Limits...
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.

Show MeSH

Related in: MedlinePlus

Fractionation of neutrophil granule extract by reverse phase  HPLC. Neutrophil granule extract (1.2 mg of protein) was loaded onto a  C4 Delta-Pak Radial-Pak cartridge column (8 × 100 mm) equilibrated in  buffer A (0.1% TFA in water). Proteins were eluted with a linear gradient  of buffer B (0.05% TFA in acetonitrile) at a flow rate of 1 ml/min. 1-ml  fractions were collected, and 200-μl aliquots of each fraction were lyophilized for testing of monocyte chemotaxis activity using the microchamber assay. Monocyte chemotaxis to FMLP (0.1 μM) and media itself  are indicated by horizontal broken lines.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199011&req=5

Figure 1: Fractionation of neutrophil granule extract by reverse phase HPLC. Neutrophil granule extract (1.2 mg of protein) was loaded onto a C4 Delta-Pak Radial-Pak cartridge column (8 × 100 mm) equilibrated in buffer A (0.1% TFA in water). Proteins were eluted with a linear gradient of buffer B (0.05% TFA in acetonitrile) at a flow rate of 1 ml/min. 1-ml fractions were collected, and 200-μl aliquots of each fraction were lyophilized for testing of monocyte chemotaxis activity using the microchamber assay. Monocyte chemotaxis to FMLP (0.1 μM) and media itself are indicated by horizontal broken lines.

Mentions: To determine if constituents of neutrophil granules are responsible for attracting monocytes, we extracted neutrophil granules and fractionated the extract on a C4 reverse phase column. Aliquots of the fractions were tested for monocyte chemotactic activity (Fig. 1). Fraction 5 manifested the highest monocyte activity. For further purification, this fraction was subjected to chromatography on a C18 column (Fig. 2). The major protein peak had monocyte chemotactic activity and was shown to be homogeneous (Fig. 2, inset). This protein was subjected to NH2-terminal amino acid sequence analysis and yielded a sequence of 15 amino acid residues I-I-G-G-R-E-S-R-P-H-S-R-P-Y-M. A search for the protein sequence in the data base (Protein Identification Resource) revealed that this protein is cathepsin G (EC 3.4.21.20), a serine proteinase with chymotrypsin-like specificity. For further experiments, cathepsin G purchased from Calbiochem was used.


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)

Fractionation of neutrophil granule extract by reverse phase  HPLC. Neutrophil granule extract (1.2 mg of protein) was loaded onto a  C4 Delta-Pak Radial-Pak cartridge column (8 × 100 mm) equilibrated in  buffer A (0.1% TFA in water). Proteins were eluted with a linear gradient  of buffer B (0.05% TFA in acetonitrile) at a flow rate of 1 ml/min. 1-ml  fractions were collected, and 200-μl aliquots of each fraction were lyophilized for testing of monocyte chemotaxis activity using the microchamber assay. Monocyte chemotaxis to FMLP (0.1 μM) and media itself  are indicated by horizontal broken lines.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Fractionation of neutrophil granule extract by reverse phase HPLC. Neutrophil granule extract (1.2 mg of protein) was loaded onto a C4 Delta-Pak Radial-Pak cartridge column (8 × 100 mm) equilibrated in buffer A (0.1% TFA in water). Proteins were eluted with a linear gradient of buffer B (0.05% TFA in acetonitrile) at a flow rate of 1 ml/min. 1-ml fractions were collected, and 200-μl aliquots of each fraction were lyophilized for testing of monocyte chemotaxis activity using the microchamber assay. Monocyte chemotaxis to FMLP (0.1 μM) and media itself are indicated by horizontal broken lines.
Mentions: To determine if constituents of neutrophil granules are responsible for attracting monocytes, we extracted neutrophil granules and fractionated the extract on a C4 reverse phase column. Aliquots of the fractions were tested for monocyte chemotactic activity (Fig. 1). Fraction 5 manifested the highest monocyte activity. For further purification, this fraction was subjected to chromatography on a C18 column (Fig. 2). The major protein peak had monocyte chemotactic activity and was shown to be homogeneous (Fig. 2, inset). This protein was subjected to NH2-terminal amino acid sequence analysis and yielded a sequence of 15 amino acid residues I-I-G-G-R-E-S-R-P-H-S-R-P-Y-M. A search for the protein sequence in the data base (Protein Identification Resource) revealed that this protein is cathepsin G (EC 3.4.21.20), a serine proteinase with chymotrypsin-like specificity. For further experiments, cathepsin G purchased from Calbiochem was used.

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