Complement receptor 3 mediates renal protection in experimental C3 glomerulopathy
Authors: Thomas D. Barbour, Guang Sheng Ling, Marieta M. Ruseva, Liliane Fossati-Jimack, H. Terence Cook, Marina Botto, Matthew C. Pickering
Journal: Kidney International (2016)

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh–/–), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh–/– mice. This effect was found to be dependent on CR3 expression on bone marrow–derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b–CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial. C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh–/–), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh–/– mice. This effect was found to be dependent on CR3 expression on bone marrow–derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b–CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial. C3 glomerulopathy (C3G) is a category of kidney diseases that includes dense-deposit disease, C3 glomerulonephritis, and complement factor H–related protein 5 (CFHR5) nephropathy.1Pickering M.C. D'Agati V.D. Nester C.M. et al.C3 glomerulopathy: consensus report.Kidney Int. 2013; 84: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar C3G is defined by the presence of isolated or dominant complement C3 within the glomerulus. It is frequently due to uncontrolled C3 activation via the complement alternative pathway, which may be genetic or acquired. Causes include deficiency or dysfunction of factor H (FH), the major plasma alternative pathway regulatory protein. Progression to end-stage kidney disease occurs in ∼40% of patients after 10 years, and recurrence after renal transplantation is common. At present, there is no proven treatment for C3G, although the C5 inhibitor eculizumab may have a role based on a small prospective trial and several case reports.2Bomback A.S. Smith R.J. Barile G.R. et al.Eculizumab for dense deposit disease and C3 glomerulonephritis.Clin J Am Soc Nephrol. 2012; 7: 748-756Crossref PubMed Scopus (253) Google Scholar Mice with homozygous deficiency of FH (Cfh–/–) have proved to be an informative experimental model of C3G.3Pickering M.C. Cook H.T. Warren J. et al.Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.Nat Genet. 2002; 31: 424-428Crossref PubMed Scopus (416) Google Scholar Cfh–/– mice spontaneously develop low plasma C3 and C5 levels and linear staining of C3 and C9 along the glomerular basement membrane (GBM). The development of glomerular C3 deposition is absolutely dependent on activation of C3 through the alternative pathway.3Pickering M.C. Cook H.T. Warren J. et al.Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.Nat Genet. 2002; 31: 424-428Crossref PubMed Scopus (416) Google Scholar Complement C3 along the GBM in these animals includes iC3b, a cleavage product of C3b.4Paixão-Cavalcante D. Hanson S. Botto M. et al.Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase.Mol Immunol. 2009; 46: 1942-1950Crossref PubMed Scopus (39) Google Scholar, 5Rose K.L. Paixão-Cavalcante D. Fish J. et al.Factor I is required for the development of membranoproliferative glomerulonephritis in factor H-deficient mice.J Clin Invest. 2008; 118: 608-618PubMed Google Scholar Complement receptor 3 (CR3), also known as Mac-1 (CD11b/CD18, αMβ2), is the main leukocyte receptor for iC3b.6Bajic G. Yatime L. Sim R.B. et al.Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3.Proc Natl Acad Sci U S A. 2013; 110: 16426-16431Crossref PubMed Scopus (91) Google Scholar, 7Beller D.I. Springer T.A. Schreiber R.D. Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor.J Exp Med. 1982; 156: 1000-1009Crossref PubMed Scopus (405) Google Scholar CR3 is a β2–integrin receptor expressed mainly on neutrophils, monocytes and macrophages, and dendritic cells.8Tan S.M. The leucocyte beta2 (CD18) integrins: the structure, functional regulation and signalling properties.Biosci Rep. 2012; 32: 241-269Crossref PubMed Scopus (112) Google Scholar The α-chain (CD11b), encoded by the ITGAM gene, confers binding of CR3 to a wide range of endogenous and pathogen-derived ligands. CR3 mediates diverse cellular functions including phagocytosis of iC3b-coated particles, cytotoxicity, chemotaxis, and cell adhesion,9Ross G.D. Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein.Crit Rev Immunol. 