József Fachet

Effect of lentinan and mannan on phagocytosis of fluorescent latex microbeads by mouse peritoneal macrophages: A flow cytometric study

Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytosis of BSA-coated, C3b- or monoclonal immunoglobulin (IgG2b)-coated fluorescent microspheres by resident or thioglycollate-elicited mouse macrophages in a dose-dependent manner. Analysis of flow cytometric data has shown that microbead phagocytosis of resident macrophages, which exhibit a lower basic phagocytic activity than the thioglycollate elicited ones, has been augmented by up to 900% due to lentinan. The percent ratio of phagocytes among peritoneal exudate cells, however, remained unchanged after short-term lentinan stimulation. Preincubation of the cells with lentinan resulted in increased ingestion of the microbeads. Activation of phagocytosis by lentinan is therefore due in part to the direct stimulation of the cells, however, lentinan also serves as supplementary opsonin for C3b-coated beads. Mannan inhibited the ingestion of C3b-coated microspheres by 75%, which was abolished in part when lentinan was also added to the cells. Mannan did not influence the phagocytosis of BSA-coated or IgG-coated beads. Our data, based solely on in vitro studies, suggest a beta-glucan receptor mediated activation of phagocytes by lentinan. These receptors are different from the C3b, Fc or mannose receptors. It is very likely that stimulation of phagocytic activity of macrophages by lentinan may contribute to the antitumor action of this immunopotentiating polysaccharide.

Strain differences in the cytotoxic activity and TNF production of murine macrophages stimulated by lentinan

The effect of lentinan, a glucan type immunomodulatory polysaccharide was studied on the antitumor cytotoxicity and on the TNF secretion of peritoneal macrophages in inbred H-2 congeneic mouse strains under in vivo and in vitro conditions. The cytotoxic activity and TNF secretion of murine macrophages was found to be elevated by lentinan in vitro and in vivo conditions. The effectiveness of lentinan to induce cytotoxicity and TNF secretion was highly influenced by the genotype of the host. The increased cytotoxicity of macrophages was modified by the H-2 and the background genes. The black background and the H-2a and H-2d haplotypes were associated with high responsiveness, while the albino and agouti background and the H-2b and the H-2k haplotypes with low responsiveness to lentinan treatment. The degree of TNF secretion of macrophages stimulated by lentinan was influenced by the H-2 genes only. Similarly, to the macrophage cytotoxicity the TNF secretion in the H-2a and H-2d haplotypes were found to be high, on the other hand, in the H-2b and H-2k were low.

A simple, rapid and sensitive fluorimetric assay for the measurement of cell-mediated cytotoxicity

A fluorimetric method using 4-methylumbelliferyl heptanoate (MUH) has been developed for detecting cell-mediated cytotoxicity and cell proliferation. The assay is based on the hydrolysis of the fluorochrome (MUH) by intracellular esterases of viable cells resulting in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter. Because of a similarity to the principle of the widely used colorimetric MTT assay, a comparison was made between the two assays when measuring cell proliferation and LAK cell cytotoxicity to different target cell types. The results have shown that the MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is completely comparable to the MTT method. The rapidity of the new cytotoxicity assay, 5 h in contrast to 9 h for the MTT assay, its applicability to both adherently and nonadherently growing target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious contender for replacing conventional radioactive techniques.

Effect of lentinan on the chemiluminescence produced by human neutrophils and the murine macrophage cell line C4Mφ

Lentinan, an immunopotentiating polysaccharide, stimulates the production of chemiluminescence (CL) by human neutrophils and the murine macrophage cell line C4M phi. The CL enhancing effect of lentinan opsonized in human serum is greater than that of lentinan itself. Lentinans stimulation of neutrophil CL was increased by 1/2 when opsonized in human serum inactivated at 56 degrees C to remove complement, while the CL was increased two fold by lentinan opsonized in whole serum. This indicates that C3b and immunoglobulin contribute separate signals in the activation process mediated by opsonized lentinan. The distinct roles of the C3b and Fc receptors was further illuminated by the finding that an Fc receptor-negative cell line was unresponsive to lentinan opsonized in heat inactivated serum (56 degrees C), whereas it exhibited a five fold increase in CL in response to lentinan opsonized in serum containing complement. Lentinan in an opsonized form can stimulate the production of CL via C3b and Fc receptors. This mechanism may be considered as one mode of action of lentinan and other similar immunopotentiating and antitumour glucan-type polysaccharides.

Effect of lentinan on pinocytosis in mouse peritoneal macrophages and the murine macrophage cell line C4MØ in vitro

Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxidase (HRP) or FITC-dextran by resident or thioglycollate-elicited mouse macrophages from 10 to 50% in a dose dependent manner. Pinocytosis of HRP and FITC-dextran by C4M phi cells, a murine macrophage cell line, exhibiting a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and 120%, respectively, by lentinan. Mannan inhibited the HRP uptake by peritoneal macrophages via specific mannose receptors. This inhibitory effect was partly abolished, when lentinan was also added to the cells. Mannan was not able to inhibit pinocytosis of HRP by C4M phi macrophages, indicating little or no mannose receptor activity on these cells. Pinocytosis of FITC-dextran was not affected by mannan. Lentinan, opsonized in mouse sera inhibited the uptake of HRP by peritoneal macrophages by 30-35%. Opsonized lentinan and mannan added together caused 60% inhibition of HRP uptake in peritoneal macrophages indicating a possible functional relationship between the mannose and C3b receptors. The results demonstrate that lentinan activates the pinocytic function of macrophages predominantly via specific beta-glucan receptors. These mechanisms may contribute to the antitumor and immunopotentiating action of lentinan and other glucan-type polysaccharides.