Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

Complement is a rat natural resistance factor to amoebic liver infection.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica This illness is prevalent in poor countries causing 100,000 deaths worldwide. Knowledge of the natural resistance mechanisms of rats to amoebic liver abscess (ALA) development may help to discover new pathogenic factors and to design novel therapeutic strategies against amoebiasis. In this work, histologic analyses suggested that the complement system may play a central role in rat natural resistance to ALA. E. histolytica trophozoites disappeared from rat liver within 6 h post-infection with minimal or no inflammatory infiltrate. In vitro findings indicate that rat complement was lethal for the parasite. Furthermore, hamsters became resistant to ALA by intravenous administration of fresh rat serum before infection. The amoebicidal potency of rat complement was 10 times higher than hamster complement and was not related to their respective CH50 levels. The alternative pathway of complement plays a central role in its toxicity to E. histolytica since trypan blue, which is a C3b receptor inhibitor, blocks its amoebicidal activity. These results suggest that amoebic membrane affinity, high for C3b and/or low for Factor H, in comparison with the hamster ones, may result in higher deposition of membrane complex attack on parasite surface and death.

Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

Low CR1 (C3b receptor) level on erythrocytes is associated with poor prognosis in hemodialysis patients.

BACKGROUND: Erythropoietin in patients under dialysis treatment for renal failure is low which induces anemia. Treatment with recombinant erythropoietin (rEPO) has been used routinely as a supplement treatment for these patients. Immune complexes (IC) react with complement and bind to CR1 on erythrocytes (E-CR1), and are transported to the liver and/or spleen where IC removal and degradation occurs. The erythrocytes then return to circulation where they bind to additional IC. There are some patients whose E-CR1 expression is low with chronic anemia in spite of rEPO treatment. We hypothesized that in hemodialysis (HD) patients altered host defense against infection is associated with low levels of E-CR1. We examined if low E-CR1 in dialysis patients constitutes a risk factor for reduced host defense and poor outcome. METHODS: In 95 HD patients, E-CR1 was quantified using a monoclonal E-CR1 antibody and FACS analysis followed by clinical course studies for 5 years. RESULTS: The patients were divided into three groups by E-CR1 level. Percent survival for the low E-CR1 group (53.3%) was significantly lower than the high E-CR1 group (86.4%) (p < 0.01). There were more hepatitis C virus-positive patients within the low E-CR1 group (27.3%) than in the high E-CR1 group (4.7%) (p < 0.05). Furthermore, 10 patients with the lowest E-CR1 levels had severe complications, notably infection at an arteriovenous fistula. CONCLUSION: A reduced E-CR1 level might be a risk factor for reduced host defense and can be used as a predicting factor for poor prognosis in a HD patient.

Importance of complement 3 and mannose receptors in phagocytosis of Paracoccidioides brasiliensis conidia by Nramp1 congenic macrophages lines.

Genetic factors influence susceptibility to Paracoccidioidomycosis, a Latin American endemic mycosis. The pattern of susceptibility of congenic mouse strains infected with Paracoccidioides brasiliensis resembles the pattern of the Nramp1 gene. Thus, congenic murine bone-marrow-derived macrophage lines B10R (Nramp1rGly169) and B10S (null Nramp1 protein expression, Nramp1sAsp169) were infected with P. brasiliensis conidia and compared, under opsonic and nonopsonic conditions. Opsonization increased the percentage of phagocytosis by both cell lines. B10R macrophages exhibited a higher percentage of cells with associated conidia and higher number of conidia per macrophage than B10S. Heat-inactivation and EDTA treatment of serum used for opsonization, and treatment of macrophages with anti-complement receptor 3 (CR3) decreased phagocytosis by both cell lines. alpha-methyl-d-mannoside reduced phagocytosis by B10R macrophages, suggesting that the mannose receptor participates in phagocytosis by these cells. The CR3 expression was similar on both cell lines and B10R expressed more mannose receptors, but neither cell line expressed CR1. IFNgamma decreased the conversion of conidia to the yeast form of P. brasiliensis in B10R, but not in B10S macrophages.

CR1 on erythrocytes of Brazilian systemic lupus erythematosus patients: the influence of disease activity on expression and ability of this receptor to bind immune complexes opsonized with complement from normal human serum.

