This study shows that two cannabinoids, Delta(9)-tetrahydrocannabinol (THC) and anandamide, induce dose-related immunosuppression in both the primary and secondary in vitro plaque-forming cell assays ...of antibody formation. The immunosuppression induced by both compounds could be blocked by SR144528, an antagonist specific for the CB(2) receptor, but not by SR141716, a CB(1) antagonist. These studies are novel in that they show that both anandamide and THC are active in the nanomolar to picomolar (for anandamide) range in these assays of immune function, and that both mediate their effects directly on cells of the immune system through the CB(2) receptor.
A vaccine strain of live, attenuated Salmonella typhimurium induces profound immunosuppression in inoculated mice 7 days after injection. Immunosuppression to mitogens and inability to mount ...plaque-forming responses to sheep red blood cells occurs in spite of many parameters of upregulated macrophage function and protection against challenge with virulent Salmonella. Studies show that macrophage nitric oxide mediates the immunosuppression and presumably also the early-onset protective capacity of the vaccine. A model of "bystander lymphocyte autotoxicity" is presented to explain the mechanism of immunosuppression. The model proposes that Salmonella-activated macrophages generate nitric oxide which inactivates lymphocytes in the vicinity, so they become dysfunctional. Inhibition of nitric oxide by NG-monomethyl-L-arginine reverses immunosuppression. Evidence is presented that supports a relationship between the microbial burden in the spleen, the degree of nitric oxide produced, and the extent of immunosuppression. It is proposed that this model of microbial immunosuppression mediated by nitric oxide is generalizable for understanding immunosuppression and loss of delayed-type hypersensitivity induced by other microbes, such as Mycobacteria and measles virus. The model could account for anergy during mycobacterial infections, particularly when the burden of acid-fast bacilli is high, as well as loss of skin test reactivity to tuberculin during measles infection.
This study shows that two cannabinoids, Δ
9-tetrahydrocannabinol (THC) and anandamide, induce dose-related immunosuppression in both the primary and secondary in vitro plaque-forming cell assays of ...antibody formation. The immunosuppression induced by both compounds could be blocked by SR144528, an antagonist specific for the CB
2 receptor, but not by SR141716, a CB
1 antagonist. These studies are novel in that they show that both anandamide and THC are active in the nanomolar to picomolar (for anandamide) range in these assays of immune function, and that both mediate their effects directly on cells of the immune system through the CB
2 receptor.
We previously demonstrated that an aroA- strain of Salmonella typhimurium, which provides excellent protection against virulent Salmonella challenge, also rendered immunized mice unable to mount in ...vivo and in vitro antibody responses to heterologous Ag. Coculture studies using transwell plates indicated that suppression was mediated by soluble factors. The suppressive cells were identified as belonging to the monocytic linkage. Macrophage precursors as well as mature adherent macrophages mediated the observed suppression. In the present study, the mechanism of immunosuppression was investigated. Suppression was found to be genetically nonrestricted as spleen cells from immunized C3HeB/FeJ mice (H-2k) suppressed the anti-SRBC plaque-forming cell (PFC) responses of normal spleen cells from two MHC noncompatible mouse strains, BALB/c (H-2d) and C57BL/6 (H-2b). Time course experiments demonstrated that the addition of spleen cells from immunized mice to normal splenocytes as late as day 4 of a 5-day assay was still markedly suppressive. Furthermore, suppression of the PFC responses was accompanied by a profound inhibition of the capacity of immune splenocytes to produce IL-2 in response to in vitro stimulation by Con A. Coculture studies showed that immune spleen cells were able to suppress IL-2 production by normal splenocytes in a dose-dependent fashion. However, the suppressed PFC responses of immune spleen cells could not be reversed by the exogenous addition of up to 200 U/ml of IL-2, suggesting that immune splenocytes are also defective in their ability to respond to IL-2. In marked contrast, suppression of PFC responses was reduced by more than 50% by the addition of as little as 1 U/ml of IL-4 and was completely abrogated when 5 U/ml of IL-4 were added to in vitro cultures of spleen cells from immunized mice. The antisuppressive action of IL-4 appeared to be via its inhibitory effect on activated macrophages. The implications of the above findings are discussed.
Our laboratory has shown that immunization of mice with an attenuated strain of Salmonella typhimuriuminduces profound suppression in the capacity of splenocytes to mount an in vitro antibody ...plaque-forming cell (PFC) response to sheep red blood cells (SRBC) and to proliferate in response to mitogens. In vitro addition of NG-monomethyl-L-arginine (NMMA), an inhibitor of nitric oxide (NO) synthase, to cell cultures from Salmonella-immunized mice completely blocked suppression of the PFC responses, implicating that NO is the suppressor factor. The present study quantified the role of nitric oxide in immunosuppression induced by Listeria monocytogenes, a gram positive intracellular pathogen of macrophages. Listeria infection resulted in suppression of the PFC assay at inoculating doses of greater than 6.5x10(3)colony forming units, with no suppression observed at lower doses. Suppression correlated with increased nitrite production. Addition of NMMA to spleen cell cultures taken from Listeria-infected mice completely blocked suppression of the PFC response, and returned nitrite production to baseline levels. In regard to Listeria-induced suppression of responses to the mitogen, Concanavalin A (Con A), the parameters were different from those observed for the PFC response. There was a direct correlation between the log10of the inoculating dose of Listeria and degree of immunosuppression, with suppression observed at doses as low as 1x10(3)cells. Addition of NMMA to the Con A-stimulated cultures resulted in reduced nitrite levels, but only partial restoration of the proliferative responses. Co-culture of splenocytes from Listeria inoculated mice with normal splenocytes in media with NMMA and reduced levels of L-arginine resulted in complete reversal of suppressed responses to Con A. Similar differences in ease of reversing suppression of the PFC response, as compared with responses to Con A, were previously noted using cells taken from Salmonella-infected mice. The present results show that a gram positive intracellular pathogen of macrophages, L. monocytogenes, induces immunosuppression in mouse spleen cells by a nitric oxide mediated mechanism that closely parallels that induced by the gram negative pathogen, S. typhimurium.