2000; 20: 197-222Crossref PubMed Google Scholar which play a critical role in regulating inflammation and antimicrobial immunity. In vitro and in vivo observations have also revealed a role for CR3 in toll-like receptor (TLR)–triggered innate immune responses, but the nature of this cross-talk remains controversial, with some studies demonstrating that CR3 can promote TLR-induced inflammation,10Han C. Jin J. Xu S. et al.Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.Nat Immunol. 2010; 11: 734-742Crossref PubMed Scopus (338) Google Scholar, 11Wang Z. Jiang J. Li Z. et al.A myeloid cell population induced by Freund adjuvant suppresses T-cell-mediated antitumor immunity.J Immunother. 2010; 33: 167-177Crossref PubMed Scopus (26) Google Scholar, 12Zhang M. Han Y. Han C. et al.The beta2 integrin CD11b attenuates polyinosinic:polycytidylic acid-induced hepatitis by negatively regulating natural killer cell functions.Hepatology. 2009; 50: 1606-1616Crossref PubMed Scopus (26) Google Scholar whereas others have reported negative roles for this integrin in TLR responses.13Hirahashi J. Mekala D. Van Ziffle J. et al.Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy.Immunity. 2006; 25: 271-283Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Zhou H. Liao J. Aloor J. et al.CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses.J Immunol. 2013; 190: 115-125Crossref PubMed Scopus (66) Google Scholar CR3 has been implicated in the pathophysiology of glomerulonephritis (GN). An allelic variant of ITGAM is a risk factor for systemic lupus erythematosus, including renal manifestations.15Kim-Howard X. Maiti A.K. Anaya J.M. et al.ITGAM coding variant (rs1143679) influences the risk of renal disease, discoid rash and immunological manifestations in patients with systemic lupus erythematosus with European ancestry.Ann Rheum Dis. 2010; 69: 1329-1332Crossref PubMed Scopus (67) Google Scholar Experiments in mice with homozygous deficiency of CR3 (Itgam–/–) have shown a deleterious role for CR3 in acute glomerular injury such as the heterologous nephrotoxic nephritis (NTN) model.16Hirahashi J. Hishikawa K. Kaname S. et al.Mac-1 (CD11b/CD18) links inflammation and thrombosis after glomerular injury.Circulation. 2009; 120: 1255-1265Crossref PubMed Scopus (66) Google Scholar, 17Tang T. Rosenkranz A. Assmann K.J. et al.A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis.J Exp Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar In contrast, a protective role for CR3 was demonstrated in experimentally triggered immune complex GN.18Alexander J.J. Chaves L.D. Chang A. et al.CD11b is protective in complement-mediated immune complex glomerulonephritis.Kidney Int. 2015; 87: 930-939Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 19Rosetti F. Tsuboi N. Chen K. et al.Human lupus serum induces neutrophil-mediated organ damage in mice that is enabled by Mac-1 deficiency.J Immunol. 2012; 189: 3714-3723Crossref PubMed Scopus (52) Google Scholar These differences have been attributed to "context-dependent" immune effects of CR3.19Rosetti F. Tsuboi N. Chen K. et al.Human lupus serum induces neutrophil-mediated organ damage in mice that is enabled by Mac-1 deficiency.J Immunol. 2012; 189: 3714-3723Crossref PubMed Scopus (52) Google Scholar The contribution of CR3 to the spontaneous renal pathology associated with FH deficiency is unknown. We investigated the effect of CR3 on the renal phenotype of Cfh–/– mice by intercrossing these animals with the Itgam–/– strain. We observed that CR3 deficiency exacerbated spontaneous renal disease associated with complete FH deficiency and that this was dependent on the expression of CR3 on bone marrow (BM)–derived cells. We also observed that the Itgam–/– strain were more susceptible to renal damage during accelerated NTN (ANTN). The in vitro stimulation of CR3 with iC3b-coated targets limited the proinflammatory cytokine profile triggered by lipopolysaccharide (LPS) in murine and human macrophages. In contrast, cytokine secretion was enhanced in neutrophils, indicating that the modulating effects of CR3 differ between monocytes/macrophages and neutrophils. Together our data indicate that strategies to promote the interaction of CR3 with iC3b and other potential ligands in the kidney may be a suitable therapeutic goal in C3G. The role of CR3 in experimental