the synovial fluid of RA patients. Furthermore, we detected direct binding of PEDF to apoptotic cells using flow cytometry, indicating potential for interference with recognition of these cells by complement. Conclusion: Despite the many beneficial effects PEDF exerts in the body, in the synovium of RA patients it may play a detrimental role by potentially activating the classical complement pathway.
Weiju Wu 1,∗ , Chengfei Liu 2 , Conrad A. Farrar 1 , Liang Ma 1 , Ke Li 2 , Steve H. Sacks 1 , Wuding Zhou 1 1
Medical Research Council (MRC) Centre for Transplantation, King’s College London, Guy’s Hospital, UK 2 Core Research Laboratory, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, China Collectin-11 (CL-11) is a collagen-containing lectin molecule that recognises carbohydrate residues on the cell surface. It has a wide tissue distribution and the kidney is a major site of the production. Our previous study showed that CL-11 detects stress-induces l-fucose pattern in response to ischemic insult and trigger complement activation to cause acute kidney injury. However, the impact of CL-11 on the late phase of renal ischemia reperfusion (IR) injury is unknown. In the present study, we tested whether renal inflammation and tubulointerstitial fibrosis following renal IR injury is dependent on CL-11. We employed a murine model of renal IR injury and CL-11−/− mice to determine the roles of CL-11 in renal inflammation and fibrosis. The results showed that compared to the wild littermates, CL-11−/− mice had significantly reduced renal tissue inflammation and fibrosis at day 7 and 10 after 30 min of bilateral renal IR injury, as evidenced by reduced: (i) impairment of renal function (by BUN), (ii) tissue injury (by PAS staining and histological scores), (iii) leukocyte infiltration (by flow cytometry and immunochemical staining for CD45+ , Ly6G+ , F4/80+ ), (iv) tubulointerstitial fibrosis (by Sirius red staining, RT-PCR for intrarenal collagen I and fibronectin expression), (v) intrarenal gene expression of proinflammatory (TNF-␣, IL-1, IL-6) and pro-fibrotic (TGF-) factors (by RT-PCR). To investigate cellular mechanisms that CL-11 contributes to renal inflammation and fibrosis we performed a series of in vitro experiments using freshly prepared peritoneal neutrophils or mono/macrophages and primarily cultured renal fibroblasts. Chemotaxis assay showed that CL-11 had potent effects in stimulating neutrophil and mono/macrophage migration. Macrophage cytokine secretion experiment showed renal tubular debris is able to stimulate the macrophages to express more TNF-␣, IL-1, IL-6, TGF- (by ELISA). Renal fibroblast proliferation assay showed that supernatants from the macrophages co-cultured with renal tubular debris elevate fibroblast proliferation. Therefore, our findings demonstrate pathogenic roles for CL-11 in late phase of renal IR injury and suggest a novel mechanism of CL11 contributing to renal fibrosis through enhancing leukocyte infiltration and increasing tissue inflammation, which contributes to renal fibroblast proliferation and extracellular matrix production. http://dx.doi.org/10.1016/j.imbio.2016.06.076
http://dx.doi.org/10.1016/j.imbio.2016.06.075
1157
60
61
Activation of complement by pigment epithelium derived protein in synovial fluid of rheumatoid arthritis patients
Collectin-11 deficiency reduces renal inflammation and ameliorates tubulointerstitial fibrosis following renal ischemia reperfusion injury
Leonie M. Vogt 1,∗ , Simone Talens 1 , Carsten Scavenius 2 , Jan J. Enghild 2 , Tore Saxne 3 , Anna M. Blom 1 1
Lund University, Department of Translational Medicine, Malmö, Sweden 2 Aarhus University, Department of Molecular Biology and Genetics, Aarhus, Denmark 3 Lund University, Department of Clinical Sciences, Lund, Sweden Introduction: Rheumatoid arthritis (RA) is a disease in which complement activation leading to inflammation plays a detrimental role. Evidence for complement activation in synovial joints in arthritis comes both from clinical studies and animal models. However, it is unclear which molecules trigger complement activation under these conditions, and the aim of this study was to identify these activators. Methods: C4d, the final cleavage products resulting from activation of C4, is found in high amounts in the diseased joint, and it may bind covalently to complement-activating molecules. Therefore, we used a novel, highly specific antibody against a cleavage neoepitope in C4d for affinity purification of C4d in complexes with molecules, which