Lipid modulation of mammary tumor cell cytolysis: direct influence of dietary fats on the effector component of cell-mediated cytotoxicity.

For understanding the mechanism by which fatty acids promote mammary tumor growth, experiments were designed to determine the influence of dietary fat concentration and saturation on both effector (Ef) and target (Ta) cells in an allogeneic antitumor cell-mediated immune response. Exposure of cytotoxic T-lymphocytes (CTL) to different fatty acids led to significant changes in the subsequent cytolytic capacity of these cells after both primary and secondary immunization. An increase in both saturated (SF) and polyunsaturated (PUF) fats led to decreased cytotoxic function after primary immunization. After a secondary challenge, the suppressive influence of SF was significantly greater than that of PUF, compared to that of the control diet containing essential fatty acids as the only fat source. This response was mediated by a direct effect on the CTL and not through an increase in suppressor or a decrease in Ef or helper cell frequency. In contrast, manipulation of the fatty acid environment of the Ta mammary tumor cells in vivo or in vitro had no significant effect on their susceptibility to lymphocyte-mediated cytotoxicity. Therefore, dietary fats may mediate their effect by a direct influence on the immunocompetent lymphocyte and not on the Ta mammary tumor cell.

Susceptibility of mammary tumor cells to complement-mediated cytolysis after in vitro or in vivo fatty acid manipulation.

The susceptibility of line 168 murine mammary tumor cells to complement (C)-mediated lysis was tested after in vitro treatment with several saturated or unsaturated fatty acids dissolved in different solvents or presented in the form of micelles to the cells. The lytic susceptibility of these cultured cells was compared with similar tumor cells obtained either from mice maintained on diets containing different concentrations and saturations of fatty acids or from cultures supplemented with serum from tumor-free control mice fed pair-matched diets. Although changes in dietary fat concentration and saturation resulted in alterations of the tumor cell fatty acid composition, those alterations did not influence the susceptibility of tumor cells to C-mediated lysis. However, single, or combinations of, unsaturated fatty acids dissolved in ethanol, unlike saturated fatty acids, reduced the lytic susceptibility of tumor cells in vitro. Hexane added to culture medium significantly suppressed the lytic susceptibility; however, when used as a carrier no significant differences were observed among treatments with the individual fatty acids at several concentrations. This result may be due to the effect of hexane on the cell membrane because this treatment also affected the osmotic fragility of the cells. Fatty acids as micelles did not influence the susceptibility of tumor cells to lysis. We concluded that only in vitro manipulation of fatty acids in some vehicles influenced the susceptibility of target tumor cells to C-mediated lysis; this finding did not parallel the situation that occurred in vivo. Moreover, the use of different vehicles to present fatty acids to tumor cells may further alter the susceptibility to C-mediated lysis.

Effect of induction of T-cell-dependent antibody with sheep red blood cells on P-450-dependent and -independent xenobiotic metabolizing enzymes.

The effect of an antigenic challenge with sheep red blood cells (SRBC) on the activities of cytochrome P-450-dependent and -independent xenobiotic metabolizing enzymes and on lipid peroxidation in the liver was investigated. The studies were carried out using three mouse strains of C57B1/10 and three strains of C3H backgrounds which are cogenic, differing genetically at the H-2 complex. The basal levels of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (7-Ec) were different among congenic strains. The activity of 7-Ec was lower in C3H background mice than in B10 background mice. Similarly, the difference due to the strain and the H-2 locus was detected in the activities of P-450-independent enzymes such as malathion and diethyl succinate carboxylesterases, glutathione S-transferase, and epoxide hydrolases in microsomal and cytosolic fractions. The degree of immune responsiveness in these mice was determined by a plaque forming cell assay. Within the same background, the H-2b mouse strain was a high responder and the H-2k a low responder to SRBC. However, treatment with SRBC had no significant depressive effect on P-450-dependent enzyme activities except in C3H/He. Activity of AHH was suppressed in C3H/He mice. Treatment with SRBC had no effect on P-450-independent enzyme activities except for malathion carboxylesterase: the activity was increased in C3H/He and C3H.JK, whereas it was decreased in B10. The basal level of lipid peroxidation was lower in C3H/He and C3H.JK. The treatment produced a significant enhancement in lipid peroxidation in C3H/He, B10 and B10.BR (P less than 0.05) with a concomitant increase in xanthine oxidase activity (P less than 0.05). Thus, the present study revealed that a specific antigenic challenge, unlike non-specific immunostimulants (e.g. poly IC, endotoxin), does not necessarily inhibit P-450-dependent xenobiotic metabolizing enzymes even though antigen challenge increased XO activity and lipid peroxidation. The possible roles of an increase in lipid peroxidation and xanthine oxidase activity in immune response to SRBC and xenobiotic metabolizing enzymes are discussed.