spontaneous C3G was assessed by generating mice with combined deficiency of FH and CR3 (Cfh–/–.Itgam–/–). Cohorts of female Cfh–/–.Itgam–/– (n = 10) and Cfh–/– (n = 5) mice were monitored in specific pathogen–free (SPF) conditions for 8 months. While plasma and glomerular C3 remained normal in Itgam–/– mice, Cfh–/–.Itgam–/– animals developed low plasma C3 and abnormal GBM C3 accumulation comparable to that seen in Cfh–/– animals (Figure 1). However, Cfh–/–.Itgam–/– animals developed greater renal inflammation at 8 months (Table 1). At this time point albuminuria was significantly increased in Cfh–/–.Itgam–/– compared to Cfh–/– mice (median 173.2 μg/16 h, range 72.5–987.2, vs. 74.8 μg/16 h, range 33.8–141.2, P = 0.028, Mann-Whitney test) together with greater glomerular cell counts (Table 1). A single Cfh–/–.Itgam–/– mouse developed hematuria and distress necessitating killing at 6 months. In this mouse only, diffuse crescentic GN was evident on light microscopy. In a second cohort of female Cfh–/–.Itgam–/– (n = 12) and Cfh–/– (n = 12) mice housed in non-SPF conditions over an 8-month period, we noted significantly reduced survival in Cfh–/–.Itgam–/– animals (Table 1). Renal histology was not available from the 6 Cfh–/–.Itgam–/– animals that died prior to the 8-month time point. Of the 6 Cfh–/–.Itgam–/– animals culled at the 8-month time point, hematuria and albuminuria were present in 4. Glomerular cellularity was similar between the Cfh–/– and Cfh–/–.Itgam–/– groups, but the number of glomerular macrophages was significantly increased in the Cfh–/–.Itgam–/– mice (Table 1). These data indicated that CR3 deficiency increased mortality and spontaneous glomerular inflammation associated with FH deficiency and that the size of this effect was influenced by the environmental conditions. We next determined whether CR3 on BM-derived cells was responsible for this effect.Table 1Spontaneous renal disease in FH-deficient mice with or without CR3Specific pathogen–free conditionsNon–specific pathogen–free conditionsGenotypeCfh–/–.Itgam–/–Cfh–/–Cfh–/–.Itgam–/–Cfh–/–Mice, number/sex10/F5/F12/F12/FSurvival at 8 months, % (number)90% (9)100% (5)50% (6)cP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.100% (12)Plasma urea, median mmol/l (range)13.3 (9.8–44.5)11.5 (7.3–15.1)13.5 (10.1–39.2)dP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.12.9 (9.7–25.7)Glomerular cell count, median (range)53.4 (40.8–75.9)aP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.36.6 (34.9–44.4)44.2 (39.2–61.7)dP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.41.7 (35.9–56.1)Glomerular macrophage count, median (range)4.3 (0.9–9.9)1.9 (1.2–3.1)8.5 (5.1–14.5)bP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.,dP = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point.3.7 (0.9–11.0)F, female.a P = 0.003. bP = 0.0007, Mann-Whitney Test. cP = 0.006, log-rank test. dData refer to the 6 mice that were culled at the 8-month time point. Open table in a new tab F, female. Three groups of irradiated Cfh–/– mice were reconstituted with Itgam–/– (n = 8), wild-type (n = 7), or Cfh–/– (n = 9) BM-derived cells. At 8 months, Cfh–/– recipients of Itgam–/– BM-derived cells showed significantly increased plasma urea, glomerular cellularity, and glomerular macrophage counts (Figure 2). Notably, renal parameters between recipients of wild-type and Cfh–/– BM-derived cells did not differ. These data suggested that the deleterious effect of CR3 deficiency on the renal disease associated with FH deficiency was mediated by the lack of CR3 expression on BM-derived rather than intrinsic renal cells. We next explored the effects of CR3 deficiency on experimental renal disease independent of FH deficiency. Itgam–/– mice showed no evidence of an abnormal spontaneous renal phenotype at 8 months in either SPF (n = 6F) or non-SPF (n = 10F) conditions. Specifically, hematuria did not develop in these animals, and glomerular histology at 8 months by light microscopy was normal (data not shown). We then speculated that CR3 deficiency could influence the response of the kidney during experimentally triggered renal inflammation. To investigate this we performed ANTN in Itgam–/– (n = 8) and wild-type (n = 10) mice. Two days after injection of nephrotoxic serum, hematuria was detectable in 7 of the 8 Itgam–/– mice