The Journal of Immunology, Vol 143, Issue 3 979-983, Copyright © 1989 by American Association of Immunologists

ARTICLES

Human eosinophil cytotoxicity-enhancing factor. Purification, physical characteristics, and partial amino acid sequence of an active polypeptide [published erratum appears in J Immunol 1991 Dec 15;147(12):4408]

DS Silberstein, MH Ali, SL Baker and JR David
Department of Medicine, Harvard Medical School, Boston, MA 02115.

Medium conditioned by PMA/LPS-stimulated U937 cells was processed for the purification of an eosinophil cytotoxicity-enhancing factor (ECEF) by the following sequence: 1) phenyl-Sepharose chromatography; 2) DEAE- cartridge chromatography; 3) preparative SDS-gel electrophoresis; and 4) reversed-phase HPLC. This resulted in the isolation of a 10 kDa polypeptide with ECEF activity. Purified material from 21 different preparations enhanced eosinophil killing of antibody-coated Schistosoma mansoni schistosomula by a mean of 206% (increase from 13.2 +/- 7.9% to 40.4 +/- 20.2% of targets killed, p less than 0.0001). Activity was maximal at a concentration of 20 ng ECEF polypeptide/ml and half- maximal between 0.8 and 4 ng/ml. Antibody specific for the 10 kDa polypeptide precipitated ECEF activity from a crude preparation and, by Western blot analysis, reacted only with a 10 kDa species in that preparation. The following N-terminal amino acid sequence was determined for the purified polypeptide: Val-Lys-Gln-Ile-Glu-Ser-Lys- Thr-Ala-Phe-Gln-Lys-Ala-Leu- -Ala- Gly- -Lys-Leu....Computer search showed that this sequence is unrelated to other known protein sequences. Thus, the ECEF polypeptide is a newly defined monokine, with the ability to enhance eosinophil cytotoxic function in vitro. This monokine may be an important regulator of eosinophil function in inflammation in vivo.

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