Search

The steroids secreted by the adrenal cortex are crucial for survival in that they are necessary for the response to a threatening environment. The main adrenal steroids are those such as mineralocorticoid, glucocorticoid and some sex steroids, mainly androgens. This chapter discusses susceptibility of individuals to infections caused by steroids, such as glucocorticoids. Steroid therapy has been suggested for a variety of complications related to human immunodeficiency virus infection, including pneumocystis infections and esophageal and oral ulcerations, because steroid treatment decreases both morbidity and mortality due to Pneumocystis carinii pneumonia. The mechanisms of increased susceptibility to infections induced by steroids are complicated, since steroids affect the function of a wide variety of host cells, including immune cells. The effect of steroids on neutrophils appears to be different than the effects on other leukocytes, since a single dose of steroid induces a significant decrease of circulating monocytes, eosinophils and basophils, but peripheral blood neutrophils increase two or three-fold rather than decrease.

Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET), and they suppress the function of LPS-stimulated dendritic cells (DCs). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). LT, not ET, was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-alpha in cells from BALB/c and B6 mice but increased IL-1beta in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-alpha, but increased IL-6 and IL-1beta in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET generally decreased marker expression across all groups. We conclude that the suppression of cytokine production by anthrax toxins is dependent on variables, including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the stimuli or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.

Nicotine has a multitude of biological actions in the central and peripheral nervous systems where nicotinic acetylcholine receptors are found. Nicotinic acetyl- choline receptors have also been identified on immune cells, but the effects of nico- tine on immune responses are not well characterized. These studies tested the hy- potheses that nicotine has an effect on both T-lymphocyte proliferation and the pro- duction of cytokines by activated T cells, processes that are necessary for effective T-cell-mediated immune responses. In addition, the effects of nicotine on these im- mune responses in aging animals and the effects of nicotine exposure prior to immu- nostimulation were investigated. Murine splenocytes were exposed to nicotine and stimulated with concanavalin A (ConA). The highest concentration of nicotine (128 µg/ml) significantly depressed proliferation of T cells both when nicotine and ConA were added concurrently and when nicotine was added 3 hr prior to ConA. Nicotine, added concurrently with ConA at concentrations between 0.25 and 64 µg/ml, signifi- cantly inhibited the production of IL-10 by splenocytes from young adult mice, whereas the inhibition of production of IL-10 by splenocytes from old mice was sig- nificantly inhibited, but the response was more variable, depending on the nicotine concentration. In contrast, the production of IFN-g by splenocytes from either young adult or old mice was not affected when nicotine (0.016-64 µg/ml) was added concur- rently with ConA. Pre-exposure to 1 µg/ml of nicotine for 3 hr significantly enhanced the production of IFN-g by splenocytes from young adult mice, whereas pre-exposure to 0.016 µg/ml of nicotine tended to but did not significantly enhance IFN- g produc- tion. Nicotine is now being used as an over-the-counter drug by people who differ in age and general immunocompetence. Therefore, the effects of nicotine on immune responses, independent from the effects of the other chemicals found in tobacco, need to be investigated. (P.S.E.B.M. 2000, Vol 224:141-146)

The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella -induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.

Chlamydia pneumoniae is the aetiological cause of a wide variety of chronic inflammatory diseases and may be associated with neurological disease. Microbiological and immunological aspects of the interaction between C. pneumoniae and the central nervous system (CNS) are not well understood because of the lack of a suitable infection model for neuronal studies. In the present study, an in vitro C. pneumoniae infection model was developed in the established microglial cell line EOC 20. Infection of the cells resulted in obvious induction of proinflammatory cytokines. The infection also selectively induced matrix metalloproteinase-9 (MMP-9) but not MMP-2. Moreover, beta interferon, which is known to modulate CNS disease, inhibited induction of MMP-9 following C. pneumoniae infection. These results support the view that C. pneumoniae infection may be associated with marked alteration of the ability of microglial cells to enhance cytokine production as well as induction of an MMP.

