Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion.

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice.

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.

CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues.

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

Characterization of human complement receptor type 2 (CR2/CD21) as a receptor for IFN-alpha: a potential role in systemic lupus erythematosus.

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.

Prevention of collagen-induced arthritis in mice transgenic for the complement inhibitor complement receptor 1-related gene/protein y.

The objective of these studies was to examine collagen-induced arthritis (CIA) in C57BL/6 mice transgenic for the rodent complement regulatory protein complement receptor 1-related gene/protein y (Crry) (Crry-Tg), a C3 convertase inhibitor. The scores for clinical disease activity and for histological damage in the joints were both significantly decreased in Crry-Tg mice in comparison to wild-type (WT) littermates. The production of both IgG1 and IgG2a anti-collagen Abs was reduced in the Crry-Tg mice, although spleen cell proliferation in response to collagen type II was not altered. The production of IFN-gamma, TNF-alpha, and IL-1beta by LPS-stimulated spleen cells was decreased, and IL-10 was increased, in cells from Crry-Tg mice in comparison to WT. The steady-state mRNA levels for IFN-gamma, TNF-alpha, and IL-1beta were all decreased in the joints of Crry-Tg mice in comparison to WT. The synovium from Crry-Tg mice without CIA contained the mRNA for the Crry transgene, by RT-PCR, and the synovium from transgenic mice with CIA exhibited little deposition of C3 protein by immunohistological analysis. These results suggest that suppression of CIA in Crry-Tg mice may be due to enhanced synthesis of Crry locally in the joint with decreased production of proinflammatory cytokines.

Administration of a soluble recombinant complement C3 inhibitor protects against renal disease in MRL/lpr mice.

Complement receptor 1-related gene/protein y (Crry) in rodents is a potent membrane complement regulator that inhibits complement C3 activation by both classical and alternative pathways. To clarify the role of complement in lupus nephritis, MRL/lpr mice were given Crry as a recombinant protein (Crry-Ig) from 12 to 24 wk of age. Control groups were given saline or normal mouse IgG. Sera and urine were collected biweekly. Only 1 of 20 (5%) Crry-Ig-treated mice developed renal failure (BUN > 50 mg/dl) compared with 18 of 38 (47.4%) mice in control groups (P = 0.001). BUN levels at 24 wk were reduced from 68.8 +/- 9.7 mg/dl in control groups to 38.5 +/- 3.9 mg/dl in the Crry-Ig-treated group (P < 0.01). Urinary albumin excretion at 24 wk was also significantly reduced from 5.3 +/- 1.4 mg/mg creatinine in the control groups to 0.5 +/- 0.2 mg/mg creatinine in the Crry-Ig-treated group (P < 0.05). Of the histologic data at 24 wk, there was a significant reduction in scores for glomerulosclerosis and C3d, IgG, IgG3, and IgA staining intensity in glomeruli in complement-inhibited animals. Crry-Ig-treated animals were also protected from vasculitic lesions. Although there was no effect on relevant autoimmune manifestations such as anti-double stranded DNA titers or cryoglobulin IgG3 levels, circulating immune complex levels were markedly higher in complement-inhibited animals. Thus, inhibition of complement activation with Crry-Ig significantly reduces renal disease in MRL/lpr lupus mice. The data support the strategy of using recombinant complement C3 inhibitors to treat human lupus nephritis.

Inhibiting the complement system does not reduce injury in renal ischemia reperfusion.

The complex pathogenesis of ischemia reperfusion injury (IRI) includes endothelial expression of adhesion molecules, leukocyte recruitment and activation, reactive oxygen species production, and apoptotic and necrotic cell death. A role for complement in IRI of different organs, including kidney, has been proposed on the basis of results of experiments that used pharmacologic inhibitors as well as animals that were deficient in individual complement proteins. Here, renal IRI in mice was examined. Animals that were deficient in C3 had partial protection from IRI induced by 27.5 min of bilateral renal ischemia, followed by 20 h of reperfusion (blood urea nitrogen [BUN] values, 46.6 +/- 6.9 and 68.4 +/- 7.9 mg/dl in C3 -/- and C3 +/+ mice; n = 7 and 8, respectively; P = 0.033). Given the reduction in IRI in C3 -/- mice, it was investigated, by use of the rodent C3 convertase inhibitor CR1-related gene/protein y-Ig (Crry-Ig), whether exogenous administration of a complement inhibitor could lessen renal injury. Despite the use of Crry-Ig in high doses, there was no significant reduction of injury induced by 20 to 30 min of ischemia followed by up to 30 h of reperfusion. Histologic examination revealed acute tubular necrosis and neutrophilic infiltration, both of which correlated significantly with BUN values (P < 0.001). Of interest, C3 deposition around renal tubules was significantly less in animals with IRI, compared with that in unmanipulated controls (P < 0.001). In Crry-Ig-treated animals, C3 deposition was inversely proportional to BUN values (r = -0.63; P < 0.001), which presumably indicates that severe vascular IRI allowed access of the 160 kD Crry-Ig to the interstitium. Thus, renal IRI in mice may have a partial complement dependence, yet pharmacologic inhibition of the complement system does not seem to be effective, likely because of the presence of other mediator systems that operate in parallel.

Transgenic expression of a soluble complement inhibitor protects against renal disease and promotes survival in MRL/lpr mice.

To investigate the role of complement in lupus nephritis, we used MRL/lpr mice and a transgene overexpressing a soluble complement regulator, soluble CR1-related gene/protein y (sCrry), both systemically and in kidney. Production of sCrry in sera led to significant complement inhibition in Crry-transgenic mice relative to littermate transgene negative controls. This complement inhibition with sCrry conferred a survival advantage to MRL/lpr mice. In a total of 154 animals, 42.5% transgene-negative animals had impaired renal function (blood urea nitrogen > 50 mg/dl) compared with 16.4% mice with the sCrry-producing transgene (p < 0.001). In those animals that died spontaneously, MRL/lpr mice with the sCrry-producing transgene did not die of renal failure, while those without the transgene did (blood urea nitrogen values of 46.6 +/- 9 and 122 +/- 29 mg/dl in transgene-positive and transgene-negative animals, respectively; p < 0.001). Albuminuria was reduced in those transgenic animals in which sCrry expression was maximally stimulated (urinary albumin/creatinine = 12.4 +/- 4.3 and 36.9 +/- 7.7 in transgene-positive and transgene-negative animals, respectively; p < 0.001). As expected in the setting of chronic complement inhibition, there was less C3 deposition in glomeruli of sCrry-producing transgenic mice compared with transgene-negative animals. In contrast, there was no effect on glomerular IgG deposition, levels of anti-dsDNA Ab and rheumatoid factor, or spleen weights between the two groups. Thus, long-term complement inhibition reduces renal disease in MRL/lpr mice, which translates into improved survival. MRL/lpr mice in which complement is inhibited still have spontaneous mortality, yet this is not from renal disease.