Correlation between Tumor Cell Cytotoxicity and Serine Protease Activity of Peritoneal Macrophages from Mice Treated with Bakers' Yeast Mannans

Abstract

Peritoneal exudate cells (PEC) from C3H/He mice given bakers' yeast mannans, WNM and WAM025, showed marked cytolytic activity against MM46 tumor cells in vitro. Because the cytotoxicity of the PEC was strongly inhibited by actinomycin D, it was assumed that the cytolytic factor was a proteinous material(s). Of the macrophages and polymorphonuclear leucocytes (PMN ) separated from the PEC, only the former cells were found to display cytolytic activity. Quinacrine, a phospholipase A2, inhibitor, and sodium azide, a myeloperoxidase inhibitor, did not reduce the cytotoxicity of macrophages from mice treated with acidic mannan (WAM025). In contrast, diisopropylfluorophosphate, a serine protease inhibitor, inhibited the cytolysis of the MM46 target cells by WAM025-treated macrophages. Chymostatin, a chymotrypsin inhibitor, and elastatinal, an elastase inhibitor, also inhibited the cytolytic effect. It is therefore concluded that serine proteases secreted into the medium from the macrophages activated by mannans participated in the cytolytic destruction of neoplasmic cells.Macrophage activation for nonspecific tumoricidal activity against target tumor cells can be induced by various immunopotentiators. In. our previous reports, it was found that mannan of bakers' yeast was able to enhance the immunological response of mice to transplanted tumor cells and to lethal infection with pathogenic microbes.1-4) In order to identify the competent cells in acidic mannan (WAM025)-treated peritoneal exudate cells (PEC), we carried out an in vitro cytolysis assay by macrophages and polymorphonuclear leucocytes (PMN) separated from the PEC by the Percoll gradient centrifugation technique.5) The results indicated that WAM025-treated macrophages were able to display a marked cytolytic effect, while PMN from the same source did not show the effect. Because the chemical entity involved in the cytolysis of tumor cells by WAM025-treated macrophages remained to be identified, we attempted to characterize the cytolytic factor elicited in mouse macrophages by the administration of the yeast mannans.The relevancy of serine protease release to tumoricidal activity of macrophages has been reported in mice treated with BCG6,7) and Nocardia rubra cell-wall skeleton.8) However, the real cytolytic factors of antitumor polysaccharide-treated macrophages have not been elucidated to date, although many reports have been published on identifying various lytic factors produced by cytolytically activated macrophages such as oxygen intermediates,9,10) lysosomal enzymes,11,12) proteases,6,7) complement component C3,13) tumor necrotic factor,14) prostaglandins, 15) and myeloperoxidase.16) Therefore, the present study was designed to investigate the possible role of serine proteases in the cytolysis of target tumor cells by antitumor mannan-induced macrophages.

Production of Interleukin-1 Activity of Kupffer Cells from Mice Treated with the Acidic Mannan Fraction of Bakers' Yeast.

“…It is possible that the protective effect of WAM025 depends on the production of IL-1 (Tables 1, 2) by the activated KC, in addtition to the other previously reported factors. [13][14][15]19) The results in Table 2 show that WAM025 could directly activate the production of IL-1 by KC obtained from a normal mouse.…”

“…Our data confirm the importance of serine proteases as a host-immune protective mechanism. 19,23) It is also suggested that the adjuvant effects of WAM025 [13][14][15]19) may in part be mediated via its ability to augment the number of and the IL-1 production by KC. Of the yeast polysaccharides, WAM025 should be a useful biological response modifier in the defense system of tumor-and infectious disease-bearing hosts.…”