but absent in all wild-type animals. Ten days after administration of nephrotoxic serum, when all animals were culled, the Itgam–/– mice had significantly greater hematuria, plasma urea levels, and glomerular crescents (Figure 3). Glomerular macrophage counts were no different between groups. Glomerular deposition of sheep and mouse IgG and antigen-specific plasma titers of mouse IgG did not differ between the groups (data not shown). These data indicated that CR3 deficiency exacerbated renal injury during ANTN. Since iC3b is abundantly present within the glomerulus in experimental C3G and complement is activated during ANTN, we hypothesized that the interaction between iC3b and CR3 on myeloid cells within the kidney resulted in an anti-inflammatory response that in turn reduced the severity of the renal injury. To investigate this we assessed the effect of iC3b-coated guinea pig red blood cells (gRBCs) on LPS-induced cytokine secretion by murine myeloid cells (both monocytes and macrophages at day 2 and day 7 of differentiation in vitro). Preincubation with iC3b-gRBCs resulted in reduced secretion of interleukin-6 (IL-6), but enhanced secretion of IL-10 (Figure 4). These cytokine changes were CR3-dependent since they were not observed when we used macrophages from Itgam–/– mice (Supplementary Figure S1A online). We then examined human cells preincubated with iC3b-coated beads (Figure 5). We initially noticed that human monocyte-derived macrophages display a progressive loss of CD11b expression during differentiation in vitro, most likely due to adherence to plastic. By day 7, the expression of CD11b, including its active form assessed with the CBRM1/5 antibody, was barely detectable (Supplementary Figure S1B). Therefore, we chose to study monocytes and monocyte-derived macrophages at day 2 only. In both cell types iC3b pre-ligation downmodulated the proinflammatory cytokine profile triggered by LPS and promoted an anti-inflammatory response (Figure 5). In contrast, the cytokine effect of iC3b-coated beads on TLR4-stimulated neutrophils was proinflammatory with significantly increased production of IL-8 and CCL3 (MIP-1α). These cytokine effects were observed regardless of whether the cells were exposed to iC3b-coated beads before, simultaneously, or 1 hour after LPS challenge (data not shown). We also analyzed cytokine production after overnight stimulation with iC3b-coated particles alone or medium alone. Neither of these conditions induced detectable cytokines, demonstrating no endotoxin contamination (data not shown). Taken together these data demonstrate that the cytokine modulating effects mediated by CR3 through the binding of iC3b are distinct and cell type–specific.Figure 5Cytokine response of human cells to preincubation with iC3b-coated beads followed by lipopolysaccharide (LPS) stimulation. Monocytes (n = 20) (top), day 2 monocyte-derived macrophages (n = 29) (middle), and neutrophils (n = 26) (bottom) were preincubated with iC3b-coated beads for 1 hour prior to LPS stimulation (10 ng/ml). Cytokines were quantified using a bead multiplex assay, and the levels with and without CR3 pre-engagement with iC3b are shown. Data are expressed as mean ± SEM, paired t-test. P values are indicated. IL, interleukin; TNF, tumor necrosis factor.View Large Image Figure ViewerDownload (PPT) Progressive renal disease in Cfh–/– mice is associated with accumulation of C3 activation fragments including iC3b along the GBM. Here we show increased severity of the spontaneous glomerular phenotype in 8-month-old Cfh–/– mice with coexisting deficiency of CR3, the leukocyte receptor for iC3b. Disease exacerbation was more marked in non-SPF compared to SPF housing conditions. This protective effect was dependent on CR3 expression on BM-derived cells. Glomerular macrophages were increased in Cfh–/– animals reconstituted with Itgam–/– BM-derived cells, suggesting that these macrophages were contributing to the protective effect. In order to determine whether CR3-dependent glomerular protection also occurred outside the setting of FH deficiency, we investigated the response of Itgam–/– mice to ANTN. Early hematuria developed exclusively in Itgam–/– mice, persisting until day 10 when these animals had evidence of severe renal injury with crescentic GN. These observations indicated that CR3 mediated a protective effect during subacute renal inflammation, that is, at a time when macrophages are present within the glomeruli. CR3-mediated signaling is usually viewed as proinflammatory, and prevention of CR3–iC3b interaction by antibody or genetic deletion has been shown to decrease the severity of inflammatory responses in several animal models including heterologous NTN.16Hirahashi J. Hishikawa K. Kaname S. et al.Mac-1 (CD11b/CD18) links inflammation and thrombosis after glomerular injury.Circulation. 