Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor

Cannabis is one of the most widely used drugs throughout the world. The psychoactive constituent of cannabis, delta 9-tetrahydrocannabinol (delta 9-THC), produces a myriad of pharmacological effects in animals and humans. For many decades, the mechanism of action of cannabinoids, compounds which are structurally similar to delta 9-THC, was unknown. Tremendous progress has been made recently in characterizing cannabinoid receptors both centrally and peripherally and in studying the role of second messenger systems at the cellular level. Furthermore, an endogenous ligand, anandamide, for the cannabinoid receptor has been identified. Anandamide is a fatty-acid derived compound that possesses pharmacological properties similar to delta 9-THC. The production of complex behavioral events by cannabinoids is probably mediated by specific cannabinoid receptors and interactions with other neurochemical systems. Cannabis also has great therapeutic potential and has been used for centuries for medicinal purposes. However, cannabinoid-derived drugs on the market today lack specificity and produce many unpleasant side effects, thus limiting therapeutic usefulness. The advent of highly potent analogs and a specific antagonist may make possible the development of compounds that lack undesirable side effects. The advancements in the field of cannabinoid pharmacology should facilitate our understanding of the physiological role of endogenous cannabinoids.

Cannabis: pharmacology and toxicology in animals and humans.

w 3 x cannabinoid receptor agonist y -cis-3- 2-Hydroxy-4- 1,1-dimethylheptyl phenyl -trans-4- 3-hydroxypropyl cyclohexanol H CP . 55,940 revealed no residual cannabinoid binding sites in the spleen of the cannabinoid CB receptor knockout mice, while binding in the 2 central nervous system was unchanged. Cannabinoid CB receptor knockout mice, which appear healthy, are fertile and care for their 2 . offspring. Fluorescence activated cell sorting FACS analysis showed no differences in immune cell populations between cannabinoid CB receptor knockout and wildtype mice. We investigated the immunomodulatory effects of cannabinoids in cannabinoid CB receptor 2 2 deficient mice using a T cell co-stimulation assay. D 9 Tetrahydrocannabinol inhibits helper T cell activation through macrophages derived from wild type, but not from knockout mice, thus indicating that this effect is mediated by the cannabinoid CB receptor. In contrast, 2 central nervous system effects of cannabinoids were not altered in these mice. Our results suggest that cannabinoid CB receptor-specific 2 ligands may be clinically useful in the modulation of macrophage immune function while exhibiting no central nervous system activity. Furthermore, we conclude that the cannabinoid CB receptor knockout mouse is a useful animal model in which to study the role of the 2

Immunomodulation by cannabinoids is absent in mice deficient for the

Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

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Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells

The use of recreational drugs of abuse has generated serious health concerns. There is a long-recognized relationship between addictive drugs and increased levels of infections. Studies of the mechanisms of actions of these drugs became more urgent with the advent of AIDS and its correlation with abused substances. The nature and mechanisms of immunomodulation by marijuana, opiates, cocaine, nicotine, and alcohol are described in this review. Recent studies of the effects of opiates or marijuana on the immune system have demonstrated that they are receptor mediated, occurring both directly via specific receptors on immune cells and indirectly through similar receptors on cells of the nervous system. Findings are also discussed that demonstrate that cocaine and nicotine have similar immunomodulatory effects, which are also apparently receptor mediated. Finally, the nature and mechanisms of immunomodulation by alcohol are described. Although no specific alcohol receptors have been identified, it is widely recognized that alcohol enhances susceptibility to opportunistic microbes. The review covers recent studies of the effects of these drugs on immunity and on increased susceptibility to infectious diseases, including AIDS.

Microbial Infections, Immunomodulation, and Drugs of Abuse

Infection of BALB/c mice with a sublethal concentration of Legionella pneumophila causes an acute disease that is resolved by innate immune responses. The infection also initiates the development of adaptive Th1 responses that protect the mice from challenge infections. To study the early responses, cytokines induced during the first 24 h after infection were examined. In the serum, interleukin-12 (IL-12) was detectable by 3 h and peaked at 10 h, while gamma interferon was discernible by 5 h and peaked at 8 h. Similar patterns were observed in ex vivo cultures of splenocytes. A transient IL-4 response was also detected by 3 h postinfection in ex vivo cultures. BALB/c IL-4-deficient mice were more susceptible to L. pneumophila infection than were wild-type mice. The infection induced higher serum levels of acute-phase cytokines (tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6), and reducing TNF-α levels with antibodies protected the mice from death. Moreover, the addition of IL-4 to L. pneumophila-infected macrophage cultures suppressed the production of these cytokines. Thus, the lack of IL-4 in the deficient mice resulted in unchecked TNF-α production, which appeared to cause the mortality. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is induced by IL-4 during Listeria monocytogenes infection, was detected at between 2 and 30 h after infection. However, MCP-1 did not appear to be induced by IL-4 or to be required for the TNF-α regulation by IL-4. The data suggest that the early increase in IL-4 serves to regulate the mobilization of acute phase cytokines and thus controls the potential harmful effects of these cytokines.