The primary polyphenol in green tea extract is the catechin epigallocatechin gallate (EGCG). Various studies have shown significant suppressive effects of catechin on mammalian cells, either tumor or normal cells, including lymphoid cells. Previous studies from this laboratory reported that EGCG has marked suppressive activity on murine macrophages infected with the intracellular bacterium Legionella pneumophila (Lp), an effect mediated by enhanced production of both tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma). In the present study, primary murine bone marrow (BM)-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms, such as Lp, were stimulated in vitro with the microbial stimulant lipopolysaccharide (LPS) from gram-negative bacteria, the cell wall component from gram-positive bacteria muramyldipeptide (MDP) or infected with Lp. Production of the T helper cell (Th1)-activating cytokine, interleukin-12 (IL-12) and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha),produced mainly by phagocytic cells and important for antimicrobial immunity, was determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Treatment of the cells with EGCG inhibited, in a dose-dependent manner, production of IL-12. In contrast, enhanced production of TNF-alpha occurred in a dose-dependent manner in the DC cultures stimulated with either soluble bacterial product or infected with Lp. Thus, the results of this study show that the EGCG catechin has a marked effect in modulating production of these immunoregulatory cytokines in stimulated DCs, which are important for antimicrobial immunity, especially innate immunity. Further studies are necessary to characterize the physiologic function of the effect of EGCG on TNF-alpha and IL-12 during Lp infection, and the mechanisms involved.

Nicotine, the addictive component of cigarette smoke, has been shown to have immunomodulatory effects. This drug alters proinflammatory cytokine production by immune cells, including lymphocytes, monocytes, and macrophages. The present study focuses on the effects of nicotine on infection by Chlamydia pneumoniae (Cpn), a ubiquitous intracellular pathogen which causes acute and chronic inflammatory diseases such as pulmonary infections, and may be associated with arthritis and atherosclerosis. Previous studies in our laboratory showed that lymphocytes and macrophages are susceptible to Cpn infection. The present study aimed at investigating the effect of nicotine on TGF-beta1, IL-10, IL-12, and TNF-alpha production in Cpn-infected human peripheral blood mononuclear cells (PBMCs). Cytokine levels in the supernatant were assessed by ELISA. The results showed that Cpn infection alters the expression levels of IL-10, IL-12, and TNF-alpha in a time-dependent fashion. Nicotine treatment of the Cpn-infected cells up-regulated IL-10, but not TNF-alpha and IL-12, and also resulted in significant down-regulation of TGF-beta1 production which was marked in the Cpn-infected control cells. The combined action of nicotine and Cpn on cytokine production may have an impact in chronic inflammatory diseases.

T helper cell type 1 (Th1)-polarizing cytokines are induced by Legionella pneumophila infection and are suppressed by pretreatment with marijuana cannabinoids (CB). Glucocorticoids and prostaglandin E2(PGE2) are also reported to suppress Th1 polarization and are induced by Δ9-tetrahydrocannabinol (THC), so their role in the suppression of polarizing cytokines was examined. Injection of L. pneumophila or THC alone into BALB/c mice induced a rapid and transient rise in serum corticosterone (CS), and the injection of both agents significantly augmented the CS response, demonstrating that THC increased CS in Legionella-infected mice. Pretreatment with the CB receptor 1 (CB1) antagonist SR141716A had no effect on the THC-induced CS response, but CB2 antagonist (SR144528) treatment increased the CS response. To see if increased CS contributed to the down-regulation of Th1 cytokines, mice were pretreated with the steroid antagonist RU486 before THC injection and Legionella infection. The results showed that RU486 did not attenuate the THC-induced suppression of serum interleukin (IL)-12 or interferon-γ (IFN-γ). In addition to CS, THC injection increased urinary PGE2 metabolites, and the CB1 antagonist attenuated this increase. Although L. pneumophila infection increased urinary PGE2, THC pretreatment did not enhance this response; in addition, treatment with the cyclooxygenase inhibitor, indomethacin, did not block the THC-induced suppression of IL-12 and IFN-γ. These results suggest that the elevation of CS and PGE2 does not account for the THC-induced attenuation of the Th1 cytokine response, and it is concluded that other suppressive mediators are induced by THC or that the drug acts directly on immune cells to suppress cytokine production.

