The biological activity of human C5a was compared to its anaphylatoxin inactive form, C5ades Arg, using highly purified components. C5a was isolated from yeast-activated human serum and was converted to C5ades Argby treatment with porcine carboxypeptidase B immobilized on Sepharose 6B. Both purified C5 fragments were homogeneous by polyacrylamide gel electrophoresis of radiolabeled peptides and by immunologic techniques. C5a caused increased vascular permeability in guinea pig skin with as little as 2 ng of injected material, whereas no detectable effect was seen with as much as 5000 ng of C5ades Arg. Both C5a and C5ades Argstimulated cytochalasin B-treated human neutrophils to release the granule constituents myeloperoxidase, lysozyme, and β-glucuronidase, but not the cytoplasmic enzyme lactic dehydrogenase. However, 85- to 175-fold more C5ades Argwas required to give the same amount of release. A similar dichotomy of biologic effectiveness was seen for generation of the oxygen metabolite, superoxide anion, O2-, with or without cytochalasin B pretreatment of the cells. In contradistinction to earlier reports, C5ades Argwas chemotactic for human neutrophils in the absence of added serum between 100 to 10, 000 ng/ml, whereas C5a was active from 3 to 500 ng/ml. A 24-fold lower concentration of C5a was required to obtain a peak chemotactic response as measured by a leading front technique (5 ng/ml) as compared to a two-filter assay (120 ng/ml). In the absence of a gradient of the peptides, C5a and C5ades Arginduced enhanced random migration, i.e., exhibited chemokinetic activity. Both C5a and C5ades Argdesensitized neutrophils to further stimulation by C5a, as measured by granule secretion and chemotaxis. However, 25-to 50-fold more C5ades Argthan C5a was required for desensitization. The ability of C5ades Argto cross-desensitize neutrophils suggests that C5ades Argbinds to the same neutrophil receptor as C5a but at a lower binding affinity. The studies reported here show that C5ades Argis capable of interacting with neutrophils in a manner similar to that of C5a and that the difference between the two polypeptides is quantitative, not qualitative. Thus, the biologic activity of C5ades Argand the presence of the serum carboxypeptidase B, which converts C5a to C5ades Arg, as well as the recently described helper factor for enhanced C5ades Argchemotaxis combine to suggest that C5ades Argmay be an important phlogistic mediator in vivo. © 1980.
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