Release of Neutrophil and Eosinophil Chemotactic Factor from Sensitized Skin in vitro Cutaneous Anaphylactic Reactions

NCF ECF Chemotactic factor HPLC LTB4 Skin, anaphylactic reaction Tsunemi Numata, MD, Department of Dermatology, Hiroshima University School of Medicine, Kasumi 1-2-3, Minamiku, Hiroshima 734 (Japan) Histamine is considered to play a critical role in anaphylaxis and produce wheal and flare reactions in skin. On the other hand, it is known that inflammatory cells such as neutrophils and eosinophils migrate into the site of anaphylactic reactions in skin. The cellular infiltrations might produce a prolonged inflammatory reaction after immediate wheal and flare reactions in the cutaneous anaphylaxis. In order to analyze the mechanisms of the neutrophil and eosinophil migration into the site of anaphylactic reactions in skin, in vitro antigen-evoked release of neutrophil chemotactic factor (NCF) and eosinophil chemotactic factor (ECF) from skin was investigated. The abdominal skin of guinea pig sensitized with egg albumin was cleaned of subcutaneous tissue and cut into 0.5-mm-thick slices. The slices were incubated with 50 μg/ml egg albumin at 37 °C for 20 min, and NCF and ECF activity in the supernatant were measured with a modification of Boyden technique [1]. using guinea pig neutrophils and eosinophils as indicator cells, respectively. The antigen-evoked NCF and ECF activities were detected in the supernatant. The results of Sephadex G-10 or G-25 gel filtration of the supernatant indicated that the molecular weight of the NCF and ECF were between 300 and 1,300. The release of the NCF and ECF was abolished in the absence of calcium in the reaction mixture. Furthermore, the release of NCF and ECF was suppressed by preincu-bating the slices with nordihydroguaiaretic acid (50 μM), a 5-lipoxygenase inhibitor, and enhanced with indo-methacin (8.5 μM), a cyclooxygenase inhibitor. The NCF and ECF partially purified by Sep-Pak C18 cartridge had the same retention time as leukotriene B4 (LTB4) on reverse-phase HPLC. The significant release of LTB4 by antigen from sensitized guinea pig skin was ascertained by radioimmunoassay.