Studies on the effects of marijuana smoking have evolved into the discovery and description of the endocannabinoid system. To date, this system is composed of two receptors, CB1 and CB2, and endogenous ligands including anandamide, 2-arachidonoyl glycerol, and others. CB1 receptors and ligands are found in the brain as well as immune and other peripheral tissues. Conversely, CB2 receptors and ligands are found primarily in the periphery, especially in immune cells. Cannabinoid receptors are G protein-coupled receptors, and they have been linked to signaling pathways and gene activities in common with this receptor family. In addition, cannabinoids have been shown to modulate a variety of immune cell functions in humans and animals and more recently, have been shown to modulate T helper cell development, chemotaxis, and tumor development. Many of these drug effects occur through cannabinoid receptor signaling mechanisms and the modulation of cytokines and other gene products. It appears the immunocannabinoid system is involved in regulating the brain-immune axis and might be exploited in future therapies for chronic diseases and immune deficiency.

Legionella pneumophila is a ubiquitous intracellular bacterium found widely in the environment and is the cause of sporadic outbursts of opportunistic infection, mainly in immunocompromised individuals, including young children as well as aged persons. The host response to this organism is similar to responses to other opportunistic intracellular microbes and features both innate and adoptive immune mechanisms. Innate immunity includes the responses of a variety of host cells and cytokines, including those produced by macrophages stimulated by microbial antigens. Adoptive immunity consists of activated lymphocytes and the cytokines they produce, such as interferon and other cytokines that activate macrophages to restrict the growth and spread of intracellular bacteria. The role of cytokines specifically in resistance and immunity to Legionella is exemplified by studies concerning the nature and mechanism whereby interferon produced by activated T lymphocytes influences macrophages to resist infection by this bacterium, not only by restricting growth but also killing this bacterium. This cytokine is considered to have a key role in activating macrophages in adoptive immunity to Legionella and other intracellular bacteria. In particular, interferon is known to have a crucial role in activating macrophages to resist infection by L. pneumophila . This review also describes newer findings that demonstrate that various cytokines that define Th1 vs Th2 helper cell activity also are important in regulating resistance versus susceptibility to this ubiquitous microorganism. Copyright 2002, Elsevier Science (USA). All rights reserved.

Bartonella henselaeis responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual,B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma. We hypothesize thatB. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1β (IL-1β), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to liveB. henselae.Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1β induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest thatBartonellamodulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis.B. henselaeinduction of VEGF, IL-1β, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.

Even though cigarette smoking has been shown to suppress immune responses in the lungs, little is known about the effect of cigarette smoke components on respiratory infections. In the present study, the effects of cigarette smoke condensate (CSC) on bacterial replication in alveolar macrophages and the immune responses of macrophages to infection were examined. Furthermore, a possible immunotherapeutic effect of epigallocatechin gallate (EGCg), a major form of tea catechins, on the CSC-induced suppression of antimicrobial activity and immune responses of alveolar macrophages was also determined. The treatment of murine alveolar macrophage cell line (MH-S) cells with CSC significantly enhanced the replication ofLegionella pneumophilain macrophages and selectively down-regulated the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) induced by bacterial infection. The treatment of macrophages with EGCg not only overcame the CSC-induced suppression of antimicrobial activity but also strengthened the resistance of macrophages to infection. EGCg also markedly up-regulated the CSC-suppressed IL-6 and TNF-α production by macrophages in response to infection. The results of exogenous TNF-α treatment and neutralization treatment with anti-TNF-α and anti-gamma-interferon (IFN-γ) antibodies and the determination of IFN-γ mRNA levels indicate that CSC-suppressed macrophages can be activated by EGCg to inhibitL. pneumophilagrowth by up-regulation of TNF-α and IFN-γ production. Thus, this study revealed that CSC selectively alters the immune responses of macrophages toL. pneumophilainfection and leads to an enhancement of bacterial replication in macrophages. In addition, the tea catechin EGCg can diminish such suppressive effects of CSC on alveolar macrophages.

Inflammation of the lung is characterized by the influx of increased numbers of various leukocytes including polymorphonuclear leukocyte (PMN) neutrophils. In addition to cells, numerous studies have pointed to the role of tumor necrosis factor-alpha in the inflammatory process. This study addresses a previously unrecognized interaction between neutrophil-derived myeloperoxidase (MPO) and resident alveolar macrophages (AMø). Rat AMø exposed to either enzymatically active recombinant MPO or enzymatically inactive MPO (iMPO) exhibited an increased respiratory burst (RB). When iMPO was employed, the enhancement of the RB was greater than that observed with MPO. Although the RB was greater with iMPO, macrophage (Mø)-mediated intracellular candidic activity was equivalent for both MPO and iMPO. It is known that pro- inflammatory cytokines contribute to the inflammatory process. When rat AMø were exposed to both forms of myeloperoxidase, iMPO demonstrated greater upregulation of cytokine genes as well as product. These data suggest that at the site of inflammation, neutrophil-derived MPO and iMPO stimulate AMø, resulting in an increased inflammatory and cytotoxic state, and thereby contributing to the general lung inflammatory response.