Induction of interleukin-4 (IL-4) by Legionella pneumophila infection in BALB/c mice and regulation of tumor necrosis factor alpha, IL-6, and IL-1β

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.

Tumor Necrosis Factor Induces Resistance of Macrophages to Legionella pneumophila Infection

Differing effects of delta-9-tetrahydrocannabinol (THC) on murine spleen cell populations dependent upon stimulators.

Virulent Legionella pneumophila repli- cate readily in thioglycollate-elicited peritoneal macrophages from genetically permissive A/J mice, but avirulent L. pneumophila do not. The pro- duction of cytokines by macrophages infected with L. pneumophila has been studied, but the correla- tion of bacterial virulence with immune responses of macrophages, such as proinflammatory cytokine production, is not well understood. In this regard, production of the cytokines tumor necrosis factor a (TNF-a), interleukin (IL)-1a, IL-1b, and IL-6 were examined in macrophage cultures infected in vitro with virulent vs. avirulent L. pneumophila. Infection of macrophages from A/J mice with the virulent L. pneumophila up-regulated mRNA ex- pression for these cytokines, whereas avirulent bacteria resulted in only a slight or no detectable increase in cytokine mRNA. Similarly, virulent L. pneumophila induced the macrophages to pro- duce relatively high levels of TNF-a, IL-1a, IL-1b, and IL-6 proteins as measured by enzyme-linked immunosorbent assays, whereas avirulent bacteria induced only low or often undetectable amounts of these cytokines. Thus, these results show the mu- rine macrophages from susceptible A/J mice are readily infected with virulent L. pneumophila in vitro and stimulated to produce the proinflamma- tory acute-phase cytokines TNF-a, IL-1a, IL-1b, and IL-6, but avirulent L. pneumophila did not. Such differences in induction of these proinflamma- tory cytokines by macrophages in response to virulent vs. avirulent L. pneumophila infections may be an important factor in the pathogenesis induced by these intracellular bacteria. J. Leukoc. Biol. 67: 863-868; 2000.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1002/jlb.67.6.863

Murine macrophages differentially produce proinflammatory cytokines after infection with virulent vs. avirulent Legionella pneumophila.

Infection of macrophages from genetically susceptible A/J mice with Legionella pneumophila induces high levels of various cytokines in serum as well as in cultures of spleen or peritoneal cells from the mice. However, modulation of receptor expression for these cytokines during infection has not been studied in detail, even though these receptors on macrophages have a critical role in inflammatory responses during the infection. In the present study, the differential expression of mRNA for TNF and IL-1 receptors as well as receptor antigens during infection of macrophages with virulent vs. avirulent L. pneumophila was investigated. Mouse thioglycollate-elicited peritoneal macrophages showed by RT-PCR constitutive steady-state levels of mRNA for TNF-type I and -type II receptors as well as IL-1 type I receptor. However, IL-1 type II receptor mRNA was not expressed in thioglycollate-elicited macrophages. Infection of macrophages with virulent bacteria caused an upregulation of IL-1 type I and TNF type I receptor mRNA, but had no effect on TNF type II receptor message. Avirulent L. pneumophila infection caused much less induction of these receptor mRNAs. The amount of receptor antigen of IL-1 type I on the surface of macrophages was also increased by infection with virulent L. pneumophila determined by flow cytometric analysis. These results indicate that L. pneumophila infection not only causes induction of various cytokines, but also modulation of certain cytokine receptors, which may regulate the susceptibility to infection.

Shannon McHugh

Papers

Induction of interleukin-4 (IL-4) by Legionella pneumophila infection in BALB/c mice and regulation of tumor necrosis factor alpha, IL-6, and IL-1β

Tumor Necrosis Factor Induces Resistance of Macrophages to Legionella pneumophila Infection

Differing effects of delta-9-tetrahydrocannabinol (THC) on murine spleen cell populations dependent upon stimulators.

Murine macrophages differentially produce proinflammatory cytokines after infection with virulent vs. avirulent Legionella pneumophila.

Differential expression of IL-1 and TNF receptors in murine macrophages infected with virulent vs. avirulent Legionella pneumophila