Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA)

Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA).

Background

Anti-neutrophil cytoplasmic autoantibodies (ANCA) induce neutrophil activation in vitro with release of injurious products that can mediate necrotizing vasculitis in vivo. The importance of ANCA IgG F(ab′)₂-antigen binding versus Fcγ receptor engagement in this process is controversial. We propose that ANCA-antigen binding affects cell signaling pathways that can result in changes of gene expression.

Methods

Microarray GeneChip analysis and real-time, quantitative PCR (TaqMan^®) was used to probe for transcripts in leukocytes from patients (in vivo gene expression study) and in leukocytes treated with ANCA IgG or ANCA-F(ab′)₂ (in vitro gene expression study).

Results

Microarray gene chip analysis showed that ANCA IgG and ANCA-F(ab′)₂ stimulate transcription of a distinct subset of genes, some unique to whole IgG, some unique to F(ab′)₂ fragments, and some common to both. DIF-2, COX-2, and IL-8 were identified as genes responsive to ANCA signaling and were selected for in depth evaluation. In vitro DIF-2 and IL-8 were increased by both ANCA IgG and F(ab′)₂, but COX-2 only by MPO-ANCA F(ab′)₂. In vivo DIF-2 levels were increased in leukocytes of ANCA patients, which correlated strongly with disease activity and ANCA titer. DIF-2 was not increased in patients in remission or in disease control patients (systemic lupus erythematosus and IgA nephropathy). COX-2 gene expression was significantly increased in patients with active disease, while IL-8 was increased in remission.

Conclusions

The data indicate that leukocyte genes are activated in vitro by both ANCA Fc and ANCA F(ab′)₂ pathways and that in vitro activation mimics changes in circulating leukocytes of patients with ANCA disease. Increased levels of DIF-2 in patient leukocytes strongly correlate with severity of disease in kidney tissue. The observations indicate a previously unrecognized role for DIF-2 in ANCA-mediated inflammation, which raises the possibility that DIF-2 has an important role in other types of inflammation.