We studied, using RT-PCR, the relative expression of cannabinoid receptor (CBR) mRNA in peripheral blood mononuclear cells (PBMC) from different donor groups. Cells from normal donors expressed a CB2 mRNA level threefold higher than CB1 across all age, gender or ethnicity groups, and amplicons were of the same size in all donors. However, cells from marijuana users expressed higher levels of CBR mRNA, but with a preserved CB1/CB2 ratio of 1:3. CBR gene products were also studied following short-term mitogen activation in vitro. CB1 expression decreased following mitogen stimulation when compared to the time-matched medium only cells while the expression of CB2 mRNA remained unchanged. These studies suggest that marijuana smoking and immune activation can alter the basal levels of CB1 and CB2 in PBMCs.

Legionella pneumophila is a ubiquitous opportunistic pathogen that causes serious infection in humans, especially in immunocompromised individuals. Adaptive immunity consists of activated T cells, cytokines such as gamma interferon (IFN-γ), and activated macrophages that restrict the growth and spread of bacteria within its intracellular environment. Innate immunity to the bacteria, on the other hand, is perhaps even more complex and includes a variety of different host cells and cytokines. The innate immunity to L. pneumophila infection limits the growth of the bacteria, which generally occurs in macrophage. Therefore, the regulation of L. pneumophila growth in macrophages by proinflammatory cytokines, such as TNF-α, may be a main event in the early phase of infection. TNF-α, which is mainly produced by macrophages, is a potent cytokine and has extensive properties affecting the regulation of immunological and inflammatory cells. In general, the adaptive immunity to bacterial infections is characterized by the memory of the previous antigen stimulation, which is not necessarily the infection, and the transferable nature of the memory with lymphocytes.

Interleukin-12 (IL-12), one of the key cytokines in regulating the development of Th1 response, is a heterodimeric cytokine composed of two disulfide-linked p35 and p40 subunits. Both subunits have to be expressed within the same cell to produce biologically active p70 heterodimer. The production of IL-12 by monocytes/macrophages is induced by exposing responsive cells to a variety of microbial products such as lipopolysaccharide (LPS). The suppression of IL-12 by Legionella pneumophila infection may be occurring at the level of gene transcription. The analysis of the steady-state levels of IL-12 p35 and IL-12 p40 mRNA determined by reverse transcriptase-PCR (RT-PCR) showed that L. pneumophila infection induced the suppression of mRNA accumulation for the IL-12 p40 gene in response to LPS stimulation, but not the IL-12 p35 gene, which was constitutive in all macrophages treated. L. pneumophila infection induced production of MCP-1 protein and up-regulated the LPS-induced production of MCP-1 protein regardless of the virulence of L. pneumophila and its viability in the macrophages. The experimental mouse infection with L. pneumophila resulted in certain levels of IL-12. Since IL-12 production is regulated by multiple mechanisms, including cytokines produced by other cells, it can be speculated that the suppressed IL-12 by infection may be overcome by other systems in vivo, such as the host defense system.

Humankind has suffered many afflictions-some from the plagues of invisible microbes, some from natural disasters, and some from man's inhumanity to man. The author and his research colleagues used Legionella pneumophila as a model system and studied immune responses of mice to this infectious organism. They developed a susceptible versus resistant mouse model to study Legionella and investigated the role of B cells, T cells, and macrophages, as well as cytokine responses to infection by this bacteria. In addition to studying the immune response to Legionella, they soon began to examine the effect of marijuana on the immune response system. These studies originated with the recognition that drug abusers were especially prone to HIV infection and the development of AIDS. It is obvious that further investigation of the host-parasite relationship as it relates to immune responses against microorganisms will result in what was thought to be an unattainable understanding of how the human species can survive in the "sea of microbes" that can use humans as their hosts.

