Anaphylactic and chemotactic response of mammalian cells to zymosan-activated shark serum

Abstract

Human anaphylatoxins, C3a and C5a, are small proteins which are generated by proteolytic cleavage of the complement components C3 and C5 during activation of complement. Complement activity via the classical and alternative pathways has been described for the nurse shark (Ginglymostoma cirratum) and studies have demonstrated chemotactic activity of shark leucocytes in response to activated shark serum. However, anaphylatoxins have not been reported in an elasmobranch. This investigation was undertaken to examine activated shark serum for anaphylatoxins by studying the anaphylactic and chemotactic response of mammalian cells. Shark (G. cirratum) serum, activated with zymosan (10mgml−1) for 3h at 30°C, was fractionated on Sephadex G-100 and G-25 columns, and fractions were pooled and assayed for spasmogenic and chemotactic activity.In vitrochemotaxis assays were set up in blind well chemotaxis chambers using human leucocytes as responding cells. A significant (P<0·05) migratory response was obtained with several serum pools. Spasmogenic activity was measured by smooth muscle contraction of rat ileum, and was present in serum fractions containing low molecular size (<17·5kDa) proteins/peptides, which also induced chemotaxis of human leucocytes. No spasmogenic or chemotactic activity was demonstrated in fractions of normal (non-activated) shark serum. This study shows that mammalian cells respond to factors generated in activated shark serum and results suggest that the chemotactic and anaphylactic activity in shark serum might be due to a biologically active peptide(s) derived from the cleavage of complement component(s), perhaps analogous to mammalian C3a and/or C5a.