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Volume 271, Number 6, Issue of February 9, 1996 pp. 2935-2940
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Defensin-1, Defensin-2, and CAP37/Azurocidin as T-cell Chemoattractant Proteins Released from Interleukin-8-stimulated Neutrophils

(Received for publication, August 16, 1995; and in revised form, November 30, 1995)

Oleg Chertov ,  Dennis F. Michiel ,  Luoling Xu ,  Ji Ming Wang ,  Kenji Tani ,  William J. Murphy ,  Dan L. Longo ,  Dennis D. Taub ,  Joost J. Oppenheim

Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH(2)-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH(2)-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1-100 ng of defensins and 1.0-100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells.



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