In this chapter, primary murine (BALB/c) bone marrow-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms like Legionella, were infected in vitro with the bacteria. In related studies, it was recently reported that epigallocatechin gallate (EGCG) inhibited interleukin-12 (IL-12) production by lipopolysaccaride-treated DCs. EGCG has also been reported to decrease LPS-induced TNF- α production in a dose-dependent manner in the murine macrophage cell line RAW 264.7 and to similarly inhibit LPS-induced TNF- α production in elicited BALB/c mouse peritoneal macrophages, effects attributed in part through blocking NF- κ activation. In cultured human peripheral blood mononuclear cells, EGCG was also reported to stimulate production of TNF- α. All of these reports suggest that cytokine modulatory effects of catechins have varied effects depending upon the immune cell subpopulation studied. However, it is likely that concentrations of catechins at tissue sites are higher than in the blood. The short half-life of catechins in vivo might be overcome, however, by repeated administration, which is feasible given the reported low toxicity of this catechin and given that even high doses, such as 1,600 mg, are well tolerated by human subjects. In conclusion, authors present evidence that EGCG can inhibit IL-12 production while enhancing TNF- α production in bone marrow-derived DCs infected with Legionella.

Although nicotine is thought to be one of the major immunomodulatory components of cigarette smoking, how nicotine alters the host defense of the lung and, in particular, immune responses of alveolar macrophages, which are critical effector cells in the lung defense to infection, is poorly understood. Nicotinic acetylcholine receptors (nAChRs) are the receptor for nicotine and may be involved in the modulation of macrophage function by nicotine. In this study, therefore, nicotine-induced suppression of antimicrobial activity and cytokine responses of alveolar macrophages mediated by nAChRs to Legionella pneumophila, a causative agent for pneumonia, were examined. The murine MH-S alveolar macrophage cell line cells expressed the messages for α4 and β2 subunits of nAChRs, but not α7 subunits, determined by RT-PCR. The nicotine treatment of MH-S alveolar macrophages after infection with L. pneumophila significantly enhanced the replication of bacteria in the macrophages and selectively down-regulated the production of IL-6, IL-12, and TNF-α, but not IL-10, induced by infection. These effects were completely blocked by a nonselective antagonist, d-tubocurarine, for nAChRs, but not by a selective antagonist, α-bungarotoxin, for α7-nAChRs. Furthermore, the stimulation of nAChRs with another agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, showed the same effects, which were blocked by the antagonist d-tubocurarine, on the bacterial replication and cytokine regulation with that of nicotine. Thus, the results revealed that nAChRs, the major exogenous ligands of which are nicotine, are involved in the regulation of macrophage immune function by nicotine and may contribute to the cigarette-induced risk factors for respiratory infections in smokers.

The obligate intracellular pathogen Chlamydia (Chlamydophila) pneumoniae is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between C. pneumoniae and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to C. pneumoniae infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to C. pneumoniae infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with C. pneumoniae . Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to C. pneumoniae infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for C. pneumoniae , i.e., lymphocytes.

Nicotine, the addictive component of tobacco, is thought to be at least partially responsible for the deleterious effects of smoking such as heart disease and cancer. Evidence shows that nicotine is an immunomodulator and that one of its possible mechanisms is regulation of apoptosis, or programmed cell death, in immune cells. This study examined the effects and the mechanisms of action of nicotine on dexamethasone (DEX)-induced apoptosis in murine immune cells by examining the expression of levels of the 17-kDa active caspase-3, a marker of apoptosis. Thymocytes and splenocytes from adult BALB/c female mice were Incubated with concentrations of nicotine correlating to those found in the blood and tissue of smokers (0.01 μg/ml [0.022 μM] and 1 μg/ml [2.2 μM]), concurrently with 100 nM DEX, to induce apoptosis. Cytosolic protein fractions were analyzed by Western blotting with polyclonal antibodies that recognize the active form of caspase-3. The data showed that nicotine significantly blocked the formation of the DEX-induced 17-kDa caspase-3 subunit expression. This downregulation ranged from 65% to 100% of the active caspase-3 expressed in cultures treated with DEX alone. Addition of d-tubocurarine chloride (dTC), a general nicotinic receptor antagonist, inhibited nicotine downregulation of the DEX-lnduced active caspase-3 expression, providing evidence that this action of nicotine was receptor-mediated. These data support that nicotine is an important immunomodulator at the level of immune cell apoptosis, a process thought to be a contributory mechanism of autoimmunity, cardiovascular disease, and carcinogenesis.

