Oxidative damage of cells involved in lipid and protein oxidation is one of the possible means of cellular damage that can take place in vivo. [1][2][3] Binding of naturally occuring autologous anti-band 3 IgG to the cell surface sialylated poly-N-acetyllactosaminyl sugar chains of band 3 glycoprotein aggregates generated during aging or oxidation of human erythrocytes has been suggested to be involved in the removal of the cells from circulation.
4)Direct recognition and phagocytic removal of damaged cells is an important function of macrophages in maintaining homeostasis.5) Although the mechanisms underlying macrophage adhesion to the cells are not known, this phenomenon implies that macrophages have an ability to recognize and phagocytose the damaged cells. 6) We have shown that mouse macrophages selectively bind oxidatively damaged erythrocytes in the absence of serum, 7,8) and this recognition is found to be due to sialylated poly-N-acetyllactosaminyl sugar chains of band 3 protein 9) and sialosaccharide chains of glycophorin 10,11) on the oxidized erythrocytes. Polymorphonuclear leukocytes (PMNs) are known to attack xenobiotics by generation of reactive oxygen species.
12)Moreover, it has been observed that in the inflammatory sites, PMNs are phagocytosed by macrophages. [13][14][15][16] Hence, it is conceivable that macrophage phagocytosis of PMNs in the inflammatory process is due to PMN oxidation caused by reactive oxygen species generated by themselves. In the present study, in order to examine this possibility, a model experiment to determine whether mouse PMNs oxidized by several oxidizing agents were effectively recognized by thioglycollate-induced mouse peritoneal macrophages was conducted. It was found that macrophage recognition was increased by the mild oxidation of PMNs. The membrane sites on the oxidized PMNs were suggested to be clustered sialylated poly-N-acetyllactosaminyl sugar chains.
MATERIALS AND METHODS
MaterialsHanks' balanced salt solution (HBSS) and N-2-hyroxyethyl-piperazine-NЈ-2-ethanesulfonic acid (HEPES) (pH 7.2) were obtained from Nissui Pharmaceutical Co., Tokyo, and Dojindo Laboratories, Kumamoto, respectively. Thioglycollate medium and proteose-peptone medium were obtained from Difco Laboratories, Detroit, MI, U.S.A. RPMI 1640 medium and penicillin-streptomycin solution were obtained from Gibco Laboratories, Grand Island, NY, U.S.A. Glycophorin A Glycophorin A was prepared from human erythrocyte membranes and purified according to the methods described elsewhere. 17,18) Sialic acid content in glycophorin A thus prepared was 15-18% as estimated by the Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Received June 16, 2000; accepted September 27, 2000 Mouse thioglycollate-induced peritoneal macrophages effectively, in the absence of serum, recognized mouse polymorphonuclear leukocytes (PMNs) mildly oxidized with diamide, superoxide (hypoxanthine/xanthine oxidase) or t-butylhydroperoxide, or modified with N-ethylmaleimide (NEM). The recognitio...
