Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Rα in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Rα production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.     

Cigarette smoke attenuation of poly I:C-induced innate antiviral responses in human PBMC is mainly due to inhibition of IFN-beta production

The cellular response to dsRNA or its synthetic analog polyinosinic-polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-beta, but not IFN-alpha, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections.     

IL-4 production is increased in cigarette smokers.

Cigarette smoking has been associated with both increases in serum levels of total IgE and an increased risk of developing allergic-like symptoms. IL-4 and interferon-gamma (IFN-gamma) have reciprocal roles in the regulation of IgE synthesis, and as such prompted us to evaluate, in smokers, the production of these two cytokines. We demonstrate that phytohaemagglutinin (PHA)-induced IL-4 production by peripheral blood mononuclear cells (PBMC) of smokers (n = 19) is significantly higher than that of non-smokers (n = 10, P < 0.005). In addition, PBMC from heavy smokers, defined by the number of cigarettes smoked per day, produced significantly higher levels of IL-4 than those of light smokers. No difference between the groups was found for IFN-gamma production. Our data suggest an imbalance in cytokine production occurring in individuals who smoke. This imbalance, favouring IL-4 production, may be part of the mechanism responsible for the observed increases in serum IgE and allergic-like symptoms associated with cigarette smoking.     

Toll样配体(R-848)与IL-12对人NK细胞亚群IFN-γ产生的作用

目的:研究Toll样配体(R-848)与IL-12对人NK细胞IFN-γ产生的作用和细胞亚群分析。方法:分离人外周血PBMC和纯化的NK细胞,分别与R-848、IL-12或R-848和IL-12共同培养。利用ELISA法检测培养上清中IFN-γ的水平,再利用流式检测并分析产生IFN-γ的NK细胞亚群。结果:正常人PBMC分别与不同浓度的Toll样配体R-848、LPS、CpG培养后,均以剂量依赖的方式诱导IFN-γ的产生,但以R-848的效果最佳。细胞亚群分析的结果表明,R-848对CD4 T和CD8 T细胞IFN-γ的表达无明显作用,但显著地促进CD56 细胞表达IFN-γ。同样地,在IL-12刺激之下,CD56bright和CD56dimNK细胞表达IFN-γ。当R-848和IL-12与PBMC和纯化NK细胞孵育后,对CD56bright和CD56dimNK细胞IFN-γ的表达具有协同作用。结论:Toll样配体与NK细胞Toll样受体结合后,促进CD56brightNK细胞亚群IFN-γ的产生,而且Toll样配体与IL-12具有协同作用,提示Toll样受体与细胞因子在调控NK细胞的生物活性中发挥着十分重要的作用。     

Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts

BACKGROUND: Although cigarette smoking is known to have detrimental effects on the immune system, the nature of the immunosuppressive agent or agents is poorly understood. OBJECTIVE: The purpose of the current study was to evaluate the effects of cigarette smoke extracts from high-tar (unfiltered Camel), medium-tar (Marlboro), and low-tar (Carlton) cigarettes on the in vitro production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha. METHODS: The concentrations of hydroquinone and catechol in cigarette smoke extracts were determined by using HPLC. Human PBMCs were treated with cigarette smoke extracts, hydroquinone, or catechol, and stimulated with anti-CD3 and phorbol-12-myristate-13-acetate. Cytokine levels in the supernatants were quantified by ELISA. RESULTS: Pretreatment of PBMCs with cigarette smoke extracts derived from a single high- or low-tar cigarette suppressed the production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha by greater than 90% without significant loss of cell viability. Nicotine, at a concentration comparable with that found in the highest-tar cigarettes (200 microg/mL), suppressed the production of IL-2, IFN-gamma, and TNF-alpha by only 21% to 38%. Catechol (50 micromol/L) inhibited production of IL-2 and IL-1beta by 62% to 73% but had little effect on TNF-alpha or IFN-gamma production. In contrast, hydroquinone inhibited the production of all 4 cytokines with IC(50) values ranging from 3 micromol/L(IL-1beta) to 29 micromol/L (IFN-gamma). However, HPLC determination of the hydroquinone concentrations in cigarette smoke extracts from single Camel (33+/-4 micromol/L), Marlboro (13+/-2 micromol/L), and Carlton (<1 micromol/L) cigarettes clearly demonstrated that the potent inhibitory effects of the low-tar cigarettes could not be accounted for by either hydroquinone or catechol. CONCLUSION: These studies indicate that cigarette smoke contains potent inhibitors of cytokine production, at least one of which is present even in low-tar cigarettes.     