BACKGROUND: Because it has been increasingly recognized that Chlamydia pneumoniae may be linked to some chronic inflammatory diseases, including atherosclerosis, detection of this pathogen in blood from patients may be valuable in the diagnosis of such diseases. However, the prevalence of chlamydia in the blood of healthy donors has not yet been extensively studied. STUDY DESIGN AND METHODS: The presence of C. pneumoniae in PBMNCs obtained from healthy persons who donated blood for blood transfusion was assessed by a PCR that was specific for the C. pneumoniae 16S rRNA gene and by the use of staining with FITC-conjugated chlamydia MoAb. RESULTS: Twenty-one (8.9%) of 237 blood samples tested showed the presence of C. pneumoniae DNA and antigen in the PBMNCs. There was no significant difference in the presence of chlamydia in blood according to sex or to age between 20 and 59 years of age. However, a possible seasonal variation in the presence of chlamydia in blood from healthy donors was suggested by the results obtained. CONCLUSION: A significant percentage of healthy donors carry C. pneumoniae, which may be a risk factor for some chronic diseases.

Cannabinoid CB(2) receptor is reported to be expressed in varying amounts in different human immune subpopulations. To examine the expression pattern of CB(2) in the mouse, immune cell subpopulations were purified and studied by semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR). CB(2) mRNA was most abundant in splenic B cells, followed by macrophages and T cells. Furthermore, CB(2) was expressed in thioglycollate-elicited peritoneal macrophages, but not in resident peritoneal macrophages. In addition to these studies on receptor expression at basal activity, CB(2) mRNA expression was also studied following immune cell activation. Bacterial lipopolysaccharide stimulation downregulated CB(2) mRNA expression in splenocyte cultures in a dose-response manner, while stimulation through cluster of differentiation 40 (CD40) using anti-CD40 antibody upregulated the response and costimulation with interleukin-4 attenuated the anti-CD40 response. These results demonstrate that CB(2) mRNA expression differs among mouse immune subpopulations similar to what is observed in human immune cells. Furthermore, the results suggest that the signaling pathways activated by lipopolysaccharide and anti-CD40 might have different effects on CB(2) mRNA expression.

Epigallocatechin gallate (EGCg) is a major form of tea catechin and has a variety of biological activities, including antitumor as well as antimicrobial activity against some pathogens. Although the biological activities of EGCg have been extensively studied, its immunological effects are not well known. In the present study, the ability of EGCg to modulate macrophage immune functions in an in vitroLegionella pneumophilainfection model of macrophages was examined. The study showed that EGCg inhibited the growth ofL. pneumophilain macrophages at a concentration as low as 0.5 μg/ml without any direct antibacterial effect on the organisms. The EGCg selectively upregulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) and downregulated IL-10 production of macrophages induced byL. pneumophilainfection in a dose-dependent manner, but did not alter IL-6 production even at a high dose. The upregulation of the levels of macrophage gamma interferon (IFN-γ) mRNA by EGCg was also demonstrated. Treatment of macrophage cultures with anti-TNF-α and anti-IFN-γ monoclonal antibodies markedly abolished the anti-L. pneumophilaactivity of macrophages induced by the EGCg treatment. These results indicate that EGCg selectively alters the immune responses of macrophages toL. pneumophilaand leads to an enhanced anti-L. pneumophilaactivity of macrophages mediated by enhanced production of both TNF-α and IFN-γ. However, the enhancement of in vitro anti-L. pneumophilaactivity by EGCg may not be directly mediated by IL-10 and IL-12 production modulation. Thus, the results of this study revealed the immunomodulatory effect of EGCg on macrophages, which have a critical role in infections.

