Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity.
A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.
Schröder
J M
JM
Department of Dermatology, University of Kiel, Federal Republic of Germany.
Mrowietz
U
U
Morita
E
E
Christophers
E
E
D000818:Animals; D002630:Chemotactic Factors / Q000302:isolation & purification* / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005966:Glucuronidase / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000032:analysis* / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008809:Mice, Inbred C3H; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology; D013481:Superoxides / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002630:Chemotactic Factors; D008070:Lipopolysaccharides; D010455:Peptides; D013481:Superoxides; D005966:Glucuronidase; D013755:Tetradecanoylphorbol Acetate