Ongoing Immunoglobulin Class Switch DNA Recombination in Lupus B Cells: Analysis of Switch Regulatory Regions

Abstract

Inflammation and tissue damage in systemic lupus erythematosus (SLE) are mediated by class-switched autoantibodies reactive with nucleic acids, nucleic acid-binding proteins, phospholipids and other self-antigens. While some healthy individuals produce IgM antibodies with specificities similar to those of lupus patients, immunoglobulin class switching to mature downstream isotypes appears to be required for the generation of pathogenic autoantibodies. To characterize the cellular and molecular basis of pathogenic autoantibody production in SLE, we studied the capacity of peripheral blood B cells of naïve phenotype from patients with SLE, rheumatoid arthritis (RA) or healthy control subjects to spontaneously switch to IgG and IgA. In addition, we determined the DNA sequences of the upstream evolutionary conserved sequence (ECS)-Iγ promoter regulatory regions that control germline I_H-C_H transcription and class switch DNA recombination (CSR) to IgG₁, IgG₂ and IgG₄. IgM⁺IgD⁺ B cells from patients with SLE, but not those from RA or healthy control subjects, underwent spontaneous CSR, as assessed by expression of germline Iγ1-Cγ1, Iγ2-Cγ2, Iγ3-Cγ3, Iγ4-Cγ4 and Iα1-Cα1 transcripts, mature (switched) V_HDJ_H-Cγ1, V_HDJ_H-Cγ2, V_HDJ_H-Cγ3 and V_HDJ_H-Cα1 transcripts and secreted IgG and IgA. Although polymorphic DNA sequences were identified in the ECS-Iγ1, ECS-Iγ2 and ECS-Iγ4 promoter regions, the transcription factor-binding sites that mediate germline Iγ-Cγ transcription were conserved in patients and controls. However, distinct patterns of nuclear protein binding to an ECS-Iγ promoter sequence that contains both positive and negative regulatory elements were observed in SLE patients and controls. These results support a role for exogenous signals, such as through CD40 ligation, rather than altered genomic sequence, in the increased production of class switched autoantibodies in SLE.

• Immunoglobulin
• DNA
• Lupus B cells
• SLE
• Polymorphism
• BLyS, B lymphocyte stimulator
• C, constant region
• CSR, class switch DNA recombination
• D, diversity region
• ECS, evolutionarily conserved sequence
• EMSA, electrophoretic mobility shift assay
• H, heavy chain
• I, intervening/initiation of transcription region
• Ig, immunoglobulin
• J, joining region
• PBMC, peripheral blood mononuclear cells
• RA, rheumatoid arthritis
• S, switch region
• SLE, systemic lupus erythematosus
• SRE, S regulatory element
• Th, T helper cell
• V, variable region