Hypocomplementaemia and low expression of CR1 on erythrocytes (E) of patients with systemic lupus erythematosus (SLE) are associated with defective clearance of circulating immune complexes (IC) and so they may have pathogenic significance. Here, we investigated whether the reduced CR1/E in SLE patients per se might affect the binding of IC to CR1/E. First, we analysed the expression of CR1 on E of active (n=30) and inactive (n=34) SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry. Both groups of patients had a significantly reduced CR1/E expression compared with healthy controls (n=40). It was also observed that the number of E bearing CR1 was reduced in both groups of SLE patients studied. Second, we determined the functional activity of CR1/E by measuring the binding to E of FITC-bovine serum albumin (BSA)/rabbit anti-BSA complexes, formed at equivalence, which were opsonized with complement from normal human serum (NHS). On the other hand, we did not find differences between the patient and control groups in the ability of E to bind IC/NHS. There was also a positive correlation between the CR1/E expression and the number of E bearing CR1 in control and inactive SLE groups, which was not observed in the group of active SLE patients. Considering the involvement of low levels of complement and CR1/E expression on complex processing, in this in vitro model the results show that an effective coating of the complexes with complement is sufficient to bind them preferentially to CR1 over normal levels of receptor expression.

The C4A and C4B isotypic forms of human complement fragment C4b have the same intrinsic affinity for complement receptor 1 (CR1/CD35).

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, (1101)PCPVLD and (1101)LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were Cs converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of C4b, once again there was little difference in the K(D) values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction.

Localization of Knops system antigens in the long homologous repeats of complement receptor 1.

BACKGROUND: The Sl(a) (Knops system) located on complement receptor 1 (CR1) has been associated with malarial rosetting, a process associated with severe malarial infections. Moreover, the long homologous repeats (LHRs) B and C of CR1 were implicated in rosette formation. As a step toward mapping the location of Knops system antigens, truncated CR1 proteins have been expressed and their ability to inhibit antibodies to the high-incidence Knops system antigens was assessed. STUDY DESIGN AND METHODS: Individual LHRs (A, B, C, and D) of CR1 of the common CR1*1 (F) allotype were expressed as secreted forms in 293T cells. Their abilities to specifically neutralize Knops system antibodies were tested by both hemagglutination and flow cytometry. RESULTS: Three examples of anti-Kn(a) (n = 6) were almost completely inhibited by LHR-C and three by LHR-D. Two examples of anti-McC(a) (n = 2) and seven examples of anti-Sl(a) (n = 8) were inhibited by LHR-D. Both examples of anti-Yk(a) (n = 2) were partially inhibited by LHR-D. CONCLUSION: The high-incidence Knops system antigens reside within LHR-D and to a lesser extent within LHR-C. Because of the role of Sl(a) antigen in malaria rosetting, these results indicate that LHR-D may represent an additional malaria interaction region in CR1.

Comparison of the suppressive effects of soluble CR1 and C5a receptor antagonist in acute arthritis induced in rats by blocking of CD59.

We investigated the effects of suppression of complement activation at C3 level and inhibition of C5a on acute synovitis in rats. Acute synovitis was induced in Wistar rats by intra-articular (i.a.) injection into one knee of 0.3 mg of MoAb 6D1 (anti-rat CD59 antibody). In the treatment groups, soluble CR1 (sCR1) or C5a receptor (C5aR) antagonist was administered intra-articularly or intravenously and effects on the course of the acute synovitis were monitored. Synovitis induced by 6D1 was characterized by joint swelling, thickening of synovial tissue, cellular infiltration and deposition of membrane attack complex (MAC) on the synovial surface. Neither inflammatory change nor MAC deposition was found in rats which received an i.a. injection of sCR1 to suppress complement activity in the joint. Intra-articular injection of sCR1 did not reduce plasma complement activity. Intravenous administration of sCR1 suppressed plasma complement activity but had no effect on the course of the arthritis and synovitis with MAC deposition was observed. Neither i.a. nor i.v. injection of C5aR antagonist had any suppressive effects on inflammatory change or MAC deposition in synovium. The data show that inflammatory change induced by 6D1 was mediated by local complement activation and was not accompanied by systemic complement activation. C5a generation was not responsible for the observed inflammation, suggesting that other complement activation products, possibly MAC, mediate the inflammatory change observed in this model of acute synovitis in rats.

A soluble recombinant multimeric anti-Rh(D) single-chain Fv/CR1 molecule restores the immune complex binding ability of CR1-deficient erythrocytes.

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the alpha-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.

The roles of complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) in the regulation of the immune complex-elicited respiratory burst of polymorphonuclear leukocytes in whole blood.