卡介苗(BCG)通过诱导IL-12产生和受体表达促进人NK细胞功能

目的:研究卡介苗(BCG)对人自然杀伤细胞(NK)功能的作用及其机制.方法:分离抗结核抗体阴性志愿者外周血PBMC、纯化NK细胞, 分别与BCG、 IL-12、 BCG+IL-12、 BCG+抗IL-12Rβ1 mAb(2B10)培养.利用ELISA方法检测培养上清液IFN-γ、 IL-12p40含量;利用ELISpot方法检测IFN-γ、颗粒酶B产生细胞的频率;利用四甲基偶氮唑盐(MTT)比色法测定杀伤功能.利用流式细胞术检测NK细胞IL-12Rβ1的表达.结果:BCG呈剂量依赖的方式诱导PBMC产生IFN-γ.在BCG刺激条件下, PBMC颗粒酶B分泌细胞数明显高于不加任何刺激剂组(P<0.05).BCG增强PBMC杀伤活性.BCG不能诱导纯化NK细胞产生IFN-γ, 但与IL-12同时刺激则表现出协同作用.纯化NK细胞经BCG刺激后杀伤活性与未刺激相比差异无统计学意义.BCG呈剂量依赖方式诱导PBMC产生IL-12、并促进NK细胞不同亚群表达IL-12Rβ1.2B10抗体抑制BCG对PBMC产生IFN-γ和分泌颗粒酶B的诱导作用.结论:BCG间接地促进NK细胞的生物学活性, 其部分机制是通过诱导单核细胞产生内源性IL-12、并上调NK细胞表达IL-12R.     

Effect of Pseudomonas aeruginosa exotoxin A on IFN-gamma synthesis: expression of costimulatory molecules on monocytes and activity of NK cells.

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Regulation of gamma interferon production by natural killer cells in scid mice: roles of tumor necrosis factor and bacterial stimuli.

CB-17 scid mice exhibit a T-cell-independent but gamma interferon (IFN-gamma)-dependent immunity to Listeria monocytogenes. In this study, we analyzed the specific cellular interactions involved in this process. scid mouse-derived natural killer (NK) cells cultured with heat-killed (HK) L. monocytogenes and macrophages secreted IFN-gamma. No IFN-gamma was produced in cultures containing HK L. monocytogenes but lacking macrophages. However, medium derived from macrophages incubated with HK L. monocytogenes or other microorganisms stimulated IFN-gamma production by isolated NK cells. Treatment of macrophage-conditioned supernatants with neutralizing monoclonal anti-tumor necrosis factor (TNF) significantly reduced their capacity to stimulate NK cells to produce IFN-gamma. Yet, purified recombinant TNF-alpha by itself was unable to stimulate NK cells. Thus, TNF was necessary but not sufficient to induce maximal IFN-gamma production by NK cells. Sonicated L. monocytogenes stimulated production of IFN-gamma by NK cells that was resistant to anti-TNF. Stimulation was markedly enhanced by the addition of recombinant TNF-alpha. These studies demonstrated that activation of scid NK cells for secretion of IFN-gamma requires two signals: TNF-alpha and a second product which may be of bacterial origin and may require processing by mononuclear phagocytes. We suggest that the T-cell-independent production of IFN-gamma by NK cells provides the host with a rapid mechanism to temporarily heighten nonspecific resistance to infection until such time as T-cell-dependent sterilizing immune responses can be generated.     

