Human eosinophil cytotoxicity-enhancing factor. Purification, physical characteristics, and partial amino acid sequence of an active polypeptide.
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.
Silberstein
D S
DS
Department of Medicine, Harvard Medical School, Boston, MA 02115.
Ali
M H
MH
Baker
S L
SL
David
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000032:analysis; D003603:Cytotoxins / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D004306:Dose-Response Relationship, Immunologic; D004804:Eosinophils / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D011817:Rabbits
D003470:Culture Media; D003603:Cytotoxins; D007106:Immune Sera; D010455:Peptides