De novo synthesis of proteinase 3 by cytokine primed circulating human polymorphonuclear neutrophils and mononuclear cells.
OBJECTIVE
When polymorphonuclear neutrophils (PMN) and peripheral blood monocytes (PBMC) are stimulated with tumor necrosis factor alpha (TNF-alpha), preexisting granule stored proteinase 3 (PR3) is translocated to the surface 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 with TNF-alpha. They were harvested at different intervals and subjected to total RNA and protein analysis. PR3 mRNA was identified by reverse transcription polymerase chain reaction, Northern blot, and sequencing. De novo PR3 synthesis was evaluated by metabolic labeling with [35S] methionine followed by immunoprecipitation using anti-neutrophil cytoplasmic antibodies from serum of patients with active Wegener's granulomatosis and mouse monoclonal 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 comparison, in primed PBMC, PR3 mRNA appears at 6 h, peaks at 12 h, and disappears at 24 h. Immunoprecipitation of metabolically labeled PR3 revealed new synthesis of PR3 by both cell types, a process that was inhibited by cycloheximide.


CONCLUSIONS
Primed PMN and PBMC can express PR3 mRNA and synthesize new PR3 protein, providing an alternative source to membrane PR3. Whether that small amount of inducible PR3 has a primary structure, a localization, or a role different from those of preformed PR3 stored in granules remains to be clarified.
Zhou
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Department of Medicine, Centre universitaire de santé de l'Estrie, Faculty of Medicine, Université de Sherbrooke, Quebec, Canada.
Richard
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Ménard
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D001324:Autoantigens / Q000096:biosynthesis* / Q000235:genetics; D015152:Blotting, Northern; D014890:Granulomatosis with Polyangiitis / Q000097:blood*; D018922:HL-60 Cells / Q000201:enzymology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008213:Lymphocyte Activation; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology*; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D001324:Autoantigens; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D012697:Serine Endopeptidases; D053146:Myeloblastin