A specific cytolytic T cell response induced by subcutaneous challenge with allogeneic rat epididymal spermatozoa.

Groups of virgin female rats were killed on days 2-5 after footpad injection with either thoracic duct lymphocytes or epididymal spermatozoa from allogeneic male rats and the cellular response in the regional lymph nodes determined by the specific cytoadhesion technique. After challenge with both thoracic duct lymphocytes and epididymal spermatozoa a specific cytolytic T cell response to the male strain histocompatibility antigens was evident on the second day and increased until the fifth day. The weight and total lymphocyte content of the nodes were significantly greater than the unimmunized control values throughout the observation period. These results provide further evidence for the expression of histocompatibility antigens by epididymal spermatozoa.

Alterations of alveolar macrophage function and level of bronchopulmonary protease inhibitors in O,O,S-trimethyl phosphorothioate-induced lung injury.

O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity present in widely used organophosphorus insecticides, has been shown to induce pneumotoxicity after oral administration. To date very little is known about the pathogenesis of the injury. Protease-anti-protease imbalance has been proposed as a mechanism of various lung injuries; thus, the effect of OOS-TMP on alpha 1-protease inhibitor capacity, pulmonary alveolar macrophage (PAM) esterases, cytotoxic activity and on specific esterase inhibitors in the bronchopulmonary lavage fluid and serum were measured. OOS-TMP (20 mg/kg) administered orally to rats produced a 27% increase in PAM esterase activity 6 h after treatment. The activity then declined to 63% of control value on day 3 and had not recovered to any significant extent on day 7. The cytotoxic activity of PAM was significantly increased at 6 h and 24 h following treatment. Chymotrypsin inhibitory capacity (CIC) of the lavage fluid was decreased by 45% at 6 h but recovered rapidly and reached control levels by 24 h. Trypsin inhibitory capacity (TIC) of serum was affected to a lesser extent such that no change was detected after 6, 12 or 24 h. These data, early elevation of PAM esterase levels with a concomitant increase in cytotoxic activity and decreased TIC and CIC in bronchopulmonary lavage fluid, support the view that pathogenesis of OOS-TMP produced lung injury could be due to increased protease levels.

Modulation of cellular and humoral immune responses by O,S,S-trimethyl phosphorodithioate, an impurity of commercial malathion.

We studied the effect of O,S,S-trimethyl phosphorodithioate (OSS-Me) on macrophage function and the immune responses of both T- and B-cells using an in vitro system. The involvement of glutathione (GSH) in the bioactivation of OSS-Me to suppress such responses was investigated after either direct culture with spleen or B-cells and/or co-culture of B-cells with either B- or T-cells that were or were not preincubated with OSS-Me and glutathione enriched cytosol. Our results show that OSS-Me preincubated with GSH suppresses immune responses through an inhibitory effect on the cell function. Both cytotoxic T-lymphocyte (CTL) generation and antibody production were impaired. Antigen presentation by macrophages was also inhibited. In mixing experiments of pretreated and normal cells, the inhibitory effect was also observed on macrophages and helper T-cells responding to a T-dependent antigen. We concluded that the immunosuppressive effect of OSS-Me is due to impairment of cell function and not mediated through generation of regulatory suppressor T-cells. This inhibitory effect was only detectable in the presence of glutathione in the preincubation mixture, suggesting that an OSS-Me glutathione adduct might be responsible for the toxic effect.

Dietary fatty acid modulation of murine T-cell responses in vivo.

The effect of dietary fat concentration and saturation on T-cell functions in vivo were investigated by using delayed-type hypersensitivity (DTH) and graft-versus-host (GVH) reactions. These were selected because they circumvent the problem of fatty acid flux from the lymphocyte during in vitro assays. The DTH reaction to allogeneic line B16-BL6 melanoma cells was suppressed in BALB/c mice fed a diet containing 20% saturated fat (coconut oil) or polyunsaturated fat (safflower oil) compared to control mice fed a diet with the minimum of essential fatty acids (EFA). Likewise, DTH responsiveness of mice fed an EFA-deficient diet was less than that of mice fed the EFA control diet. The GVH reaction of C57BL/6 spleen cells injected into irradiated BALB/c mice was suppressed in those fed 20% polyunsaturated fat. Serum levels of linoleic acid increased commensurate with the levels of polyunsaturated fat in the diet. Likewise, previous work has demonstrated that levels of linoleic acid in whole lymphocytes changed in direct relation to the levels of fatty acids in serum and the diet. Thus, T-cell functions in vivo may be differentially affected by the degree of saturation or the concentration of dietary fat. Moreover, linoleic acid appears to play a pivotal role in modulating cellular immune responses.

Gestational immunosuppression is mediated by specific Lyt 2+ T cells.