Effect of Salmonella typhi wild type and O-antigen mutants on human natural killer cell activity

We investigated the effect of glutaraldehyde-fixed Salmonella typhi Ty2 (Vi(-)) wild-type (World Health Organization's vaccine strain) and mutant strains MEI028 (rough, O-antigen(-)) and MEI012 [smooth (O-antigen(+)95%), immunomagnetically isolated NK cell preparations. Incubation of PBMC with each and every one of the S. typhi strains studied consistently and significantly, increased this cellular immune function, as well as the supernatant level of the various cytokines tested e.g. IFN-gamma, TNF-alpha, IL-10 and IL-12 (ELISA). In similar experiments, a significant increase in the cytolytic activity of HPNK cells was elicited by S. typhi Ty2 but not by mutant strain MEI028; neither of the cytokines assayed (IFN-gamma and TNF-alpha) was detected in the supernatant.Our results suggest that S. typhi O-antigen plays an essential role in a mechanism resulting in the direct activation of NK cell activity in HPNK cell preparations. However, the relative quantitative significance of this antigen in the direct stimulation of NK cell cytotoxicity expression in PBMC samples is less clear, as it appears that in this case bacterial-induced monocyte-released cytokines plays a most important role. Incubation with S. typhi Ty2 or MEI028 elicited significant expression of CD69, an early marker of NK cell activation, in PBMC but not in HPNK cell samples (flow cytometry); in similar experiments, the expression of CD16/56 and activation marker CD25 remained essentially unchanged.     

Inefficient protection of human TAP-deficient fibroblasts from autologous NK cell-mediated lysis by cytokines inducing HLA class I expression

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.     

IL-10 enhances NK cell proliferation, cytotoxicity and production of IFN-gamma when combined with IL-18.

Previous studies have shown that IL-10 inhibits the accessory cell functions required for production of IFN-gamma by T cells and NK cells. Our results show that although IL-10 did not induce the production of IFN-gamma by NK cells, it did enhance the ability of IL-18 to stimulate NK cell production of IFN-gamma. In addition, IL-10 augmented NK cell proliferation and cytotoxic activity when combined with IL-18. However, IL-10 did not affect the ability of IL-12 to stimulate NK cells to produce IFN-gamma or proliferate, but there was an additive effect with IL-12 to increase NK cell cytotoxic activity. Interestingly, the type I IFN, whose receptors (R) are related to the IL-10R, also enhanced the effects of IL-18 on NK cell production of IFN-gamma and NK cell cytotoxicity. The ability of IL-10 to elevate the production of IFN-gamma appeared to be specific for NK cells since IL-10 had no effect on the production of IFN-gamma by Th1 clones stimulated with IL-18 or IL-12 in the presence of a monoclonal antibody specific for CD3. These latter results correlated with lower mRNA levels for the alpha and beta chains of the IL-10R in Th1 cells than observed in NK cells. Thus, the ability of IL-10 and IL-18 to up-regulate NK cell function, but not Th1 cell activity, appears to be based on expression of the IL-10R.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

Regulation of NK cell function in vivo by the dose of tumour transplanted in the peritoneum

Antigen dose is known to regulate T cell activation and anergy. Similarly, dose of antigen also regulates NK cell lytic potential and phenotype development. Resident peritoneal cells of rat contain a small population of NK and NKT cells. Inoculation of AK-5 tumour cells intraperitoneally modulate the cytotoxic function of NK and NKT cells present in the peritoneal exudate cells (PEC) in a dose dependent manner. Low dose of tumour causes activation of NK and NKT cell cytotoxic function and enhanced NK and NKT cell population in PEC, whereas, high doses of tumour cause inactivation of NK and NKT cell cytotoxic function and depletion of the two sub-populations in the peritoneum. Different doses of tumour inoculation in the peritoneal cavity did not suppress the cytotoxic function of NK cells from spleen suggesting that a direct interaction between NK cells and tumour cells is required for the suppression of NK cell cytotoxic function. Tumour inoculation induced secretion of IL-2, IL-12, IFN-gamma and TNF-alpha by tumour infiltrating mononuclear cells (TIM) in ascitic fluid as well as in serum. The levels of IL-2, IL-12, IFN-gamma and TNF-alpha secretion were higher in animals, which rejected tumours as compared with the animals that failed to reject the tumours. Injection of anti IL-12 and anti IFN-gamma antibody reduced the survival rate of tumour injected animals, however, anti IL-2 antibody had no effect on the survival of animals. Following incubation with AK-5 tumour cells, activated NK cells upregulated perform expression, whereas, there was upregulation of CD95 expression in inactivated NK cells.     

Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists

NK cells limit the emergence of cancers and viral infections by surveillance of 'missing-self' and 'induced-self' ligands, and by direct recognition of pathogen-associated molecules. We examined individual roles for Toll-like receptors (TLRs)-7 and -8 in human NK-cell activation using synthetic, small molecule agonists of either TLR-7 (imiquimod and 3M-001), TLR-8 (3M-002) or both TLR-7/8 (3M-003 and R-848) for comparison with known ligands of TLR-2 to -9. Tracking cytokine production in PBMC initially revealed that a subset of TLR agonists including polyinosinic-polycytidylic acid (poly I:C), 3M-002, 3M-003, R-848 and single-stranded RNA trigger relatively high levels of IFN-gamma expression by NK cells. Isolated NK cells did not express TLR-7 or TLR-8. Unlike MALP-2 and poly I:C, 3M-001-3 did not induce expression of either CD69 or IFN-gamma by purified NK cells suggesting indirect activation. IL-18 and IL-12p70 were primarily required for induction of IFN-gamma by both synthetic and natural TLR-8 ligands, while type I IFN was required for induction of CD69 on NK cells by the TLR-7 agonist 3M-001. In addition to expression of IFN-gamma and CD69, relative induction of NK-cell cytotoxicity by TLR-7 and TLR-8 agonists was compared. Immune response modifiers (IRMs) with a TLR-8 agonist component (3M-002 and 3M-003) stimulated greater levels of K562 cytolysis than achieved with 3M-001 or IL-2 (1000 units ml(-1)). In vivo NK-cell cytotoxicity was also enhanced by R-848, but not in type I IFNR-deficient mice. We conclude that IRMs can modulate NK-cell function both in vitro and in vivo and that distinct indirect pathways control human NK-cell activation by TLR-7 and TLR-8 agonists.     

Epigallocatechin gallate,a potential immunomodulatory agent of tea components,diminishes cigarette smoke condensate-induced suppression of anti-Legionella pneumophila activity and cytokine responses of alveolar macrophages

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 of Legionella pneumophila in macrophages and selectively down-regulated the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) 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-alpha production by macrophages in response to infection. The results of exogenous TNF-alpha treatment and neutralization treatment with anti-TNF-alpha and anti-gamma-interferon (IFN-gamma) antibodies and the determination of IFN-gamma mRNA levels indicate that CSC-suppressed macrophages can be activated by EGCg to inhibit L. pneumophila growth by up-regulation of TNF-alpha and IFN-gamma production. Thus, this study revealed that CSC selectively alters the immune responses of macrophages to L. pneumophila infection 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.     

Natural killer cell tumoricidal activity in cigarette smokers and in silicotics

Cigarette smoking and asbestos exposure have been reported to alter the tumor-killing activity of blood natural killer (NK) cells. However, NK cell function in relation to silica dust exposure has not been examined. We studied blood NK cell number, percentage, and tumoricidal activity in 120 hardrock miners, 57 of whom had radiographic evidence of silicosis, and in 33 community controls. There was a significant increase in leukocyte count and lymphocyte count in current smokers compared with former and never smokers, but these cell counts were normal in silicotics and those with silica dust exposure. No significant differences in NK cell (CD16+) number or percentage were found by flow cytometric analysis of smoking or dust exposure groups. Surprisingly, NK tumoricidal activity was significantly higher in cigarette smokers compared with former smokers and never smoker control subjects. Although tumoricidal activity was elevated in silicotics, this was accounted for by their cigarette smoking. These results suggest that cigarette smoking is associated with elevation of blood NK cell tumoricidal activity on a per cell basis, in the absence of any alteration in the absolute number of NK cells in blood.     

Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro.

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG-ODN) on cultured cells from allergic patients and non-atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG-ODN led to a significant increase of IFN-gamma. By intracellular cytokine staining, IFN-gamma production could be attributed to NK cells and inhibition experiments indicated an IL-12-dependent mechanism. Moreover, CpG-ODN increased mRNA expression of IL-12 and IL-18 in PBMC. In this respect, no significant difference between allergic and non-atopic individuals was observed. Monocyte-derived dendritic cells were identified as one IL-12- and IL-18-producing source. In addition, stimulation of PBMC derived from atopic patients with CpG-ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG-ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen-specific IgE levels remained unchanged. Our data suggest that CpG-ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.