Normal Mouse Peritoneal Cells Research Articles

Secretion of the C3 component of complement by peritoneal cells cultured with encapsulated Cryptococcus neoformans

Two isolates of Cryptococcus neoformans were identified as being widely divergent in pathogenic potential after intratracheal infection of mice. These isolates differed in their ability to upregulate capsule synthesis when grown under tissue culture conditions, and this property correlated with virulence. We postulated that differential capsule synthesis may cause differential stimulation of macrophages to produce products such as complement components. To test this hypothesis, heat-killed yeast cells were incubated with normal mouse peritoneal cells (PC) before the level of C3 secreted was determined. Cryptococcal stimulants were grown on mycological agar, which does not promote capsule synthesis, or in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, which stimulates capsule synthesis, to determine the role that the capsule plays in the induction of C3 secretion. C3 levels were elevated in cultures containing cryptococci grown in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, and the level of C3 detected was correlated with the amount of capsule expressed by the yeast cell stimulant. Nonencapsulated mutants of C. neoformans did not stimulate C3 secretion. Purified capsular polysaccharide (glucuronoxylomannan [GXM]) also stimulated the PC to secrete C3. Two signals were required before GXM stimulated C3 secretion. The second signal was identified as endotoxin present in small amounts (0.06 ng per ml) in tissue medium. Endotoxin may provide a priming stimulus for PC to express receptors or other cytokines needed for effective stimulation of C3. These experiments show that enhancement of C3 secretion by C. neoformans is due to GXM and is correlated with the virulence of the cryptococcal isolate.

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages.

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.

Tetrahydrocannabinol-induced suppression of macrophage spreading and phagocytic activity in vitro.

The effects of tetrahydrocannabinol (THC) on several parameters of macrophage function in vitro were assessed. Delta 9 THC added to cultures of normal mouse peritoneal cells in vitro affected the ability of the cells to spread on glass surfaces and also had some effect on their ability to phagocytize yeast. These effects were dose related. A concentration of 20 micrograms of THC almost completely inhibited macrophage spreading, but it also decreased viability and the total number of these cells. Doses of 10 or 5 micrograms of THC also inhibited spreading but had little effect on cell viability or number. A dose of 1.0 microgram of THC had some inhibitory effect on spreading and the lowest dose affecting spreading appeared to be about 0.05 micrograms per culture. Higher doses of THC were necessary to inhibit phagocytosis of yeast particles as determined by direct microscopic examination or use of radiolabeled yeast as the test particles. These results indicate that several readily measured functions of macrophages may be suppressed by THC.

Immunopharmacological actions of neurotropin (4). Effect of neurotropin on mouse peritoneal macrophages

It was reported that neurotropin (NSP), an extract isolated from the inflamed skins of rabbits inoculated with vaccinia virus, activates murine T cell functions participating in cell-mediated immunity. The present study was undertaken to examine the effect of NSP on plastic dish-adherent macrophages (M phi) from ddY mice in vitro. Total activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase in resident peritoneal M phi was slightly enhanced when the M phi were cultured with NSP (10-1000 micrograms/ml) for 48 and 96 hr, but no enhancement was noted in 24 hr culture. Intracellular activity of lactate dehydrogenase (LDH) was also strongly enhanced in a dose-dependent manner by culturing with NSP for 48 and 96 hr. The enhanced LDH activity in the M phi cultured with NSP for 96 hr was completely inhibited by cycloheximide, an inhibitor of protein synthesis. In addition, consumption of glucose in the culture media by the M phi was also enhanced by culturing with NSP for 96 hr. Intracellular activity of LDH and glucose consumption of plastic dish-nonadherent cells from normal mouse peritoneal cells, however, was not enhanced by NSP in 96 hr culture. In regard to allogeneic M phi-mediated cytostatic activity to P815-X2 mastocytoma, NSP had no effect on cytostatic activities of the resident and thioglycollate-induced M phi, although NSP by itself dose-dependently inhibited the growth of P815-X2 mastocytoma without affecting cell viability. These results suggest that NSP biochemically activates mouse peritoneal M phi in vitro, but the M phi activated by NSP can not inhibit the growth of P815-X2 mastocytoma.