Female BALB/c mice were tested during the first week of pregnancy for their lymphocyte-mediated cytotoxic response to paternal alloantigens. Spleen or uterine regional lymph node cells were not spontaneously cytotoxic against concanavalin A-activated paternal target lymphocytes. Female mice immunized i.p. with paternal H-2-matched or third-party allogeneic cells on the fifth day and tested on the 12th day of pregnancy demonstrated total suppression of cell-mediated cytotoxicity to paternal alloantigens and partial suppression to third-party alloantigens. A generalized non-specific immunosuppression to alloantigens seems to be associated with pregnancy, which may indicate that soluble factors were involved in mediating the suppressive effect. Cocultures of spleen cells from virgin mice and the whole population of spleen or regional lymph node cells from allogeneic pregnant female mice demonstrated specifically suppressed responses to alloantigens. Similar cocultures with Thy 1.2- and Lyt 2.2-depleted populations restored the cytotoxicity levels of activated spleen cells. We conclude that antigen-specific Lyt 2+ T cells were activated during pregnancy to regulate the female T-cell response to paternal alloantigens.

Immunosuppressive effect of an impurity of malathion: inhibition of murine T- and B-lymphocyte responses by O,O,S-trimethyl phosphorothioate.

The effect of O,O,S-trimethyl phosphorothioate (OOS-TMP) on immune responses such as antigen presentation, antibody production, and cytotoxic T-lymphocyte (CTL) function was examined in vitro. The roles of non-enzymatic and enzymatic glutathione (GSH) conjugation of OOS-TMP in these responses were studied. Antibody responses to T-dependent and T-independent antigens were evaluated after (i) direct culture with spleen or B cells; (ii) cocultivation of B cells with T cells with and without preincubation of OOS-TMP with GSH fortified cytosol. Antigen presentation by macrophages was also assessed after such treatment as compared to untreated controls. OOS-TMP preincubated with GSH had an inhibitory effect on the cytotoxic T lymphocyte and the direct hemolytic plaque forming cell responses. This was found to be mediated by a direct inhibitory effect on macrophages, T, and B cells of the immune system and not through the generation of regulatory suppressor T cells. Thus, the mode of suppressive action of OOS-TMP in vitro is due to inhibition of lymphocytic proliferation. This is only possible in the presence of glutathione which was determined to be a prerequisite for the induction of OOS-TMP suppressive effect.

Seminal plasma abrogates the postcoital T cell response to spermatozoal histocompatibility antigens.

The T-cell responses to spermatozoal histocompatibility antigens were studied in the uterine regional lymph nodes of female rats by a specific cytoadhesion assay. The antigenic challenge was introduced either by artificial insemination with syngeneic or allogeneic epididymal spermatozoa or coitus. The uterine regional T-cell response to allogeneic spermatozoa following artificial insemination was measureable after 2 days and continued undiminished until day 5. In contrast, the measurable response to allogeneic coital challenge peaked on day 3 and disappeared by days 4-5. Since the difference between the two stimuli involved the presence of seminal plasma, these results indicate a possible immunoregulatory (suppressive) effect of seminal plasma on the cellular immune reaction of the female to the male alloantigens after mating.

Seminal plasma inhibits lymphocyte response to T-dependent and -independent antigens in vitro.

The effect of seminal plasma, epididymal spermatozoa, or whole semen on antibody producing cells was examined in vitro after (i) direct culture with spleen or B cells, and (ii) cocultivation of B cells with T cells previously incubated with seminal plasma. Seminal plasma, and not epididymal spermatozoa, have an inhibitory effect on the direct hemolytic plague forming cell response. This was mediated by a direct inhibitory effect on the B cell and not through the generation of suppressor T cells as demonstrated by responses to T-independent and -dependent antigens. Thus, the mode of suppressive action of seminal plasma in vitro is probably different from that previously reported in vivo.

Suppressive effect of O,O-dimethyl, S-ethyl phosphorothioate on immune response.

The effect of O,O-dimethyl, S-ethyl phosphorothioate (OO-Me S-Et) on macrophage function and the responses of T- and B-cells of the immune system were investigated using an in vitro model. The role of glutathione (GSH) enhancement of OO-Me S-Et suppression of immune responses was studied after either direct culturing with spleen or B-cells alone or coculture of B-cells with either B- or T-cells with or without preincubation with OO-Me S-Et and glutathione enriched cytosol. The results indicated that OO-Me S-Et, when preincubated with GSH, suppresses immune responses through an inhibition of B- and T-cell functions. Both cytotoxic T-lymphocyte (CTL) generation and antibody production were impaired. In mixing experiments of pretreated and normal cells, the inhibitory effect was also observed on macrophages and helper T-cells responding to a T-dependent antigen. It appears that the immunosuppressive effect of OO-Me S-Et is due to impairment of collaboration of T- and B-cells, i.e., mainly due to impairment of T-cell functions. The immunotoxic effect was only detectable when glutathione was present in the preincubation mixture.