Inhibition of the classical and lectin pathway of the complement system by recombinant LAIR-2.
ABSTRACT: Activation of complement may cause severe tissue damage in antibody-mediated allograft rejection and other antibody-mediated clinical conditions; therefore, novel potent complement inhibitors are needed. Previously, we described binding of the inhibitory receptor LAIR-1 and its soluble family member LAIR-2 to collagen. Here, we investigated binding of LAIR-1 and LAIR-2 to the complement proteins C1q and MBL, which both have collagen-like domains, and evaluated the effect of this binding on complement function. We demonstrate specific binding of recombinant LAIR proteins to both C1q and MBL. Surface plasmon resonance experiments showed that LAIR-2-Fc protein bound C1q and MBL with the highest affinity compared to LAIR-2-HIS. We, therefore, hypothesized that LAIR-2-Fc is a potent complement inhibitor. Indeed, LAIR-2-Fc inhibited C4 fixation to IgG or mannan, reduced activation of C4 by aggregated IgG in plasma and inhibited iC3b deposition on cells. Finally, LAIR-2-Fc inhibited complement-mediated lysis of cells sensitized with anti-HLA antibodies in an ex vivo model for antibody-mediated transplant rejection. Thus, LAIR-2-Fc is an effective novel complement inhibitor for the treatment and prevention of antibody-mediated allograft rejection and antibody-mediated clinical conditions.
Project description:C1q, the first component of complement, and leukocyte-associated Ig-like receptor 1 (LAIR-1; CD305), an inhibitory receptor expressed on hematopoietic cells, have both been associated with arrest of monocyte-derived dendritic cell (DC) differentiation and inhibition of Toll-like receptor activity in plasmacytoid DCs. Defects in both molecules have been implicated in susceptibility to, and progression of, systemic lupus erythematosus. Inhibitory signaling partners for C1q on monocytes and DCs remain undefined. Because C1q contains collagen-like motifs and LAIR-1 is a universal collagen receptor, we hypothesized that C1q is a functional ligand for LAIR-1. Binding analyses in cell-free systems and on the cell membrane demonstrate that C1q and its collagen tail associate with LAIR-1 and LAIR-2 (CD306), a soluble inhibitor of LAIR-1. Both C1q and its collagen tail trigger phosphorylation of LAIR-1 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in monocytes. Functional analyses show that C1q-mediated inhibition of monocyte-DC differentiation and C1q-mediated inhibition of IFN-? production by plasmacytoid DCs were both reversed by LAIR-2. Moreover, C1q-mediated inhibition of DC differentiation was reversed by LAIR-1 siRNA. Thus, C1q is a functional ligand for LAIR-1 restricting immune cell differentiation and activation. The discovery of C1q interactions with LAIR-1 and LAIR-2 lends much needed insight into molecular mechanisms operating to prevent the loss of tolerance, particularly in systemic lupus erythematosus.
Project description:Background:Antibody-mediated rejection (AMR) is a major cause of kidney allograft failure. Its molecular mechanisms are multifaceted and may include a role of complement activation via the classical pathway. Here, we investigated whether noninvasive complement monitoring adds predictive power to the diagnosis of AMR in the setting of donor-specific antibody (DSA) positivity. Methods:In this cross-sectional study, 741 kidney transplant recipients with stable graft function ?180 days posttransplantation were screened for the presence of human leukocyte antigen (HLA) alloantibodies. Eighty-three of 111 DSA-positive recipients underwent protocol biopsies and were tested for blood and urinary levels of complement proteins (C1q, C4, C3) and activation products (C4d, C3a, C5a, C5b-9). Results:Forty-seven recipients were diagnosed with AMR, and 21 were C4d-positive. While biopsy-confirmed AMR (and C4d) associated with DSA-binding strength (IgG mean fluorescence intensity of the immunodominant DSA versus AMR; area under the receiver operating characteristic curve: 0.76), tested complement markers did not have any predictive value for rejection (area under the receiver operating characteristic curve: 0.49-0.56). There were, however, tight correlations between complement activation products in urine and protein/creatinine ratio (? = 0.44-0.64; P < 0.001). Analysis of death-censored graft survival over a median of 60 months revealed no independent associations with levels of complement markers in blood or urine. Conclusions:Complement patterns in blood and urine failed to identify AMR in late biopsies and may have no relevant diagnostic value in this particular context.