Since current studies indicate the possible involvement of Chlamydia pneumoniae in the pathogenesis of multiple sclerosis (MS), demonstration of C. pneumoniae in the cerebrospinal fluid (CSF) of patients with MS is highly desirable. However, there is controversy concerning the detection of C. pneumoniae in CSFs from MS patients due to the lack of a standard protocol for extraction and detection of C. pneumoniae DNA. In this regard, we attempted to establish a highly effective extraction protocol for C. pneumoniae DNA from CSFs utilizing a commercial kit and a PCR detection method. The extraction and PCR detection protocol established in this study succeeded in detecting as few as 20 C. pneumoniae organisms in 200 μl of mock CSF. The use of this protocol to detect C. pneumoniae DNA in CSFs revealed that 68% of CSF samples obtained from patients with MS were positive (11 out of 16 samples) for chlamydia DNA. Thus, the protocol established here is sensitive enough to detect chlamydia DNA from CSFs and can be used by other laboratories for evaluation of the presence of chlamydiae in CSFs because the protocol is based on the use of a commercial kit.

Many advances have been made in the last few years concerning our understanding of the receptors and ligands composing the cannabinoid system. Likewise, the science surrounding cytokine biology has advanced enabling us to measure these proteins more precisely as well as understand and interpret the meaning of changes in their levels. Scientists wishing to study the health consequences of smoking marijuana as well as understand the possible role of endogenous cannabimimetic ligands in immune regulation have continued to study the influence of these substances on the regulation and development of the cytokine network. Research has shown that two major cannabinoid receptor subtypes exist and that subtype 1 (CB1) is expressed primarily in the brain whereas subtype 2 (CB2) is expressed primarily in the periphery. A variety of ligands for these receptors based on the cannabinoid structure have been synthesized and studied as well as low affinity compounds, noncannabinoid ligands, and endogenous ligands derived from fatty acid eicosanoids. Highly selective receptor antagonists have also been introduced and studied. Synthetic, low affinity ligands such as (+)-HU-211 and DMH-11C have been shown to cause anti-inflammatory effects possibly through inhibiting the production and action of TNF-alpha and other acute phase cytokines. In addition, suppression of TNF and other cytokines such as GM-CSF, IL-6, IFNgamma, and IL-12 has also been seen following exposure to high affinity and psychoactive ligands such as marijuana and THC. However, some of these ligands have also been shown to increase rather than decrease interleukins such as IL-1, IL-4, IL-10, and IL-6, cytokines such as TNF-alpha, and chemokines such as IL-8, MIP-1, and RANTES. The endogenous ligand, anandamide, has been shown in culture to either suppress the proliferation response to prolactin or enhance the response to cytokines such as IL-3 and IL-6. This eicosanoid has also been shown to increase the production of interleukins and other cytokines. Cannabinoid receptors have been shown to be involved in some but not all of these effects. It is clear that psychoactive and nonpsychoactive compounds have demonstrated effects in vivo and in vitro on the production and function of a variety of cytokines. Depending upon the model system, these effects are often conflicting, and the involvement of cannabinoid receptors is unclear. However, enough evidence exists to suggest that the cannabinoid system significantly impacts the functioning of the cytokine network, and this association may provide clues to the mechanisms of certain immune diseases and form the basis for new immunotherapies.

The cDNA expression array technique is a powerful tool to determine, at one time from many genes, specific gene messages modulated by infection. In the present study, we identified genes modulated in response to virulent versus avirulent Legionella pneumophila infection of the alveolar macrophage cell line MH-S by the cDNA expression array technique. Many macrophage genes were found to be modulated after 5 h of in vitro infection with L. pneumophila . In particular, it was found that the monocyte chemotactic protein 3 (MCP-3) gene expression was significantly induced by infection with virulent L. pneumophila but not with avirulent L. pneumophila . In contrast, other chemokine genes, such as macrophage inflammatory protein (MIP) 1α, were induced by both virulent and avirulent L. pneumophila . Reverse transcription (RT)-PCR assay of total RNA isolated from macrophages infected with the bacteria for 5 or 24 h confirmed the differential induction of the chemokine genes by virulent versus avirulent L. pneumophila . Thus, the cDNA expression array technique readily revealed differential induction by L. pneumophila infection of select chemokine genes of macrophages from more than 1,100 genes. These results also indicate that certain chemokine genes may be selectively induced by virulent bacteria.

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.Key words: Legionella pneumophila, cytokine receptors, macrophages.

