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Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.

Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, Wang JM, Howard OM, Sayers TJ, Oppenheim JJ - J. Exp. Med. (1997)

Bottom Line: 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 chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Support Program, Science Applications International Corporation Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.

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 microg/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 alpha1 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 microg/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.

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Chemotactic activity of cathepsin G, azurocidin, and thrombin for human monocytes. Monocyte migration was evaluated using a 48-well microchamber technique (11) as described in Materials and Methods.  The results are expressed as the chemotaxis index which represents the ratio of the number of cells in HPF in the test to control samples (media) *P  <0.05; ***P <0.001. FMLP (10 nM) is included as a positive control.
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Figure 3: Chemotactic activity of cathepsin G, azurocidin, and thrombin for human monocytes. Monocyte migration was evaluated using a 48-well microchamber technique (11) as described in Materials and Methods. The results are expressed as the chemotaxis index which represents the ratio of the number of cells in HPF in the test to control samples (media) *P <0.05; ***P <0.001. FMLP (10 nM) is included as a positive control.

Mentions: Since azurocidin/CAP37, a serine proteinase-like protein from neutrophils, as well as thrombin were previously reported to be chemotactic for monocytes (6, 14), we compared the chemotactic activity of cathepsin G for monocytes with that of azurocidin/CAP37 and thrombin. The monocyte chemotactic activity of cathepsin G appeared to be dose dependent with an optimal concentration of 0.5–1 μg/ml (Fig. 3). In other experiments using higher doses of cathepsin G (up to 10 μg/ml) a bell-shaped dose response was obtained (not shown). Cathepsin G appeared to be a much more potent chemoattractant for monocytes than either azurocidin or thrombin.


Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.

Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, Wang JM, Howard OM, Sayers TJ, Oppenheim JJ - J. Exp. Med. (1997)

Chemotactic activity of cathepsin G, azurocidin, and thrombin for human monocytes. Monocyte migration was evaluated using a 48-well microchamber technique (11) as described in Materials and Methods.  The results are expressed as the chemotaxis index which represents the ratio of the number of cells in HPF in the test to control samples (media) *P  <0.05; ***P <0.001. FMLP (10 nM) is included as a positive control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199011&req=5

Figure 3: Chemotactic activity of cathepsin G, azurocidin, and thrombin for human monocytes. Monocyte migration was evaluated using a 48-well microchamber technique (11) as described in Materials and Methods. The results are expressed as the chemotaxis index which represents the ratio of the number of cells in HPF in the test to control samples (media) *P <0.05; ***P <0.001. FMLP (10 nM) is included as a positive control.
Mentions: Since azurocidin/CAP37, a serine proteinase-like protein from neutrophils, as well as thrombin were previously reported to be chemotactic for monocytes (6, 14), we compared the chemotactic activity of cathepsin G for monocytes with that of azurocidin/CAP37 and thrombin. The monocyte chemotactic activity of cathepsin G appeared to be dose dependent with an optimal concentration of 0.5–1 μg/ml (Fig. 3). In other experiments using higher doses of cathepsin G (up to 10 μg/ml) a bell-shaped dose response was obtained (not shown). Cathepsin G appeared to be a much more potent chemoattractant for monocytes than either azurocidin or thrombin.

Bottom Line: 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 chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Support Program, Science Applications International Corporation Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.

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 microg/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 alpha1 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 microg/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.

Show MeSH
Related in: MedlinePlus