were specifically bound to C4d, from pooled synovial fluid of four RA patients. As a control, pre-immune IgG from the same rabbit was coupled to a second column. Using mass spectrometry analysis, molecules eluted from both columns were identified. Those present only in the eluates from the anti-C4d column were subsequently studied for their ability to activate the classical complement pathway. Results: Besides immunoglobulins and several components of the classical pathway, one of the C4d binding molecules identified was pigment epithelium derived protein (PEDF). PEDF is a broadly expressed multifunctional member of the serine proteinase inhibitor (serpin) family. It plays critical roles in many physiological and pathophysiological processes, including neuroprotection, angiogenesis, fibrogenesis and inflammation. We expressed PEDF in eukaryotic cells and purified it using affinity chromatography. Upon subsequent testing with normal human serum, PEDF immobilized in a microtiter plate was found to induce C4b and C3b deposition. C1q-depleted serum, heat inactivated serum, and serum containing 20 mM EDTA all completely abrogated the complement activating potential of PEDF, indicating that PEDF operates through the classical pathway. Further, using specific antibodies we detected complexes between PEDF and C4d in the synovial fluid of RA patients. Furthermore, we detected direct binding of PEDF to apoptotic cells using flow cytometry, indicating potential for interference with recognition of these cells by complement. Conclusion: Despite the many beneficial effects PEDF exerts in the body, in the synovium of RA patients it may play a detrimental role by potentially activating the classical complement pathway.
Weiju Wu 1,∗ , Chengfei Liu 2 , Conrad A. Farrar 1 , Liang Ma 1 , Ke Li 2 , Steve H. Sacks 1 , Wuding Zhou 1 1
Medical Research Council (MRC) Centre for Transplantation, King’s College London, Guy’s Hospital, UK 2 Core Research Laboratory, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, China Collectin-11 (CL-11) is a collagen-containing lectin molecule that recognises carbohydrate residues on the cell surface. It has a wide tissue distribution and the kidney is a major site of the production. Our previous study showed that CL-11 detects stress-induces l-fucose pattern in response to ischemic insult and trigger complement activation to cause acute kidney injury. However, the impact of CL-11 on the late phase of renal ischemia reperfusion (IR) injury is unknown. In the present study, we tested whether renal inflammation and tubulointerstitial fibrosis following renal IR injury is dependent on CL-11. We employed a murine model of renal IR injury and CL-11−/− mice to determine the roles of CL-11 in renal inflammation and fibrosis. The results showed that compared to the wild littermates, CL-11−/− mice had significantly reduced renal tissue inflammation and fibrosis at day 7 and 10 after 30 min of bilateral renal IR injury, as evidenced by reduced: (i) impairment of renal function (by BUN), (ii) tissue injury (by PAS staining and histological scores), (iii) leukocyte infiltration (by flow cytometry and immunochemical staining for CD45+ , Ly6G+ , F4/80+ ), (iv) tubulointerstitial fibrosis (by Sirius red staining, RT-PCR for intrarenal collagen I and fibronectin expression), (v) intrarenal gene expression of proinflammatory (TNF-␣, IL-1, IL-6) and pro-fibrotic (TGF-) factors (by RT-PCR). To investigate cellular mechanisms that CL-11 contributes to renal inflammation and fibrosis we performed a series of in vitro experiments using freshly prepared peritoneal neutrophils or mono/macrophages and primarily cultured renal fibroblasts. Chemotaxis assay showed that CL-11 had potent effects in stimulating neutrophil and mono/macrophage migration. Macrophage cytokine secretion experiment showed renal tubular debris is able to stimulate the macrophages to express more TNF-␣, IL-1, IL-6, TGF- (by ELISA). Renal fibroblast proliferation assay showed that supernatants from the macrophages co-cultured with renal tubular debris elevate fibroblast proliferation. Therefore, our findings demonstrate pathogenic roles for CL-11 in late phase of renal IR injury and suggest a novel mechanism of CL11 contributing to renal fibrosis through enhancing leukocyte infiltration and increasing tissue inflammation, which contributes to renal fibroblast proliferation and extracellular matrix production. http://dx.doi.org/10.1016/j.imbio.2016.06.076
http://dx.doi.org/10.1016/j.imbio.2016.06.075
1157