Specific association of Type I c-Abl with Ran GTPase in lipopolysaccharide-mediated differentiation.

Each of several isoforms of c-Abl may be involved in different biological functions. Type I c-Abl has been shown to be involved in LPS-induced differentiation and Type IV c-Abl, apoptosis. Ran has recently been shown to be involved in LPS endotoxin signal transduction. Here we show that Type I c-Abl associates with Ran. Formation of this complex is specific, as Ran did not associate with the highly homologous Type IV c-Abl isoform. In non-stimulated lymphoid B cells, Type I c-Abl tyrosine kinase is inactive, whereas Type IV kinase is active. Formation of Type I c-Abl/Ran complex and activation of Type I c-Abl kinase activity are LPS dose-dependent. This complex is detectable in B cells of endotoxin-sensitive inbred mice but absent in B cells of endotoxin-resistant mice. These findings therefore suggest that Type I c-Abl and Ran are important targets in lipopolysaccharide-induced biological responses of hematopoietic cells.

Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages.

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment. ELISA data showed that both IL-12 p40 and p70 were increased by morphine. The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria. The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.

Implications of Salmonella-induced nitric oxide (NO) for host defense and vaccines: NO, an antimicrobial, antitumor, immunosuppressive and immunoregulatory molecule.

Attenuated Salmonella induce immunosuppressive, microbicidal and tumoricidal macrophages in mice. All three effects are mediated by activated macrophages producing nitric oxide (NO). NO is induced by the innate immune response pathway involving IL-12, NK cells and IFN-gamma in response to infection. NO has beneficial and detrimental effects on the host.

Decreases in macrophage mediated antitumor activity with aging.

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 (M phi) and predicted that the change in aged mice was caused by an intrinsic M phi defect which develops with advancing age. To directly test our hypothesis, we examined the antitumor activity of resident peritoneal M phi, purified and activated in vitro with IFN gamma plus LPS. Paralleling the results seen in vivo, M phi from aged mice exhibited reduced antitumor activity in comparison with M phi from younger mice. Moreover, there was reduced capacity of in vitro activated M phi 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 M phi from aged mice have an intrinsic defect which prevents them from fully expressing their antitumor potential.

Opioid modulation of immune responses: effects on phagocyte and lymphoid cell populations.

The literature describing effects of morphine on cells of the immune system points to the clear conclusion that morphine given in vivo suppresses a variety of immune responses that involve the major cell types in the immune system, including natural killer (NK) cells, T cells, B cells, macrophages and polymorphonuclear leukocytes (PMNs). Depression of NK cell activity has been reported in humans, monkeys and rodents. Similarly, responses of T cells are depressed by morphine, as assessed by inhibition of induction of delayed-type hypersensitivity reactions and cytotoxic T-cell activity, modulation of T-cell antigen expression, and depression of responses to T-cell mitogens. Effects on T cells have been reported in humans, monkeys and rodents. Effects of morphine on B-cell activity have mainly been tested in rodents using assays of antibody formation, which also require macrophages and T cells, preventing a conclusion as to the cell type being affected. Consistent effects on phagocytic cell function have been reported in rodents given morphine. In contrast, studies on immunomodulatory effects of morphine added to cells of the immune system in vitro have shown robust effects on some of these cell types, but not others. There is a rich literature demonstrating downregulation of phagocytic cell function by morphine, particularly for human peripheral blood mononuclear cells (PBMCs) and PMNs. Phagocytosis, chemotactic responses, interleukin production, and generation of activated oxygen intermediates and arachidonic acid products have all been reported to be inhibited. On the contrary, the literature does not support direct effects of morphine on NK cell function, is inconclusive concerning effects on B cells, and provides limited evidence for effects on T cells. The divergence between the in vivo and in vitro data suggests that effects on some cells in the immune system observed after in vivo morphine are probably not direct, but mediated. In aggregate, the literature supports the existence of an in vivo neural-immune circuit through which morphine acts to depress the function of all cells of the immune system. Further, there is strong evidence that morphine can directly depress the function of macrophages and PMNs, and modulate expression of one type of T-cell surface marker. There is, however, little evidence for direct effects of morphine on NK cells and B cells. A further complication emerges from reports of immunopotentiation of immune function in in vitro assays using endogenous opioids. The possibility of different receptors for endogenous and exogenous opioids or of interactions among the activated opioid receptors may account for these opposing effects.