Predominant synthesis of 5-hydroxyeicosatetraenoic acid by a cloned mastocytoma P-815 line, 2-E-6 cells

Since mouse mast tumor P-815 cells produce the slow reacting substance of anaphylaxis, their 5- lipoxygenase activity was examined by determining the conversion of arachidonic acid to 5- hydroxyeico- satetraenoic acid (HETE). Mast tumor cells from mouse ascites fluid synthesized 12-HETE as a major and 5-HETE as a minor metabolite. Once the cells were transferred to an in vitro culture system, the predominant synthesis of 12-HETE was abolished and synthesis of 5-HETE was greater than that of 12-HETE. 2-E-6 cells, obtained by cloning the tumor cells, synthesized a negligible amount of 12-HETE, but produced a large amount of 5-HETE. When the 2-E-6 cells were inoculated into mice and harvested again from the ascites fluid, their ratio of 5-HETE to 12-HETE synthesis was similar to that of normal mouse peritoneal cells; that is, 12-HETE synthesis was much greater than 5-HETE synthesis. It is concluded that the predominant synthesis of 12-HETE in mast tumor cells was derived from natural peritoneal cells, which have very high 12- lipoxygenase activity. The cloned mastocytoma, 2-E-6 cells, should be useful in investigating regulation of 5- lipoxygenase activity.

Mechanism of selective nonspecific cell-mediated cytotoxicity of virus-infected cells

STUDIES of mechanisms of resistance to herpes simplex virus (HSV) in mice have demonstrated antibody-dependent cell-mediated cytotoxicity (AD-CMC) of HSV-infected cells exposed in suspension cultures to anti-HSV sera and normal mouse peritoneal cells (PC) or human peripheral blood leukocytes. In this system it was impossible to demonstrate T-cell-mediated cytotoxicity1–3. In an attempt to adjust the assay conditions to permit detection of such cytotoxicity by immune spleens or PC, the “monolayer” system of Gardner et al.4 was examined in syngeneic, allogeneic and xenogeneic combinations. Target cell monolayers were infected with HSV and exposed to immune or normal mouse PC. The latter produced unexpectedly high levels of nonspecific release of 51Cr by the monolayer HSV-infected cells. Because nonspecific or non-selective killing of human tumour cells5 by lymphocytes from normal patients has been a major problem in human tumour immunology, we have investigated the mechanism of this nonspecific cell-mediated cytotoxicity in the HSV system. Our results indicate that selective nonspecific killing is a consequence of the cross linking of effector and target cells through aggregated immunoglobulins.

Precommitment of normal mouse peritoneal cells by erythrocyte antigens in relation to auto-antibody production.

WE have reported that peritoneal cells (PCs) from unstimulated mice can, after 2 h of incubation in the appropriate medium, start to secrete anti-sheep erythrocyte (SRBC) antibodies1,2. We extended this observation by showing that PCs cultured in standard conditions for 4–6 d in the absence of SRBC, can form numerous haemolytic plaques, demonstrable by the ordinary techniques of local haemolysis (in Agarose, in liquid layer or in cellulose gum3). The immunological nature of this plaque-forming activity was established by the following criteria: complement dependency, inhibition by anti-mouse IgM serum and immunological specificity. Thus, we concluded3,4 that PCs of normal adult mice had been stimulated previously by an unknown immunogen sharing common or cross-reacting determinants with SRBC, and that tissue culture conditions would derepress the built-in capacity of these cells to produce antibodies. Treatment of autologous erythrocytes by the proteolytic enzyme bromelain revealed that normal mouse spleen cells regularly produce antibodies against their own erythrocytes5,6. This led us to investigate the behaviour of normal PCs towards SRBC or mouse red blood cells (MRBCs) treated by bromelain (BrMRBCs). We report here that mouse PCs in culture develop a plaque-forming activity against isologous BrMRBC as well as against SRBC, and we show that these two types of erythrocytes share some common antigen.

<italic>Brief Communication:</italic> Mouse Mononuclear Cell Chemotaxis: Description of System

The mononuclear cell chemotaxis assay was adapted for use with normal mouse peritoneal cells. Mouse peritoneal macrophages responded well to endotoxinactivated mouse serum and to chemotactic factors produced by mouse spleen cell cultures stimulated with mitogens or specific antigen. The assay was quantitative, reproducible, and applicable to several mouse strains.