Female but Not Male Mice Deficient in Soluble IgM Are Susceptible to Chemically Induced Glomerular Injury.

B cell-targeted therapies are effective for treating multiple different kidney diseases in humans and also protect mice from Adriamycin nephropathy. Because glomerular IgM is frequently seen in both humans and mice with "nonimmune" forms of glomerular disease, we hypothesized that natural IgM binds to epitopes displayed in the injured glomerulus, exacerbating injury. To test this hypothesis, we induced Adriamycin nephropathy in BALB/C mice that cannot secrete soluble IgM (sIgM-/- mice) and compared them with BALB/C controls. Contrary to our prediction, we found that female sIgM-/- mice developed higher mortality and more severe kidney injury after injection of Adriamycin. The absence of soluble IgM did not reduce glomerular complement activation, and IgG was seen deposited within the injured glomeruli. Furthermore, we discovered that female sIgM-/- mice have higher levels of anti-cardiolipin IgG, and that IgG from these mice binds to epitopes in the injured kidney. These findings indicate that natural IgM may prevent generation of autoreactive IgG. Circulating levels of anti-cardiolipin IgG decreased after induction of kidney injury in female mice, consistent with deposition of the Abs in injured tissues. Better understanding of the mechanisms by which the immune system modulates and amplifies kidney injury may enable the development of targeted therapies to slow kidney disease progression.

Highly pathogenic natural monoclonal antibody B4-IgM recognizes a post-translational modification comprised of acetylated N-terminal methionine followed by aspartic or glutamic acid.

The natural monoclonal antibody B4-IgM recognizes murine annexin 4 (mAn4) and exacerbates ischemia-reperfusion injury in many mouse models. During apoptosis, the intracellular mAn4 protein translocates to the membrane surface, remaining attached to the outer membrane leaflet where it is recognized by the anti-mAn4 B4-IgM antibody. B4-IgM does not recognize human annexin 4 (hAn4). However, the B4-IgM antibody epitope was detected by Western blot of unknown human proteins and by flow cytometry on all studied human cell lines undergoing apoptosis and on a minor subset of healthy cells. The B4-IgM antibody also recognizes the epitope on necrotic cells in cytoplasmic proteins, apparently entering through pores large enough to allow natural antibodies to penetrate the cells and bind to the epitope expressed on self-proteins. Using proteomics and site-directed mutagenesis, we found that B4-IgM binds to an epitope with post-translationally modified acetylated N-terminal methionine, followed by either glutamic or aspartic acid. The epitope is not induced by apoptosis or injury because this modification can also occur during protein translation. This finding reveals an additional novel mechanism whereby injured cells are detected by natural antibodies that initiate pathogenic complement activation through the recognition of epitopes that are shared across multiple proteins found in variable cell lines.

Noninvasive Detection of iC3b/C3d Deposits in the Kidney Using a Novel Bioluminescent Imaging Probe.

SIGNIFICANCE STATEMENT: Histologic quantification of complement C3 deposits in kidney biopsies provides prognostic information in patients with glomerulonephritis. Unfortunately, kidney biopsies are invasive procedures that cannot be performed regularly and only provide a snapshot of a small portion of one kidney at the time of sampling. We have developed a method to noninvasively detect specific C3 fragment deposition throughout both kidneys, using a monoclonal antibody targeting tissue-bound iC3b/C3d linked to a bioluminescent resonance energy transfer construct that emits near-infrared light. In a mouse model of glomerulonephritis, the probe detected iC3b/C3d in kidneys of live mice by bioluminescent imaging. This demonstrates that noninvasive imaging with an anti-iC3b/C3d probe can be used to monitor inflammation in the kidneys.

Factor H related proteins modulate complement activation on kidney cells.

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney.

Natural antibody and complement activation characterize patients with idiopathic nephrotic syndrome.