Inhibition of T cell superantigen responses following treatment with the kappa-opioid agonist U50,488H.

Previous work in our laboratory has shown that cytokine production by primary murine macrophages, and macrophage cell lines, is inhibited following treatment with the kappa-opioid agonist U50,488H. Furthermore, we have found that the participation of both accessory cells and T cells in an antibody response is suppressed by this compound. We have utilized the superantigen staphylococcal enterotoxin B (SEB) to further examine the effects of U50,488H on accessory and T cell function. The results showed that the proliferative response of lymph node T cells to SEB presented by activated macrophages was significantly inhibited by the kappa-opioid agonist at concentrations as low as 100 nM. However, suppression of the T cell response to SEB presented by resting macrophages required 100 times the concentration of U50,488H. On the other hand, the production of IL-2 in response to lymph node T cell stimulation with SEB was not altered by the opioid treatment. Additional experiments utilizing the opiate antagonist naloxone and the kappa-selective antagonist nor-binaltorphimine (norBNI) were performed in order to further characterize the opioid receptor involved in the suppressive activity of U50,488H. Results showed that both naloxone and norBNI were able to block the inhibitory activity of U50,488H. Further analysis showed that the proliferative response of thymic T cells was more sensitive to the effects of U50,488H, and the response with both activated and resting macrophages was suppressed. In addition, the production of IL-2 by the thymic T cells was also inhibited by the opioid treatment. The mechanism of suppression of superantigen-induced T cell responses is discussed.

Morphine alters macrophage and lymphocyte populations in the spleen and peritoneal cavity.

We have previously shown that subcutaneous implantation of a 75 mg morphine pellet results in suppression of the ability of murine splenocytes to mount an antibody response to sheep red blood cells, due in part to a reduction of macrophage function. The present studies used flow cytometry to examine whether the decrement in macrophage function in the spleens of morphine-treated mice results from a reduction in macrophage numbers. Parallel analysis was carried out on non-elicited peritoneal cells. In the spleen, morphine resulted in a reduction in the relative proportion of macrophages and B-cells, with a concomitant increase in the proportion of T-cells. Alteration in the ratio of CD4+ to CD8+ T-cells was not observed. In contrast, in the peritoneal cavity, morphine increased the number of macrophages and reduced the number of B-cells. Naltrexone blocked all of the changes in cellular composition. These results support the conclusion that an important mechanism in the immunosuppression seen in the spleens of mice implanted with morphine pellets is a differential reduction in the number of macrophages and B-cells as compared with T-cells. Further, these studies show that subsets of cells of the immune system are differentially affected by morphine in different anatomical compartments.

Inhibition of primary murine macrophage cytokine production in vitro following treatment with the kappa-opioid agonist U50,488H.

Previous work in our laboratory has shown that both mu- and kappa-opioid agonists exhibit immunosuppressive activity for antibody responses in vitro. Our earlier work has suggested that both accessory cells and T cells may be altered following treatment with the kappa-opioid agonist U50,488H. We intend to further determine the identity of the immune cell population(s) which are affected by opioid treatment, and to determine the nature of the opioid receptor type expressed on these cells. In this study, non- elicited peritoneal macrophages were treated simultaneously with the kappa-agonist U50,488H and lipopolysaccharide (LPS), and the levels of the cytokines interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha were determined. The results show that U50,488H had a suppressive effect on the production of TNF-alpha and IL-1 at concentrations as low as 1 nM, while IL-6 was suppressed at concentrations as low as 10 nM. Additional experiments utilizing the opiate antagonist naloxone and the kappa-selective antagonist norbinaltorphimine (norBNI) were performed in order to further characterize the opioid receptor involved in the cytokine suppression produced by treatment with U50,488H. Results showed that naloxone was able to partially block U50,488H suppression while norBNI was able to completely reverse the suppression of IL-6 production. These results suggest that macrophage/monocyte function is significantly modulated following activation of the kappa-opioid receptor.

Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line.

To our knowledge, this is the first demonstration of kappa-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the kappa-opioid receptor is expressed by the immature T cell line R1.1. We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse kappa-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5' region upstream of the ATG start codon. Comparison of these sequences to the previously published kappa-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5' non-coding region.

Characterization of kappa-opioid receptor transcripts expressed by T cells and macrophages.

We have found that the immature T cell lines R1.1 and DPK and the macrophage lines P388D1 and WEHI-3 also express kappa-opioid receptor (KOR) mRNA. Characterization of the KOR transcripts in both brain tissue and these T cells has revealed both the normal full-length as well as a truncated form of the mRNA. Our results show that the truncated transcript lacks the second exon. Primary macrophages express this truncated form of the transcript in the absence of detectable levels of the full-length form. These results suggest a degree of heterogeneity in the expression of the opioid receptors which has not previously been reported.

Macrophage nitric oxide mediates immunosuppression in infectious inflammation.

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.

Opioids, opioid receptors, and the immune response.

It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.

Administration of mu-, kappa- or delta2-receptor agonists via osmotic minipumps suppresses murine splenic antibody responses.

Previously, our laboratory has shown that morphine given by implantation of a 75-mg slow-release pellet for 48 h suppresses murine splenic antibody responses to sheep red blood cells (SRBCs) in a plaque-forming cell (PFC) assay. However, the use of slow-release pellets for such studies is limited, as these pellets are only available in fixed doses and similar pellets for kappa and delta agonists have not been developed. In the present study, we investigated the feasibility of administering opioids via Alzet osmotic minipumps to assess their immunomodulatory effects. Groups of mice received minipumps dispensing morphine sulfate, which has primary activity at the mu opioid receptor; U50,488H, which is a kappa-selective agonist; deltorphin II, which is a delta2-selective agonist; or DPDPE, which has greater selectivity for delta1 than delta, receptors. Morphine, U50,488H and deltorphin II were all immunosuppressive, with biphasic dose-response curves exhibiting maximal (approximately 50%) suppression of the PFC response at doses of 0.5 to 2 mg/kg/day 48 h after pump implantation. Further, immunosuppression by morphine sulfate, U50,488H or deltorphin II was blocked by simultaneous implantation of a minipump administering the opioid receptor-selective antagonists CTAP (1 mg/kg/day), nor-binaltorphimine (5 mg/kg/day), or naltriben (3 mg/kg/day), respectively. DPDPE was inactive at doses lower than 10 mg/kg/day. We conclude that osmotic minipumps are a practical and useful way of administering opioids to study their effects on the immune system, and give further evidence that immunosuppression induced in vivo by opioid agonists is mediated not only via mu, but also via kappa and delta2 opioid receptors.

Morphine treatment in vitro or in vivo decreases phagocytic functions of murine macrophages.

Studies were performed to compare in vitro and in vivo effects of morphine on the phagocytic function of murine peritoneal macrophages. Macrophage monolayers were incubated with Candida albicans for 30 min in the absence of autologous serum. Morphine added in vitro was found to decrease both the phagocytic activity (percent of phagocytic cells) and the phagocytic index (average number of ingested yeasts per cell) in a concentration-dependent manner, with maximal effects of 26% and 41%, respectively, at 10(-6) M. When morphine was administered in vivo via an implanted 75-mg pellet, there was a 22% decrease in phagocytic activity and a 40% decrease in the phagocytic index. Naltrexone completely blocked the effects of morphine both in vitro and in vivo. The results suggest that morphine is capable of interacting directly with opioid receptors on macrophages, resulting in a decrease in phagocytic function.

Effects of in vivo morphine treatment on antibody responses in C57BL/6 bgJ/bgJ (beige) mice.

