De novo synthesis of proteinase 3 by cytokine primed circulating human polymorphonuclear neutrophils and mononuclear cells

Objective. When polymorphonuclear neutrophils (PMN) and peripheral blood mo nocytes (PBMC) are stimulated with tumor necrosis factor alpha (TNF-alpha), preexisting granule stored proteinase 3 (PR3) is translocated to the surfa ce of their plasma membrane. We investigated whether PR3 gene reactivation and new PR3 protein production were also features of priming by cytokine. Methods. Normal human PMN and PBMC were isolated and stimulated in vitro wi th TNF-alpha. They were harvested at different intervals and subjected to t otal RNA and protein analysis. PR3 mRNA was identified by reverse transcrip tion polymerase chain reaction, Northern blot, and sequencing. De novo PR3 synthesis was evaluated by metabolic labeling with [S-35] methionine follow ed by immunoprecipitation using anti-neutrophil cytoplasmic antibodies from serum of patients with active Wegener's granulomatosis and mouse monoclona l anti-native PR3 antibodies. Results. Resting PMN and PBMC do not express PR3 mRNA. During priming, PR3 mRNA appears in PMN at 2 h, peaks at 6 h, and has disappeared at 12 h. By c omparison, in primed PBMC, PR3 mRNA appears at 6 h, peaks at 12 h, and disa ppears at 24 h. Immunoprecipitation of metabolically labeled PR3 revealed n ew synthesis of PR3 by both cell types, a process that was inhibited by cyc loheximide. Conclusion. Primed PMN and PBMC can express PR3 mRNA and synthesize new PR3 protein, providing an alternative source to membrane PR3. Whether that sma ll amount of inducible PR3 has a primary structure, a localization, or a ro le different from those of preformed PR3 stored in granules remains to be c larified.