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) are common forms of idiopathic nephrotic syndrome. The causes of these diseases are incompletely understood, but the response of patients to immunosuppressive therapies suggests that their pathogenesis is at least in part immune mediated. Preclinical and clinical research indicates that activation of the classical pathway of complement contributes to glomerular injury in FSGS. Glomerular IgM deposits are also prominent in some patients, raising the possibility that IgM is a trigger of classical pathway activation. In the present study, we examined the pattern of complement activation in the glomeruli and plasma of patients with nephrotic syndrome. We also tested whether patients with FSGS and MCD have elevated levels of natural IgM reactive with epitopes on glomerular endothelial cells and cardiolipin. We found evidence of classical pathway activation in patients with idiopathic nephrotic syndrome compared with healthy control subjects. We also detected higher levels of self-reactive IgM to both targets. Based on these results, IgM and classical pathway activation may contribute to disease pathogenesis in some patients with FSGS and MCD.NEW & NOTEWORTHY IgM is detected in biopsies from some patients with nephrotic syndrome, although this has been attributed to passive trapping of the protein. We found, however, that IgM colocalizes with complement activation fragments in some glomeruli. We also found that affected patients had higher levels of IgM reactive to glomerular endothelial cell epitopes. Thus, IgM activates the complement system in the glomeruli of some patients with nephrotic syndrome and may contribute to injury.

Detection of pro angiogenic and inflammatory biomarkers in patients with CKD.

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-ß as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients' plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.

Complement factor H-deficient mice develop spontaneous hepatic tumors.

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

Complement fragments are biomarkers of antibody-mediated endothelial injury.

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.

Targeting the Immune Complex-Bound Complement C3d Ligand as a Novel Therapy for Lupus.

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment-derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2-/- mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d-CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/lpr model of SLE.

Endothelial Microparticles and Systemic Complement Activation in Patients With Chronic Kidney Disease.

BACKGROUND: Endothelial microparticles are associated with chronic kidney disease (CKD) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD. METHODS AND RESULTS: We analyzed complement data of 30 healthy subjects, 30 patients with stage III/IV CKD, and 30 renal transplant recipients with stage III/IV CKD, evaluating the potential correlation of complement fragments with brachial artery flow-mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post-kidney transplant CKD. Plasma Ba levels correlated significantly with lower brachial artery flow-mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro. CONCLUSION: The alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD. Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD-associated vascular disease. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02230202.

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury.

Modulation of the Alternative Pathway of Complement by Murine Factor H-Related Proteins.

Factor H (FH) is a key alternative pathway regulator that controls complement activation both in the fluid phase and on specific cell surfaces, thus allowing the innate immune response to discriminate between self and foreign pathogens. However, the interrelationships between FH and a group of closely related molecules, designated the FH-related (FHR) proteins, are currently not well understood. Whereas some studies have suggested that human FHR proteins possess complement regulatory abilities, recent studies have shown that FHR proteins are potent deregulators. Furthermore, the roles of the FHR proteins have not been explored in any in vivo models of inflammatory disease. In this study, we report the cloning and expression of recombinant mouse FH and three FHR proteins (FHR proteins A-C). Results from functional assays show that FHR-A and FHR-B proteins antagonize the protective function of FH in sheep erythrocyte hemolytic assays and increase cell-surface C3b deposition on a mouse kidney proximal tubular cell line (TEC) and a human retinal pigment epithelial cell line (ARPE-19). We also report apparent KD values for the binding interaction of mouse C3d with mouse FH (3.85 µM), FHR-A (136 nM), FHR-B (546 nM), and FHR-C (1.04 µM), which directly correlate with results from functional assays. Collectively, our work suggests that similar to their human counterparts, a subset of mouse FHR proteins have an important modulatory role in complement activation. Further work is warranted to define the in vivo context-dependent roles of these proteins and determine whether FHR proteins are suitable therapeutic targets for the treatment of complement-driven diseases.

Distinct roles for the complement regulators factor H and Crry in protection of the kidney from injury.