Project description:The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.
Project description:C1q collagen-like region (CLR) engaging and activating the LAIR-1 inhibitory immunoreceptor represents a non-complement mechanism for maintaining immune quiescence. Given the binding promiscuity of C1q's globular region (gC1q), we hypothesized that C1q concurrently associates with distinct inhibitory immunoreceptors to produce C1q-mediated modulatory networking. Like LAIR-1, CD33 inhibitory immunoreceptors are highly expressed on monocytes. Binding CD33 restricts cell activation/differentiation; however, natural ligands for CD33 remain elusive. CD33 has IgC2-like domains potentially recognized by gC1q. Thus, we asked whether C1q binds to CD33 and if C1q mediates CD33/LAIR-1 crosslinking. Our findings demonstrate that C1q and gC1q interact with CD33 to activate its inhibitory motifs, while CLR does not. Whole C1q is required to crosslink CD33 and LAIR-1 and concurrently activate CD33/LAIR-1 inhibitory motifs. While C1q binds CD33C2 domains, decreased C1q-CD33 interactions resulting from sialic acid masking of CD33C2 domains suggests a process for regulating C1q-CD33 activity. Consistent with defective self-tolerance, CD33/LAIR-1 expression is reduced in systemic lupus erythematosus (SLE) myelomonocytes. The anti-inflammatory cytokine M-CSF, but not DC growth factors, sustains CD33/LAIR-1 expression on both healthy and SLE cells suggesting further biological control of C1q-CD33/LAIR-1 processes.
Project description:BACKGROUND:The association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts. METHODS:We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course. RESULTS:qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate. CONCLUSIONS:We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.
Project description:INTRODUCTION:Complement binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients. MATERIAL AND METHODS:A total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated. RESULTS:De-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients. CONCLUSION:Detection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.
Project description:Fc gamma receptors (Fc?Rs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 Fc?Rs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37-9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08-3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.
Project description:Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 ?g intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (Fc?R) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.
Project description:Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n?=?9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n?=?30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P?<?0.001 and P?<?0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P?<?0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments.
Project description:Alloantibody can contribute significantly to rejection of heart transplants by activation of complement and interactions with a variety of effector cells, including macrophages and monocytes through activating Fc?RI, Fc?RIII, Fc?RIV, the inhibitory Fc?RIIB and complement receptors. These receptors link cellular and humoral immunity by bridging the antibody specificity to effector cells. Activating Fc?Rs are also involved in serum amyloid P component (SAP)-mediated clearance of apoptotic bodies.B10.A (H-2a) hearts were transplanted into wild-type (WT) or Fc?RIII-knockout (KO) C57BL/6 (H-2b) mouse recipients. Levels of alloantibodies and SAP in the circulation were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Intragraft cytokine mRNA expression was measured by real-time polymerase chain reaction. Intragraft deposition of C4d, von Willebrand factor, SAP, and activated caspase 3 was visualized by immunochemistry.B10.A hearts in C57BL/6 Fc?RIII-KO recipients were rejected acutely within 6 to 8 days compared with 10 to 14 days in WT. The rejection in Fc?RIII-KO was accompanied by higher levels of circulating IgM/IgG alloantibodies and SAP than in WT recipients. Histology in Fc?RIII-KO cardiac allograft recipients indicated perivascular margination of monocytes and neutrophils, vascular endothelial cell injury, and intense vasculocentric infiltrates with extensive apoptosis. Higher numbers of apoptotic cells, stronger C4d and SAP deposition, and extensive activated caspase 3 were found in areas of dense pockets of apoptotic blebs in Fc?RIII-KO.We propose that absence of Fc?RIII is associated with the lack of efficient SAP-mediated clearance of apoptotic cells through Fc?Rs. Apoptotic cells become immunogenic and induce enhanced inflammation, alloantibody production, and complement activation leading to accelerated cardiac allograft rejection.