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Alcohol consumption impairs Th1-mediated cellular immune responses and enhances serum IgE levels. It has been reported that the elevated IgE levels are associated with a Th2 polarization response, but the mechanisms for enhancing Th2 polarization by the ethanol treatment remain to be elucidated. The aim of this review is to present and discuss the mechanism of Th2 polarization response by alcohol. IL-12 production by APCs such as monocytes, macrophages, and dendritic cells (DCs) preferentially leads to Th1 polarization. Acute ethanol consumption results in a significant decrease in IL-12 production in LPS-stimulated DCs and a CD40/CD40L interaction between CD40 on the DCs and CD40 ligand expressed on activated T cells. This suggests that Th2 polarization by ethanol is caused by impaired IL-12 production from APCs. In contrast, the induction of IL-10 by LPS is enhanced by ethanol treatment, suggesting that elevated IL-10 may play a role in ethanol-induced suppression of IL-12. However, ethanol inhibited IL-12 production in LPS-stimulated DCs devoid of IL-10 (IL-10/DC), suggesting that down-regulation of IL-12 by ethanol is independent of the IL-10 levels. Furthermore, several studies report that PGE2, cAMP and linolic acid, and endogenous lipid mediators released in inflammatory conditions, also inhibit IL-12 production. These inhibitory effects are similar to the IL-12 inhibition by ethanol. In addition, increase in the levels of these lipid mediators is induced by ethanol treatment. Alternatively, cytokine signaling studies indicate that IL-12 production by DCs is negatively regulated by PI3K and GSK-3, but positively regulated by p38 MAPK, mTOR, and NF-kappa B. Thus, it seems possible that ethanol may interact on the upstream of IL-12 producing a signal pathway. In fact, ethanol alters the stability of cell membrane, and suppresses clustering of TLR4 and recruitment of signaling molecules into lipid rafts, where it associates with the Ser/Thr kinase and the adaptor proteins, and forms a signaling complex. Down-regulation of lipid raft signaling is results in the impaired IL-12 production leading to the Th1 polarization, and causes CD4+ T cells to differentiation toward the Th2 lineage.

We previously reported that oral administration of NK-4, a criptocyanine dye, enhances interleukin (IL)-12-depend- ent interferon (IFN)-γ production by lipopolysaccharide (LPS)-stimulated mouse splenocytes. These findings raised a possibility that NK-4 potentiated IFN-γ production by T cells, natural killer (NK) cells or natural killer T (NKT) cells in response to IL-12 produced by macrophage and dendritic cells. To explore this possibility, we first analyzed percentages of T, NK or NKT cells in splenocytes of mice that were administered NK-4 orally for three days. The percentage of NKT cells in splenocytes from NK-4-treated mice was significantly (p<0.05) increased compared to vehicle-treated mice. When splenocytes were stimulated with α-galactosylceramide (α-GalCer), an NKT cell ligand, IFN-γ production by splenocytes from NK-4-treated mice tended to increase, while no difference in the IL-4 production and proliferation were observed between the vehicle- and NK-4-treated mice. When IFN-γ/IL-4 ratios were calculated in individual mice, the ratios were significantly (p<0.05) elevated in NK-4-treated mice. Furthermore, IL-12 production by α-GalCer-stimulated splenocytes from NK-4-treated mice was also significantly (p<0.05) increased. These results suggest that oral administration of NK-4 increases the population of type I NKT cells with potent IFN-γ-producing activities. Since IL-12 and IFN-γ have been shown to play important roles in anti-tumor immunity as well as in the defence against bacterial infection, our results further imply that NK-4 may provide a potential therapeutic tool in cancer immunotherapy.

We investigated the effect of 5-FU and radiation in OK-432-induced cytokine production. Stimulation of peripheral blood mononuclear cells (PBMCs) with OK-432 (1 micro/ml) for 24 h induced Th1-type cytokines (IFN-gamma, TNF-alpha, IL-12, IL-18) as well as IL-10 and TGF-beta. When the PBMCs were stimulated by 5-FU (5 microg/ml) or X-ray (2 Gy) simultaneously with OK-432, production of IL-10 and TGF-beta was significantly inhibited, while no significant change in Th1 cytokine production was observed. Although OK-432 also enhanced the expression of the genes encoding SOCS-1 and SOCS-3, which are negative regulators for cytokine signaling, this was reduced by 5-FU or X-irradiation. Induction of IL-10 and TGF-beta by OK-432 was significantly decreased by adding antisense ODN for SOCS-1 or that for SOCS-3. Radiation and 5-FU induce Th1-dominant state by inhibiting the OK-432-induced production of IL-10 and TGF-beta mediated by regulation of SOCS-1 and SOCS-3 expression, and are suggested to increase anti-cancer immunity.

We have tried to identify the effective components of OK-432, a Streptococcus-derived anti-cancer immunotherapeutic agent. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting anti-cancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and Msp I revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced Th1-type cytokines, such as IFN-gamma and IL-12, and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, a peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. Anti-tumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced as compared with that in wild-type mice, while the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG-DNA in OK-432 functions as an active component in OK-432-induced anti-cancer immunity via TLR9, at least in part.