Mutations in the complement regulatory proteins are associated with several different diseases. Although these mutations cause dysregulated alternative pathway activation throughout the body, the kidneys are the most common site of injury. The susceptibility of the kidney to alternative pathway-mediated injury may be due to limited expression of complement regulatory proteins on several tissue surfaces within the kidney. To examine the roles of the complement regulatory proteins factor H and Crry in protecting distinct renal surfaces from alternative pathway mediated injury, we generated mice with targeted deletions of the genes for both proteins. Surprisingly, mice with combined genetic deletions of factor H and Crry developed significantly milder renal injury than mice deficient in only factor H. Deficiency of both factor H and Crry was associated with C3 deposition at multiple locations within the kidney, but glomerular C3 deposition was lower than that in factor H alone deficient mice. Thus, factor H and Crry are critical for regulating complement activation at distinct anatomic sites within the kidney. However, widespread activation of the alternative pathway reduces injury by depleting the pool of C3 available at any 1 location.

Annexin A2 Enhances Complement Activation by Inhibiting Factor H.

Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography-mass spectrometry, we identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media, the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but it can also improve complement-mediated bacterial clearance.

Complement Activation in Patients with Focal Segmental Glomerulosclerosis.

BACKGROUND: Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. STUDY DESIGN: Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. SETTING AND PARTICIPANTS: We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. OUTCOMES: Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). MEASUREMENTS: Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. RESULTS: Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. LIMITATIONS: Limited number of patients with samples from all time-points. CONCLUSIONS: The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.

IgM exacerbates glomerular disease progression in complement-induced glomerulopathy.

Although glomerular immunoglobulin M (IgM) deposition occurs in a variety of glomerular diseases, the mechanism of deposition and its clinical significance remain controversial. Some have theorized IgM becomes passively trapped in areas of glomerulosclerosis. However, recent studies found that IgM specifically binds damaged glomeruli. Therefore, we tested whether natural IgM binds to neo-epitopes exposed after insults to the glomerulus and exacerbates disease in mice deficient in the complement regulatory protein factor H; a model of non-sclerotic and nonimmune-complex glomerular disease. Immunofluorescence microscopy demonstrated mesangial and capillary loop deposition of IgM, whereas ultrastructural analysis found IgM deposition on endothelial cells and subendothelial areas. Factor H-deficient mice lacking B cells were protected from renal damage, as evidenced by milder histologic lesions on light and electron microscopy. IgM, but not IgG, from wild-type mice bound to cultured murine mesangial cells. Furthermore, injection of purified IgM into mice lacking B cells bound within the glomeruli and induced proteinuria. A monoclonal natural IgM-recognizing phospholipids also bound to glomeruli in vivo and induced albuminuria. Thus, our results indicate specific IgM antibodies bind to glomerular epitopes and that IgM contributes to the progression of glomerular damage in this mouse model of non-sclerotic glomerular disease.

Cyclosporine induces endothelial cell release of complement-activating microparticles.

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases.

The role of the complement system in acute kidney injury.

Acute kidney injury is a common and severe clinical problem. Patients who develop acute kidney injury are at increased risk of death despite supportive measures such as hemodialysis. Research in recent years has shown that tissue inflammation is central to the pathogenesis of renal injury, even after nonimmune insults such as ischemia/reperfusion and toxins. Examination of clinical samples and preclinical models has shown that activation of the complement system is a critical cause of acute kidney injury. Furthermore, complement activation within the injured kidney is a proximal trigger of many downstream inflammatory events within the renal parenchyma that exacerbate injury to the kidney. Complement activation also may account for the systemic inflammatory events that contribute to remote organ injury and patient mortality. Complement inhibitory drugs have now entered clinical use and may provide an important new therapeutic approach for patients suffering from, or at high risk of developing, acute kidney injury.

Renal ischemia-reperfusion injury amplifies the humoral immune response.

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

Detection of complement activation using monoclonal antibodies against C3d.

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.