hr. J. Biochem. Vol. 23, No. 10, pp. 997-1003, 1991 Printed in Great Britain. All rights reserved 0020-71 IX/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc DIFFERENCES IN THE ARGINASE ACTIVITY BY RESIDENT AND STIMULATED MURINE PERITONEAL MACROPHAGES PRODUCED AND RAT A. HRABAK, F. ANTDNI and ILDIK~CSUKA 1st Department of Biochemistry, Semmelweis University Medical School, Budapest, VIII. Puskin u. 9. H-1444, P.O. Box 260, Budapest, Hungary (Received 8 October 1990) Abstract-1. Murine macrophages showed a considerably higher in vitro arginase production in short time cultures than rat peritoneal cells. 2. The in vivo stimulation with casein or thioglycollate resulted in an enhanced in vitro enzyme production in mice. 3. The adherence is not the condition of the enzyme production. 4. The difference between the two species cannot be explained by the lack of bivalent ions, the absence of energy supply, proteolysis, the low number of macrophages or by the different cell types of the peritoneal exudate of mouse and rat. 5. The lysoxyme production of murine and rat peritoneal macrophages was also investigated and no difference was observed between the two species. INTRODUCTION It is well known that arginase is characteristic for liver and plays a central role in the protein metab- olism participating in the urea cycle. More recently, it has been also found in a number of other cell types e.g. in fibroblasts (Pohjanpelto and Hiiltta, 1983), nervous system (Grill0 et al., 1983), erythrocytes (Fukumoto et al., 1983) and macrophages (Currie, 1978; Schneider and Dy, 1983, 1985; Brusdeilins et al., 1985; Hrabak et al., 1988, 1990). Macrophages play a central role both in the afferent and efferent stages of the immune response. These functions are realized by the production and secretion of several enzymes and other factors. It has been known for years that arginase is one of these enzymes secreted by macrophages but its exact role has not been explored yet (Schneider and Dy, 1983, 1985; Dy et al., 1983; Kung et al., 1977). The correlation of immuno- genicity and omithine production was observed in peritoneal macrophages (Kriegbaum et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG al., 1987). Cytotoxic effects of macrophages may be due to the arginine depletion caused by the secreted arginase. Recently it was found that murine liver arginase. is identical with an immunosuppressive factor on lym- phocytes (Huang et al., 1990). The antitumor activity and the formation of nitric oxide (NO) are arginine- dependent in specifically activated macrophages (Hibbs et al., 1987a,b; Drapier and Hibbs, 1986). According to these results arginase should inhibit the antitumor response and NO production of macro- phages. Albina et al. (1989a,b) showed that several Abbreviations: EGTA, ethylene glycol tetra-acetic acid; FITC, fluoresceine isothiocyanate; IPA, 2-propanol; Leu-OMe, L-leucine methyl ester; NEM, N-ethyl- maleimide; PBS, phosphate buffered saline; PCA, perchloric acid; PMSF, phenyl-methyl sulfonyl fluoride; TCA, trichloroacetic acid. functions of macrophages (e.g. superoxide pro- duction, phagocytosis and protein synthesis) were down-regulated by a physiological arginine concen- tration (0.1 mM). Therefore, the function of secreted arginase cannot be excluded in the regulation of the immune response. The arginine depletion may cause either the blocking of antitumor effect or the inhi- bition of the protein synthesis of lymphocytes (Currie et al., 1979; Chen and Broome, 1980). The role of intracellular arginase in cell differentiation and pro- liferation, and hence in immune regulation, was suggested on the basis of numerous studies performed on mixed leucocyte cultures (Kung et al., 1977). The latter effect would be through the regulation of DNA synthesis and polyamines which are formed from omithine by omithine decarboxylase (Otani et al., 1982; Gehda ef al., 1983; Kahana and Nathans, 1984). More recently, the molecular cloning of human liver arginase was also performed (Haraguchi et al., 1987). The data enumerated above supported the idea that arginine plays a central role in the macrophage functions and, as a consequence, arginase is import- ant in the regulation of the immune response. In our experiments we studied and compared the production and secretion of arginase from resident and stimu- lated murine and rat peritoneal macrophages. Lysozyme, another secreted enzyme, was also investi- gated for comparison. MATERIALSANDMETHODS Animals, media and materials CFLP male mice (25-30g weight) and Wistar male rats (200-250 g weight) were purchased from LATI (G&doll&, Hungary). Hanks and Eagle media were free of phenol red dye to avoid interference in direct protein determination. Media were pun&ased from National Institute of Public Health. N-ethyl-maleimide (NEM); phenyl-methyl sulfonyl 997