2009; 120: 1255-1265Crossref PubMed Scopus (66) Google Scholar, 17Tang T. Rosenkranz A. Assmann K.J. et al.A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis.J Exp Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar Hence the findings that CR3 provided a beneficial effect in our spontaneous model of C3G in Cfh–/– mice were unexpected, and suggest that strategies to block CR3-dependent inflammation may not be beneficial as previously thought. An explanation for the contradictory role of CR3 in various disease models10Han C. Jin J. Xu S. et al.Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.Nat Immunol. 2010; 11: 734-742Crossref PubMed Scopus (338) Google Scholar, 20Bullard D.C. Hu X. Schoeb T.R. et al.Critical requirement of CD11b (Mac-1) on T cells and accessory cells for development of experimental autoimmune encephalomyelitis.J Immunol. 2005; 175: 6327-6333Crossref PubMed Scopus (67) Google Scholar, 21Ehirchiou D. Xiong Y. Xu G. et al.CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation.J Exp Med. 2007; 204: 1519-1524Crossref PubMed Scopus (127) Google Scholar, 22Shimizu K. Libby P. Shubiki R. et al.Leukocyte integrin Mac-1 promotes acute cardiac allograft rejection.Circulation. 2008; 117: 1997-2008Crossref PubMed Scopus (24) Google Scholar may lie in the predominant role played by the different innate or adaptive immune cells in these experimental conditions and the fact that CR3 operates in a cell type–specific manner. Similarly, previous data in C3-deficient mice demonstrated that C3 contributed to renal injury in the acute phase of heterologous NTN.23Sheerin N.S. Springall T. Abe K. et al.Protection and injury: the differing roles of complement in the development of glomerular injury.Eur J Immunol. 2001; 31: 1255-1260Crossref PubMed Scopus (40) Google Scholar However, C3-deficient animals developed greater renal injury during the autologous phase of heterologous NTN. In the same study C3 deficiency was also associated with exacerbation of renal injury in ANTN. We speculated that in our model of spontaneous renal pathology associated with FH deficiency the renal protective effect was mediated by an interaction between iC3b and CR3 on BM-derived macrophages. We therefore examined the CR3-dependent cytokine responses of myeloid cells to iC3b-coated targets. We studied the macrophage cytokine response to TLR4 ligation, which plays a deleterious role in several models of GN, including ANTN.24Giorgini A. Brown H.J. Sacks S.H. et al.Toll-like receptor 4 stimulation triggers crescentic glomerulonephritis by multiple mechanisms including a direct effect on renal cells.Am J Pathol. 2010; 177: 644-653Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Here, we found that the interaction of iC3b with CR3 on both murine and human monocytes and macrophages is associated with an enhanced anti-inflammatory cytokine profile (reduced IL-6 and increased IL-10 secretion) following LPS stimulation. These data are consistent with earlier studies indicating that engagement of CR3 inhibited secretion of proinflammatory cytokines by TLR4-stimulated monocytes and macrophages.10Han C. Jin J. Xu S. et al.Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.Nat Immunol. 2010; 11: 734-742Crossref PubMed Scopus (338) Google Scholar, 25Cao C. Gao Y. Li Y. et al.The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b.J Clin Invest. 2010; 120: 1971-1980Crossref PubMed Scopus (100) Google Scholar, 26Huynh L. Wang L. Shi C. et al.ITAM-coupled receptors inhibit IFNAR signaling and alter macrophage responses to TLR4 and Listeria monocytogenes.J Immunol. 2012; 188: 3447-3457Crossref PubMed Scopus (23) Google Scholar, 27Wang L. Gordon R.A. Huynh L. et al.Indirect inhibition of Toll-like receptor and type I interferon responses by ITAM-coupled receptors and integrins.Immunity. 