Identification of Human Neutrophil-derived Cathepsin G and Azurocidin/CAP37 as Chemoattractants for Mononuclear Cells and Neutrophils

Abstract

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5–1 μg/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 protein–coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by α1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 μg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.

Proteolytic Activation of Alternative CCR1 Ligands in Inflammation

“…Chymase can cleave endothelins, producing 21-and 31-residue vasoactive peptides that are chemotactic for human monocytes, neutrophils (46), and macrophages (47). With regard to other proteases, neutrophil protease cathepsin G has also been shown to be chemotactic for human neutrophils, monocytes, and macrophages in a manner likewise dependent on enzyme activity (48,49). Injection of cathepsin G into mouse skin resulted in neutrophil and macrophage recruitment (49).…”

A Potent and Selective Nonpeptide Antagonist of CXCR2 Inhibits Acute and Chronic Models of Arthritis in the Rabbit

“…Since CXCR2 is expressed by a number of leukocyte subsets in addition to neutrophils, including monocytes, macrophages, dendritic cells, T cells, NK cells, and basophils (50 -52), it is possible that the CXCR2 antagonist directly inhibits ELR ϩ chemokine-induced mononuclear cell infiltration into the joint (53). Alternatively, the antagonist may directly inhibit only neutrophil chemotaxis and/or degranulation, thereby preventing the release of mediators such as defensins, CAP37/azurocidin and cathepsin G, which function as chemotactic agents for T cells and monocytes (54,55). Consistent with the latter explanation is the fact that in each of the arthritis models described, neutrophils are the first cell type to accumulate in the synovial fluid, followed sequentially by monocytes and lymphocytes (data not shown).…”

Neutrophil-derived azurocidin alarms the immune system

“…This is even more important, knowing that monocytes are able to herald the acquired immune response. Chertov et al [33] found azurocidin to be strongly chemotactic for monocytes and to a lesser extent, for PMN. In addition, azurocidin was proven to be chemotactic for T cells.…”