We have demonstrated that immunotherapy of young (6–10 weeks old), and aged, (greater than 24 months old), tumor bearing mice with biological response modifiers enhanced survival and inhibited tumor ...growth, while treatment of aged mice had little or no effect. We hypothesized that the antitumor activity in young mice was principally mediated by activated macrophages (Mφ) and predicted that the change in aged mice was caused by an intrinsic Mφ defect which develops with advancing age. To directly test our hypothesis, we examined the antitumor activity of resident peritoneal Mφ, purified and activated in vitro with IFN
γ plus LPS. Paralleling the results seen in vivo, Mφ from aged mice exhibited reduced antitumor activity in comparison with Mφ from younger mice. Moreover, there was reduced capacity of in vitro activated Mφ from aged mice to produce TNF, IL-1 and nitric oxide, which are critical monokines and effector molecules that have been established to either directly inhibit tumor growth or cause tumor cell destruction. These studies establish that peritoneal Mφ from aged mice have an intrinsic defect which prevents them from fully expressing their antitumor potential.
An attenuated strain of Salmonella typhimurium, SL3235, developed as a prototypic typhoid vaccine, is shown to retard growth of a murine plasmacytoma, TEPC-183, and to prolong survival of ...tumor-bearing mice. Live salmonella, but not acetone-killed organisms, had antitumor activity. The immunotherapeutic effect was demonstrable when the tumor was injected intralesionally or intraperitoneally. Increased survival, longer mean time to death, and retardation of tumor growth were found when the salmonella were given intralesionally as late as the sixth day post-tumor injection. Timing of salmonella inoculation, as well as the salmonella dose, had an effect on treatment efficacy. Injection of salmonella intraperitoneally exerted a strong antitumor effect when given as late as the third day post-tumor inoculation. The highest dose (2 x 10(6)) of salmonella was less effective than doses 10- or 100-fold lower. TEPC-183 plasmacytoma is rapidly growing and highly immunosuppressive, so the ability of the salmonella to exert therapeutic activity against it is a measure of the potency of the vaccine. These observations are of interest, as they show that a genetically engineered, avirulent strain of Salmonella has immunotherapeutic properties similar to those of BCG and other biological response modifiers, and might have clinical potential as an antitumor agent.
A live, avirulent strain of Salmonella typhimurium, SL3235, was previously shown to afford protection against virulent Salmonella challenge in three mouse strains of the C3H lineage, C3H/HeJ, ...C3HeB/FeJ, and C3H/HeNCrlBR, which differ in their innate susceptibility to Salmonella infection, as well as in their responsiveness to lipopolysaccharide (LPS). Concurrent with protection, however, SL3235 was found to induce greater than 90% reduction in proliferative responses of splenocytes from immunized mice to a panel of B and T cell mitogens. Suppression appeared to be independent of susceptibility to Salmonella infection, since the mitogenic responses of hypersusceptible C3H/HeJ and C3HeB/FeJ, as well as resistant C3H/HeNCrlBR mice, were suppressed. The suppressor cell population in immunized C3HeB/FeJ mice was recently shown to be of monocytic lineage. Using transwell plates, co-culture studies indicated that suppression was mediated by soluble factors. In the present study, the effect of LPS responsiveness on susceptibility to SL3235-induced suppression was evaluated in C3H mice by studying their ability to mount plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) and in vivo antibody responses to tetanus toxoid. Comparison of PFC responses as a function of SL3235 dose in C3HeB/FeJ and C3H/HeJ mice, revealed that the latter strain was markedly more resistant to the development of suppression, as evidenced by the significantly higher (10-35-fold) SL3235 doses needed to achieve comparable suppression to those seen in C3HeB/FeJ mice. In contrast to C3HeB/FeJ mice, suppression in C3H/HeJ mice required direct cell-cell contact. In both mouse strains, suppression was alleviated by pre-treatment of immune splenocytes with either mitomycin C or x-irradiation, indicating that actively proliferating cells are required for suppressor function. Resistance of C3H/HeJ mice to SL3235-induced suppression was not due to a lesser bacterial load in vivo, since a higher number of SL3235 organisms were seen in C3H/HeJ spleens compared to C3HeB/FeJ mice. Rather, resistance of C3H/HeJ mice correlated with their reduced ability to recruit macrophages and other inflammatory cells into the spleen, as evidenced by the significantly smaller degree of splenomegaly induced in these mice following immunization with SL3235.
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