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, 53 (1-2), 99-110

Human Complement Receptor 2 (CR2/CD21) as a Receptor for DNA: Implications for Its Roles in the Immune Response and the Pathogenesis of Systemic Lupus Erythematosus (SLE)

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Human Complement Receptor 2 (CR2/CD21) as a Receptor for DNA: Implications for Its Roles in the Immune Response and the Pathogenesis of Systemic Lupus Erythematosus (SLE)

Rengasamy Asokan et al. Mol Immunol.

Abstract

Human CR2 is a B cell membrane glycoprotein that plays a central role in autoimmunity. Systemic lupus erythematosus (SLE) patients show reduced CR2 levels, and complete deficiency of CR2 and CR1 promotes the development of anti-DNA antibodies in mouse models of SLE. Here we show that multiple forms of DNA, including bacterial, viral and mammalian DNA, bind to human CR2 with moderately high affinity. Surface plasmon resonance studies showed that methylated DNA bound with high affinity with CR2 at a maximal K(D) of 6nM. DNA was bound to the first two domains of CR2 and this binding was blocked by using a specific inhibitory anti-CR2 mAb. DNA immunization in Cr2(-/-) mice revealed a specific defect in immune responses to bacterial DNA. CR2 can act as a receptor for DNA in the absence of complement C3 fixation to this ligand. These results suggest that CR2 plays a role in the recognition of foreign DNA during host-immune responses. This recognition function of CR2 may be a mechanism that influences the development of autoimmunity to DNA in SLE.

Conflict of interest statement

Conflict of interest

The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1
Figure 1A. Dose-dependent binding measured by ELISA of CR2 SCR1-15 to its plate-bound DNA ligands from viral, bacterial and mammalian origin. Binding was measured using non-blocking anti-CR2 mAb HB-5 that interacts with CR2 SCR3-4. Mean ± SD of three experiments is shown. Figure 1B. Cross-competition ELISA measuring relative ability of C3d, gp350 and DNA to block CR2 SCR1-15 interactions with plate-bound Micrococcus luteus DNA. Mean ± SD of three experiments is shown.
Figure 2
Figure 2
Surface plasmon resonance sensograms demonstrating the association and dissociation of protein ligands C3d (A) and EBVgp350 (B) as well as viral DNA fragments, 15 bp E1a DNA (C), 25 bp E1a DNA (D) and 100bp E1a DNA (E) with immobilized biotinylated CR2 SCR1-4-BCCP. Nanomolar concentrations of injected analyte are indicated at the right hand side of each sensogram. Solid lines are the result of global fitting analysis, and the colored lines are the normalized experimental sensogarm kinetic data.
Figure 3
Figure 3
Surface plasmon resonance sensograms demonstrating the association and dissociation of bacterial DNA ligands E. coli HMW DNA (A), Micrococcus luteus DNA (B) and Calf thymus chromatin (C) with immobilized biotinylated CR2 SCR1-4-BCCP. Nanomolar concentrations of injected analyte are indicated at the right hand side of each sensogram. Solid lines are the result of global fitting analysis, and the colored lines are the normalized experimental sensogarm kinetic data.
Figure 4
Figure 4
Surface plasmon resonance sensogram demonstrating the association and dissociation of structurally modified DNA including 16bp scrambled DNA (A) 50% CpG DNA (B) and 50% methylated DNA (C) with immobilized biotinylated CR2 SCR1-4-BCCP. Nanomolar concentrations of injected analyte are indicated at the right hand side of each sensogram. Solid lines are the result of global fitting analysis, and the colored lines are the normalized experimental sensogarm kinetic data.
Figure 5
Figure 5
Competition ELISA measuring relative ability of anti-CR2 mAbs to block soluble CR2 SCR1-15 binding to plate-bound DNA ligand, 25bp E1a DNA (A), PGEM plasmid DNA (B) E.coli HMW DNA (C) and Micrococcus luteus DNA (D). Binding was measured using anti-CR2 mAbs HB-5 that will bind to CR2 SCR3-4 and not compete with any of these four mAbs tested. Results demonstrate that mAbs 171, 1048, 994 and 629 are inhibitory in the same relative rank order of each ligand. Mean of three representative experiments is shown.
Figure 6
Figure 6
Titration of anti-DNA antibody production against Micrococcus luteus dsDNA immunized sera from C57BL/6 Cr2+/+, Cr2−/− and C3−/− mice. Micrococcus luteus dsDNA at 5 ug/ml was coated overnight at 4°C. Sera from immunized C57BL/6 Cr2+/+, Cr2−/− and C3−/− were serially diluted with PBS and loaded on the plate. Level of anti-DNA bound was probed with peroxidase-conjugated goat anti-mouse IgG. Mean ± SD for three experiments is shown. Cr2−/− mice re-immunized at day 14 showed reduced anti-DNA antibody production compared to Cr2+/+, p < 0.001; and C3−/− mice, p < 0.01; indicating impaired anti-DNA antibody production.

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