The binding of immune complexes (IC) to polymorphonuclear leukocytes (PMN) and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% human serum, using flow cytometry. Blockade of the complement receptor (CR)1 receptor sites for C3b on whole blood cells using the monoclonal antibody (mAb) 3D9 resulted in a 1.9-fold increase in the IC-elicited PMN RB after 5 min of incubation, rising to 3.1-fold after 40 min. This enhancement was not due to increased IC deposition on PMN. Blockade of CR3 abrogated the mAb 3D9-induced rise in RB activity and inhibited the IC binding to PMN in a whole blood cell preparation, with or without mAb 3D9, by approximately 40% from 15-40 min while reducing their RB over 40 min to approximately one third. Blockade of CR1 on either erythrocytes (E) or leukocytes, before mixing the populations, revealed that the potentiation of the RB by mAb 3D9 was associated with abrogation of E-CR1 function, whereas blockade of leukocyte-CR1 had a diminishing effect. Exposure to IC at high concentrations induced release of both specific and azurophilic granule contents from PMN. The latter was CR3 dependent in that blockade of the receptor inhibited the lactoferrin release by one third during 40 min of incubation. In conclusion, CR3 plays a significant role in the IC-mediated generation of an RB and release of specific granules by PMN, while CR1 on whole blood cells, primarily E CR1, restricts the IC-elicited RB in PMN. We propose that CR1 in whole blood promotes the degradation of IC-bound iC3b to C3dg, thereby rendering the IC inaccessible for binding to CR3.

Adherence of Salmonella typhimurium to murine peritoneal macrophages is mediated by lipopolysaccharide and complement receptors.

Adherence of Salmonella typhimurium to mouse peritoneal macrophages (Mø) was monitored using a direct microscopic assay and flow cytometry. Competitive binding studies using wild-type lipopolysaccharide and derivatives confirmed a role for this moiety in bacterial adherence. Mø pretreated with 2-deoxy-D-glucose exhibited lower binding activity than did untreated controls, suggesting involvement of either Fc or complement receptors. Pre-exposing Mø to Fc fragments, however, failed to reduce bacterial binding, thus eliminating a role for Fc receptors in this process. Mø pretreated with neutrophil elastase exhibited a diminished ability to bind S. typhimurium, suggesting involvement of complement receptor 1. Monoclonal antibodies M1/70 and M18/2, specific for epitopes on the alpha and beta chains, respectively, of complement receptor 3, also blocked this adherence. In each case we were unable to eliminate completely bacterial adhesion to Mø. Monoclonal antibodies to two additional Mø receptors, Mac-2 and Mac-3, did not block bacterial attachment. These data indicate that multiple mechanisms are involved in the initial adhesion of S. typhimurium to mouse Mø.

Reduction in erythrocyte complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55) during normal pregnancy.

RBC-membrane CR1 has been assayed in pregnancy and at 48 h postpartum. It has been shown that RBC CR1 is reduced as pregnancy progress, reaching a nadir in the third trimester and that it returns to levels approaching normal within 48 h postpartum. Further, there is a reduced expression of RBC decay accelerating factor (DAF) during pregnancy but no change in expression of RBC CD59. We suggest that the reduction in RBC CR1 and DAF may reflect increased levels of circulating immune complexes and consequent increased complement activation in pregnancy.

Antibodies to murine complement receptor 1 and 2 can inhibit the antibody response in vivo without inhibiting T helper cell induction.

Administration to mice of mAb against CR1/CR2 inhibits the primary and secondary in vivo Ab response. We have addressed the question of whether induction of T helper cells, in addition to B cells, is inhibited by this treatment. A hapten-carrier system was used, where one group of mice was immunized with a monoclonal CR1/CR2-specific Ab and Ag (SRBC or keyhole limpet hemocyanin (KLH)), another with Ag alone, and a third group left nonimmunized. After 2 to 15 wk, spleen cells from these mice were transferred to irradiated syngeneic recipients, together with Ag coupled to the hapten 4-hydroxy-5-iodo-3-nitro-phenacetyl (NIP). The number of NIP-specific, IgG-producing B cells, which in this system is an indication of T cell priming, was determined in an ELISPOT assay. Although the primary SRBC- or KLH-specific IgG response was inhibited markedly (74-98%) by CR1/CR2-specific Ab, no decrease in the number of NIP-specific IgG-producing B cells was detected after secondary immunization. In sharp contrast, the SRBC- or KLH-specific secondary IgG responses were low or undetectable. These findings suggest that CR1/CR2-specific Ab can inhibit the primary Ab response without affecting T helper cells and that the induction of B cell memory is decreased markedly by this treatment.