Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation

Abstract

We have previously reported inhibition of cell-free activation of the neutrophil superoxidegenerating NADPH oxidase by a soluble cationic protein of neutrophil granules and by low concentrations of human defensin. Subcellular fractionation carried out in the current study indicated that the inhibitory substance was derived from azurophilic granules, was released into the medium on cell stimulation, and was resistant to phenylmethylsulfonyl fluoride (PMSF). Phorbol ester was the most effective stimulus for the release of the blocking activity. The possibility was raised that granule protein(s) act in vivo as negative modulators of superoxide production. Gel filtration of granule extract revealed a markedly retarded protein peak exhibiting oxidase-blocking activity and containing lysozyme as the main protein. Because lysozyme did not exert inhibitory effects on oxidase activation, association of the inhibitory protein with lysozyme was assumed. Indeed a column of immobilized lysozyme retained a fraction of the granule extract's oxidase-blocking activity. Elution with a low-pH buffer recovered a component capable of inhibition of the NADPH oxidase in stimulated neutrophils and in the cell-free system. The main 29-kDa protein band in the eluted fraction was identified as proteinase 3, a serine protease of azurophilic granules. Enzymatically active as well as PMSF-blocked conventionally purified proteinase 3 interfered with phorbol myristate acetateinduced superoxide release. These findings support the hypothesis that exocytosed granule constituents may prevent excessive activation of the NADPH oxidase.

The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcγRIIIb and cytochrome b558

“…MhCD pretreatment reduced the rate of PMA-elicited superoxide release (4,5,6,7) and after (4V , 5V , 6V , 7V ) activation with PMA, fmet-leu-phe, or TNFa. Neutrophils isolated from different donors were used for activation with each agonist.…”

“…Cells (2 Â 10 8 /ml) in 10 mM potassium phosphatebuffered saline (pH 7.0) supplemented with 1 mM EGTA, 3.5 mM phenylmethylsulfonyl fluoride (PMSF), 15 Ag/ml leupeptin, were disrupted by a brief sonication [6]. Unbroken cells were removed by low-speed (250 Â g) centrifugation.…”

“…Human polymorphonuclear leukocytes (PMNs) were isolated from fresh buffy coats by standard procedures of dextran sedimentation, hypotonic lysis of erythrocytes, and Ficoll-Hypaque density gradient centrifugation [6,24]. Isolated cells were resuspended in Krebs -Ringer phosphate (KRP)-buffered solution (131 mM NaCl, 15.7 mM NaPi, pH 7.4, 5.2 mM KCl, 2 mM glucose, 1.3 mM MgSO 4 , 0.9 mM CaCl 2 ).…”

“…À production O 2 À production was measured as superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (0.8 mg/ml) at 550 nm in the Thermomax microplate reader (Molecular Devices) [6].…”

“…PR3 is a multifunctional protein [4], which may participate not only in killing of invading microorganisms, but also in myeloid differentiation [5], regulation of NADPH oxidase activity [6], and apoptosis [7].…”