Anti-C1q receptor/calreticulin autoantibodies in patients with systemic lupus erythematosus (SLE)

Abstract

SLE is a disease characterized by the presence of multiple autoantibodies and high levels of circulating immune complexes. We studied the presence and functional relevance of autoantibodies directed against a receptor for the collagen-like stalks of the first subcomponent of complement, also known as calreticulin (cC1qR/CaR), in patients with SLE. In a cross-sectional study it was found that higher titres of antibodies against cC1qR/CaR are present in sera of SLE patients compared with normal donors. No association between anti-cC1qR/CaR titres and SLE disease activity was found. Following gel filtration of SLE serum it was found that anti-cC1qR/CaR reactivity is associated with the peak of monomeric IgG. Purified IgG from patients was able to specifically immunoprecipitate cC1qR/CaR. Since we have shown previously that cC1qR/CaR is able to inhibit the haemolytic activity of Clq, we determined a possible pathogenic role for anti-cC1qR/CaR on complement regulation. IgG derived from SLE serum reversed the inhibitory capacity of cC1qR/CaR in a dose-dependent fashion up to 63%, whereas IgG from normal donors had no significant effect. With respect to the capacity of anti-cC1qR/CaR antibodies to activate neutrophils, it was found that incubation of normal neutrophils with F(ab')2 anti-cC1qR/CaR resulted in a very limited oxidative burst. However, cross-linking of F(ab')2 anti-cC1qR/CaR on the neutrophils clearly induced neutrophil activation. Pre-incubation of the SLE-derived F(ab')2 with cC1qR/CaR prevented activation of neutrophils up to 81+/-5%. These results suggest that the presence of anti-cC1qR/CaR antibodies in patients with SLE may modulate complement and neutrophil activation.

Fig. 1

Anti-cC1qR/CaR titres in normal human sera and SLE sera. Purified human cC1qR/CaR was coated and incubated with sera of normal donors (ND) or SLE patients. Subsequently, bound IgG was measured by incubation with an anti-human IgG MoAb conjugated to digoxigenin. The optical density was measured and plotted. —, Average; ----, ND average + 2 s.d.

Fig. 2

Dose-dependent binding of anti-cC1qR/CaR antibodies to cC1qR/CaR. Purified cC1qR/CaR or bovine serum albumin (BSA) was coated on microtitre wells and incubated with increasing concentrations of three SLE sera in the presence of 5 m NaCl. Bound IgG was detected by incubation with a MoAb anti-human IgG conjugated to digoxigenin. The optical density was measured and plotted.

Fig. 3

IgG isolated from SLE serum binds cC1qR/CaR. IgG from SLE serum was isolated by gel filtration using a Sepharose S-300 column. In the fractions IgG (□) and anti-cC1qR/CaR (▪) were determined by ELISA and C1q content (○) was determined by a haemolytic assay.

Fig. 4

Immunoprecipitation of cC1qR/CaR by purified IgG from SLE. cC1qR/CaR was conjugated to biotin and incubated with purified IgG from SLE serum or normal donor (ND) serum. Alternatively, SLE IgG that was preincubated with increasing amounts of purified cC1qR/CaR was added to cC1qR/CaR-Bio and incubated. After precipitation using Prot G, the precipitate was electrophoresed on a polyacrylamide gel and blotted on PVDF membranes. CC1qR/CaR-Bio was demonstrated by subsequent incubation with streptavidin–horseradish peroxidase (HRP) and DAB.

Fig. 5

Reversal of cC1qR/CaR activity by SLE IgG. Purified cC1qR/CaR was incubated with either increasing concentrations of nornal donor (ND) IgG (•) or SLE IgG (▪). After incubation with antibody-sensitized erythrocytes (EA) and a limited amount of C1q in C1qD serum the percentage reversal on the inhibitory effect of cC1qR/CaR was plotted.

Fig. 6

Neutrophil activation by SLE F(ab′)₂. Freshly isolated neutrophils were incubated with different dilutions of F(ab′)₂ isolated from normal donor (ND) serum or SLE serum in the presence of cytochrome C. As a positive control, neutrophils were stimulated with phorbol myristate acetate (PMA). Alternatively, neutrophils were incubated with either ND F(ab′)₂ or SLE F(ab′)₂. After washing, goat IgG anti-human IgG was added together with cytochrome C to X-link bound anti-cC1qR/CaR F(ab′)₂. After incubation, the optical density (OD) of the supernatants was measured and the percentage oxygen radical release, compared with release induced by PMA stimulation, was plotted (a). To determine the specificity, F(ab′)₂ isolated from SLE serum was preincubated with different concentrations of purified cC1qR/CaR before being added to neutrophils. After an incubation the OD of the supernatant was measured and the percentage release of oxygen radicals was calculated and plotted (b).