2010; 32: 518-530Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Pre-ligation with a synthetic CD11b agonist has also been shown to inhibit human macrophage secretion of tumor necrosis factor-α in response to synthetic TLR7/8 ligands.28Reed J.H. Jain M. Lee K. et al.Complement receptor 3 influences toll-like receptor 7/8-dependent inflammation: implications for autoimmune diseases characterized by antibody reactivity to ribonucleoproteins.J Biol Chem. 2013; 288: 9077-9083Crossref PubMed Scopus (39) Google Scholar Our data showing an anti-inflammatory effect due to iC3b ligation of CR3 on macrophages are also consistent with earlier studies showing downregulation of proinflammatory cytokines during phagocytosis of complement-opsonized apoptotic cells.29Mevorach D. Mascarenhas J.O. Gershov D. et al.Complement-dependent clearance of apoptotic cells by human macrophages.J Exp Med. 1998; 188: 2313-2320Crossref PubMed Scopus (569) Google Scholar, 30Ricklin D. Hajishengallis G. Yang K. et al.Complement: a key system for immune surveillance and homeostasis.Nat Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2460) Google Scholar In addition, our data support the notion that the cytokine modulating effects of the iC3b–CR3 interaction are cell type–specific, since the effect on human neutrophils was proinflammatory. This opposite response mediated by CR3 to LPS stimulation may explain the conflicting results in the literature, including the deleterious role of CR3 in heterologous NTN, since this model mainly depends on the initial glomerular neutrophil influx.16Hirahashi J. Hishikawa K. Kaname S. et al.Mac-1 (CD11b/CD18) links inflammation and thrombosis after glomerular injury.Circulation. 2009; 120: 1255-1265Crossref PubMed Scopus (66) Google Scholar, 17Tang T. Rosenkranz A. Assmann K.J. et al.A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis.J Exp Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar Additional immune mechanisms may have contributed to our finding of CR3-mediated protection in the ANTN model. Firstly, ANTN has been extensively characterized in C57BL/6 mice as involving a T helper 1–predominant, delayed-type hypersensitivity–like nephritogenic immune response.31Holdsworth S.R. Kitching A.R. Tipping P.G. Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis.Kidney Int. 1999; 55: 1198-1216Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar T helper 17–directed cellular immune responses have also been implicated in several murine models of crescentic nephritis, including ANTN.32Steinmetz O.M. Summers S.A. Gan P.Y. et al.The Th17-defining transcription factor RORγt promotes glomerulonephritis.J Am Soc Nephrol. 2011; 22: 472-483Crossref PubMed Scopus (72) Google Scholar Both enhanced delayed-type hypersensitivity and T helper 17 immune differentiation have previously been demonstrated in Itgam–/– mice using a model of low-dose oral antigen (ovalbumin) exposure followed by high-dose immunization with antigen and complete Freund's adjuvant (CFA).21Ehirchiou D. Xiong Y. Xu G. et al.CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation.J Exp Med. 2007; 204: 1519-1524Crossref PubMed Scopus (127) Google Scholar Secondly, CR3 may negatively regulate CFA-induced proinflammatory signaling, with renal injury in the ANTN model being dependent on the sensitization phase induced by IgG mixed with CFA. CFA is a recognized TLR agonist,33Billiau A. Matthys P. Modes of action of Freund's adjuvants in experimental models of autoimmune diseases.J Leukoc Biol. 2001; 70: 849-860PubMed Google Scholar, 34Lim S.K. Freund adjuvant induces TLR2 but not TLR4 expression in the liver of mice.Int Immunopharmacol. 2003; 3: 115-118Crossref PubMed Scopus (25) Google Scholar and increased renal injury due to TLR4 ligation specifically during sensitization has been demonstrated in ANTN studies.24Giorgini A. Brown H.J. Sacks S.H. et al.Toll-like receptor 4 stimulation triggers crescentic glomerulonephritis by multiple mechanisms including a direct effect on renal cells.Am J Pathol. 2010; 177: 644-653Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar A protective role for CR3 was also reported in a passive transfer model of GN in lupus-prone (New Zealand black × New Zealand white)F1 with selective neutrophil transgenic expression of human FcγRIIA or