Monoclonal anti‐double stranded DNA antibody is a leucocyte‐binding protein to up‐regulate interleukin‐8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils

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Abstract
Objectives. To determine whether anti‐double stranded DNA (anti‐dsDNA) autoantibody could bind and affect the functions of normal human polymorphonuclear neutrophils (PMN). Methods. Normal human PMN were incubated with different concentrations of a monoclonal mouse anti‐dsDNA antibody (12B3) or mouse isotype‐matched IgG2a. The binding of anti‐dsDNA and PMN was measured by flow cytometry and interleukin‐8 (IL‐8) gene expression in PMN was detected by enzyme‐linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction (RT–PCR). PMN apoptosis was justified by morphological changes. The cognate antigen(s) of anti‐dsDNA on the PMN surface was identified by membrane biotinylation, immunoprecipitation and Western blot. Results. The binding of PMN with anti‐dsDNA was much higher than with non‐specific mouse IgG2a (70.8 vs 2.0%). Anti‐dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced the production and mRNA expression of IL‐8 by PMN. However, anti‐dsDNA facilitated PMN apoptosis after 3 h incubation. Western blot analysis of biotinylated PMN cell lysates demonstrated that a 50–52 kDa membrane molecule is the cognate antigen of anti‐dsDNA. Conclusions. Anti‐dsDNA autoantibody up‐regulates IL‐8 gene expression and elicits activation‐induced cell death (AICD) of human PMN via binding to a 50–52 kDa membrane‐expressed molecule.