C57BL/6J bgJ/bgJ (beige) mice are less sensitive than other strains to the analgesic effects of morphine, although they have normal numbers of mu receptors. In the present study, beige mice and their normal littermates (beige+) were treated in vivo with morphine or the opioid antagonist, naltrexone and their primary in vitro antibody responses were assessed. Morphine treatment caused splenic atrophy and suppressed the primary in vitro antibody response in beige and beige+ mice. However, these effects were not blocked by naltrexone co-treatment. In these mouse strains, naltrexone decreased spleen size and antibody responses by itself, which may mask its ability to antagonize morphine. In beige mice, placebo pellet implantation suppressed the primary in vitro antibody response. Morphine did not cause a further suppression of the antibody response in beige mice compared to placebo. Because of this anomalous response to placebo treatment, the immunosuppressive effects of morphine on the antibody response/10(7) cells can not be attributed to a specific drug effect in this strain. However, when antibody responses were expressed on a per spleen basis, the overall capacity to respond to antigenic challenge was suppressed by morphine treatment.

Immunotherapy of a plasmacytoma with attenuated salmonella.

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.

Strain dependent variation of delayed-type hypersensitivity in Salmonella typhimurium infected mice.

Inherently hypersusceptible C3H/HeJ and C3HeB/FeJ mice show increased resistance to challenge with virulent S. typhimurium after immunization with a live, avirulent S. typhimurium mutant in the absence of a delayed-type hypersensitivity response, while innately resistant C3H/HeNCr1BR and CD-1 mice show both immunity and positive footpad reactions after immunization. Understanding the mechanism for this specific anergy in the hypersusceptible strains may provide clues for understanding the immune defect that causes these mice to be so exquisitely susceptible to Salmonella infection.

Immunity to Salmonella infection.

The foregoing literature review and data presentation have been set forth in the hope of clarifying some complex and confusing issues in regard to Salmonella infection. From a practical point of view, the information presented has implications for the direction to take with regard to improving the current typhoid vaccine, as the presently used acetone-killed cell preparation has considerable toxicity. The issues are important from a theoretical standpoint, because they have bearing on the nature of the concepts researchers and clinicians carry as working hypothesis with regard to the mechanisms of immunity to Salmonella infection. An incomplete appreciation of the literature seems to have led many scientists to believe that only cellular immunity can protect a mouse, and by analogy a human, against Salmonella. The logical deduction from such a premise is that only live vaccines will be effective in humans againsT S. typhi. Such a conclusion would appear unfounded, as documented in this review, for killed vaccines have been shown to be highly effective in vaccinating many mouse strains, as well as humans, against enteric fever.

Effect of herpes simplex virus type 1 infection on cytokine gene expression in activated murine peritoneal macrophages.

The intrinsic resistance to herpes simplex virus type 1 (HSV-1) of murine peritoneal macrophages (PM phi) obtained after in vivo infection of different stimuli has been investigated and shown to vary depending on the state of M phi activation. Activation of M phi by C. parvum (CP-M phi) or by an avirulent strain of S. typhimurium (Sal-M phi) increased the permissiveness of M phi to HSV-1 infection as evidenced by increased HSV-1 immediate early (IE) gene expression, synthesis of IE proteins, and the degree of cytopathic effect. HSV-1 infection was also found to sharply reduce the level of IL-1-beta mRNA in CP-M phi) and Sal-M phi, and the level of IL-3 mRNA in infected Sal-M phi, as measured by northern blot hybridization. Barely detectable levels of IL-beta mRNA were found in Sal-M phi after infection with HSV-1 when the polymerase chain reaction (PCR) assay was used to confirm the reduction of IL-1-beta mRNA. These data suggest that HSV-1 infection can modulate gene expression of some cytokines in the activated M phi.

The effect of morphine and DAGO on the proliferative response of murine splenocytes.

Morphine added in vitro to BALB/c mouse spleen cells inhibits mitogen responses. Naloxone and naltrexone, but not nor-BNI, antagonize the effect of morphine, suggesting that the kappa opioid receptor is not involved in in vitro immunomodulation of mitogen responses by morphine.