Structural requirements for the activity of an immunologically generated lipid chemotactic factor

Abstract

The predominant lipid chemotactic factor (LCFR) generated by IgGa-dependent immunological challenge of the rat peritoneal cavity was resolved from other lipid mediators released simultaneously into the peritoneal cavity and from previously described lipid chemotactic factors by sequential chromatography on Amberlite XAD-8, DE-52, silicic acid, and silica gel H plates, and by Zorbax C-18 reverse phase high pressure liquid chromatography. The incubation of purified rat peritoneal mononuclear leucocytes, but not mast cells, with calcium ionophore A23187 in vitro resulted in the generation of LCFR activity which was chromatographically identical to the LCFR released in vivo. That indomethacin inhibited the appearance of LCFR both in vivo and in vitro indicated a dependence on the cyclo-oxygenation of arachidonic acid. Because of the limited quantities of highly purified LCFR, the critical determinants of human neutrophil chemotactic activity were studied by examining the functional effects of chemical modification of specific substituents. The chemotactic activity of LCFR was diminished significantly by acetylation with acetic anhydride in pyridine or methylation with ethereal diazomethane, but was not influenced by 0.1 M periodate or acid or base hydrolysis, suggesting that LCFR is a complex fatty acid containing a hydroxyl or amino group. The addition of acetylated LCFR to the stimulus compartment at a 0.3-0.5 molar ratio with native LCFR diminished the neutrophil chemotactic response by 50%, without affecting a comparable response to f-Met-Leu-Ala-Phe. Thus LCFR is a unique lipid chemotactic factor which is distinct from other lipid mediators and accounts for the bulk of the PMN leucocyte chemotactic activity released by immunological challenge of the rat peritoneal cavity.