IDENTIFICATION OF HUMAN NEUTROPHIL-DERIVED CATHEPSIN-G AND AZUROCIDINCAP37 AS CHEMOATTRACTANTS FOR MONONUCLEAR-CELLS AND NEUTROPHILS/

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the sourer of chemota ctic factors for monocytes. To identify these putative monocyte attrac tants, we have systematically prepared neutrophil granules, lysed them , and sequentially purified the released proteins by several reverse p hase chromatography procedures. Assays for monocyte chemotactic activi ty of the chromatography fractions yielded a major peak of activity as sociated with a protein of 30 kD, according to SDS-PAGE analysis. NH2- terminal sequence of the protein revealed this to be identical to cath epsin G. The monocyte chemotactic activity of human cathepsin G was do se dependent with optimal concentration at 0.5-1 mu g/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G-induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G pr otein-coupled receptor. Enzymatic activity of cathepsin G is associate d with its monocyte chemotactic activity, since DFP-or PMSF-inactivate d cathepsin G no longer induced monocyte migration. The chemotactic ac tivity of cathepsin G can also be completely blocked by oil antichymot rypsin, a specific inhibitor of-chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattrac tant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T c ell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 mu g/ml, was chemotactic for monocytes and neutr ophils. As predicted from the in vitro chemotactic activity, subcutane ous injection of cathepsin G into BALB/c mice led to infiltration of b oth mononuclear cells and neutrophils. Thus, the transition of inflamm atory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule protein s such as cathepsin G and azurocidin/CAP37.