Peptides C3a and C5a, also known as anaphylatoxins, are derived from C components C3 and C5 and have previously been reported to exhibit chemotactic activity. Virtually no data exist, however, regarding the relative potency of C3a or C5a for stimulating directed cellular movement. The chemotactic response of polymorphonuclear leukocytes (PMN) toward purified human C3a and C5a was determined under both in vitro and simulated in vivo conditions. Leukotactic activity of human C5a and C3a was examined with a modified Boyden chamber assay over a broad concentration range. Human C5a exhibited activity only over the range of 0.04 to 1.7 × 10-8 M. Greater or lesser concentrations of C5a were incapable of stimulating cellular migration. Since C5a is rapidly converted to C5ades Arg by a carboxypeptidase in serum, we also examined this modified anaphylatoxin molecule. For this study C5aArg was formed by digesting C5a with pancreatic carboxypeptidase B (EC 18.104.22.168). Human C5ades Arg is a form of C5a that is inactive as an anaphylatoxin. C5ades Arg was active as a chemotactic factor in the in vitro assay, but only when nonactivated serum was added. Approximately 10 times more C5ades Arg than C5a is required to stimulate a leukotactic response and the magnitude of the maximum cellular response is only about 50% of that produced by intact C5a. It was observed that serum provides a definite helper role for C5ades Arg stimulated chemotaxis under the in vitro assay procedures employed. Human C3a was inactive over a concentration range from 8 × 10-10 to 8 × 10-6 M and trypsintreated human C3 was also active over a range of 4 × 10-8 to 2.4 × 10-6 M. Earlier reports that 1- to 5-min trypsin digestion of C3 generates a chemotactic activity were not confirmed by our studies with human PMN; however, previous observations of activity may have resulted from minor contamination of the C3 with C5.
Human C5a was maximally active over a narrow concentration range of 2 to 6 × 10-7 M as estimated in rabbits by utilizing a skin window assay technique (Otani and Hugli, 1977). Human C5ades Arg also stimulated neutrophil accumulation under simulated in vivo conditions but at a slightly higher concentration range of 0.2 to 1.2 × 10-6 M. The C5ades Arg did not require added serum to produce an apparent leukotactic response in vivo, but, as in vitro, activity was not observed at levels above or below the effective concentration ranges indicated for C5a and C5ades Arg C3a was inactive over a concentration range of 5.5 × 10-9 to 5.5 × 10-6 M as an attractant for rabbit neutrophils in the skin window assay. The concentrations of C5a or C5ades Arg that are required to induce chemotaxis under simulated in vivo conditions correspond to the potential serum levels of these factors (e.g., 4 to 5 × 10-7 M). It is suggested from our quantitative measurements that the predominant chemotactic activity generated in human serum during C activation is associated with the C5ades Arg molecule. Therefore, C5ades Arg exhibits the potential for playing a predominant functional role under physiologic conditions as a major humoral chemotactic factor.
This work was supported by United States Public Health Service Grant HL 20220, a Program Project Grant from the National Heart and Lung Institute, HL 16411, and Grant-in-Aid 74-864 from the American Heart Association, Inc. This is publication Number 1359 from Scripps Clinic and Research Foundation.