Helicobacter pylori (Hp) infection causes gastric ulcers and gastric carcinomas. The mechanisms of these diseases are not known but Hp induction of cytokines is believed to be involved. However, the profile as well as the involvement of cytokines induced by Hp infection is less clear. In the present study, steady state levels of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNFalpha) and TGFbeta1 mRNA of human gastric epithelial cell lines Kato III, AGS and NCI-87N in response to Hp infection were investigated. The cell cultures were infected with Hp for up to 18 h. Total RNA was extracted and analyzed by reverse transcription-PCR. The three cell lines tested expressed low constitutive levels of mRNA for IL-8 and TNFalpha. The mRNA levels of IL-8 quickly increased within 2 h in all three cells tested and reached a peak at 4 h following infection with Hp. In contrast, the levels of TNFalpha after Hp infection increased in only Kato III cells. The other cells, AGS and NCI-87N, responded with minimum increases after Hp infection. The TGFbeta1 mRNA was constitutively expressed in both AGS and NCI-87N cells, but Kato III cells expressed only low levels prior to infection. The Hp infection did not increase the levels of TGFbeta1 mRNA as well as TGFbeta1 secretion in all cells tested. These results indicate that the cytokine response to Hp infection differs according to the cells studied and the response may be linked somewhat to TGF levels of gastric cells.

The marijuana cannabinoid, Δ9-tetrahydrocannabinol (THC), suppresses immunity to Legionella pneumophila and development of Th1 activity and cell-mediated immunity. In the current study, THC effects on cytokines regulating the development of Th1 cells were examined. BALB/c mice showed significant increases in serum IL-12 and IFN-γ within hours of infection; however, the levels of these Th1-promoting cytokines as well as resistance to a challenge infection were suppressed by THC (8 mg/kg) injected 18 h before priming. The Th2-promoting cytokine, IL-4, was increased within hours of a Legionella infection and was further increased by THC treatment. These results suggested that THC injection suppressed the cytokine environment promoting Th1 immunity. In additional experiments, THC pretreatment and infection of IL-4 knockout mice showed that serum IL-12 and IFN-γ were suppressed equally in both knockout and normal mice. This suggested that the drug-induced increase in IL-4 was not responsible for the decreases in serum IL-12 and IFN-γ. However, THC treatment was shown to suppress the expression of IL-12 receptor β2 mRNA, indicating that, in addition to suppression of IL-12, THC injection suppressed the expression of IL-12 receptors. Finally, the role of cannabinoid receptors in Th1-promoting cytokine suppression was examined, and results with receptor antagonists showed that both cannabinoid receptors 1 and 2 were involved. It is suggested that suppression of Th1 immunity to Legionella is not due to an increase in IL-4 production but to a decrease in IFN-γ and IL-12. Furthermore, both types of cannabinoid receptors are involved.

Virulent Legionella pneumophila replicate readily in thioglycollate-elicited peritoneal macrophages from genetically permissive A/J mice, but avirulent L. pneumophila do not. The production of cytokines by macrophages infected with L. pneumophila has been studied, but the correlation 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 α (TNF-α), interleukin (IL)-1α, IL-1β, 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 expression 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 produce relatively high levels of TNF-α, IL-1α, IL-1β, 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 murine macrophages from susceptible A/J mice are readily infected with virulent L. pneumophila in vitro and stimulated to produce the proinflammatory acute-phase cytokines TNF-α, IL-1α, IL-1β, and IL-6, but avirulent L. pneumophila did not. Such differences in induction of these proinflammatory 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.

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.

Nitazoxanide (NTZ), a synthesized drug of the nitrothiazolide class, was initially developed as an antiparasitic compound. This compound has recently been shown to have antibacterial activities against some bacterial pathogens. In the present study, NTZ and its main metabolite tizoxanide (TIZ) were found to have strong minimum inhibitory concentrations (MICs) against both metronidazole (MTZ)-resistant strains and sensitive clinical isolates of Helicobacter pylori. The MIC90 of both NTZ and TIZ against 37 clinical isolates was 8 μg/ml. Vacuolating toxin activity of H. pylori assayed by HeLa cell vacuole formation was inhibited by NTZ at a sub-MIC. In contrast, urease production by H. pylori was not specifically affected by the sub-MIC of NTZ. An acidic pH (pH 5.0) medium reduced the antimicrobial activity of the drug in terms of growth inhibition due to the low growth rate of the bacteria, but killing activity of NTZ against the bacteria was still observed. Thus, it was apparent that both NTZ and TIZ are highly effective against H. pylori, even when the bacteria are resistant to MTZ.