Systemic and local immunological responses were studied in patients with or without preoperative administration of chemotherapeutic and/or immunotherapeutic drugs for colorectal cancer. The plasma TGFbeta and other cytokines such as IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma in the supernatant fluid of culture of peripheral blood mononuclear cell (PBMC) and regional lymph node were measured by the ELISA method. A systemic response of cytokines was as follows: the production of plasma TGFbeta increased in many cases by chemotherapeutic drugs with a significant elevation of the mean production. Productions of IFN-gamma, IL-2, IL-12 in the supernatant fluid of culture of PBMC increased in many cases by immunotherapeutic drugs, and that of IL-4, IL-6 increased in many cases by chemotherapeutic drugs. A local response of cytokines was as follows: the production of IL-2 by immunotherapeutic drugs was greater than that without immunotherapeutic drugs whereas the production of IL-10 by immunotherapeutic drugs was smaller than that without immunotherapeutic drugs.

Gefitinib (brand name: Iressa) is a drug approved for molecule-targeting therapy in lung carcinoma patients. There are still many unresolved problems concerning the safety and mechanism of action of this drug. Based on the expectation that this drug combined with immunotherapy would be highly effective, we recently conducted an experiment in tumor-bearing mice. In this experiment, however, the tumor was not significantly reduced in size by this combined therapy. However, since the tumor in this animal model was EGFR positive and because tumor growth tended to be suppressed in mice with higher immune activity, it seems likely that additional studies of this therapy will contribute to identifying the optimum drugs to be combined with gefitinib and the optimum method of dosing that will lead to satisfactory efficacy and safety of gefitinib combined with immunotherapy.

Immunity to tuberculosis (TB) required a Th1 pattern of cytokine release. In experimental models even a minor Th2 component abrogates immunity, and leads to an immunopathology that mimics the human disease. There to determine if Th2 cells are present in a peripheral blood, we analyzed the intracellular IL-4 using a flow cytometer. IL-4 producing cells were founded in CD4+ and CD4- Tells. Moreover, we studied about gamma delta (gamma delta) T cells in TB patients. V gamma 9-expressing gamma delta T cells were decreased in patients. However, the proliferative response to PPD by V gamma 9-expressing gamma delta T cells from some patients were higher levels compared with controls. The roles of gamma delta T cells of TB were not yet revealed completely.

Protection of hosts against tuberculosis depends on expression of cellular immunity. To express cellular immunity, interleukin 12 (IL-12) has been shown to play an important role. Although Mycobacterium tuberculosis is known to induce IL-12 from macrophages (M phi s), the mechanism for the induction is still unclear. To understand the mechanisms of IL-12 induction from M phi s by M. tuberculosis, the IL -12-inducing ability of substances derived from M. tuberculosis was investigated in vitro. Production of IL-12 in culture medium of M phi s was measured by ELISA system using specific antibodies. Live M. tuberculosis H37Rv induced slightly higher IL-12 production than live M. tuberculosis H37Ra upon stimulation of human or mouse alveolar macrophages (hAM phi s or mAM phi s). Heat-killed M. tuberculosis failed to induce IL-12 production of alveolar macrophages (AM phi). The responses of hAM phi s and mAM phi s to M. tuberculosis were remarkably different. mAM phi s produced five times larger amount of IL-12, compared with that from hAM phi s. Human peripheral blood mononuclear cells (PBMC) obtained by the density gradient centrifugation were also used for induction of IL-12 production. Although production levels of IL-12 from PBMC stimulated with M. tuberculosis were below the detectable level, addition of interferon-gamma (IFN-gamma) or neutralizing antibody against IL-10 augmented the production of IL-12 from PBMC, suggesting that IFN-gamma and IL-10 regulate the production of IL-12 from M phi positively and negatively, respectively. To characterize the physicochemical properties of IL-12-inducing molecules, M. tuberculosis H37Rv was disrupted by pressing with 1,000 bar and centrifuged and separated into cytosol and cell wall fraction. The culture filtrate was also examined on IL-12-inducing activity. Among the three subjects examined, cytosol was found to induce the highest production of IL-12 from mAM phi s 1 day after the stimulation. Addition of IFN-gamma to the cytosol fraction markedly increased the production of IL-12 from mAM phi s. The molecular weight of IL-12-inducing substance was shown to be more than 30kDa by fractionating with molecular filters. Treatment of 30kDa-fraction with IL-12-inducing activity by proteinase K completely abolished the activity. Furthermore, approximately 90% of IL-12-inducing activity of 30kDa-fraction was lost by proteinase K treatment even in the presence of IFN-gamma. These results indicate that the major component of IL-12-inducing activity is a protein. The identification of this IL-12-inducing active substance may provide a new therapeutic tool for tuberculosis.

Diabetics are prone to bacterial infection in part, due to polymorphonuclear neutrophil dysfunction, but the precise mechanism is not yet fully explained. Of many complications, diabetes mellitus (DM) is one of the most common diseases, which causes pulmonary tuberculosis. To elucidate the mechanism of susceptibility to tuberculosis infection in patients with diabetes mellitus, we measured IFN-gamma, IL-12 and IL-10 productions by CD4+ alpha beta T cells and autologous monocytes stimulated with live BCG in patients with pulmonary tuberculosis complicated with DM (TB + DM) or without DM (TB) and healthy controls. The levels of IFN-gamma and IL-12 production in TB patients were significantly lower than those in the control. These cytokine productions were also lower in TB + DM patients than in TB patients significantly. The level of IL-10 production in TB patients were highest among these three groups. The production of this cytokine in TB + DM patients was lowest. The level of IFN-gamma production was significantly lower in TB + DM patients under poor DM control than in those patients under good DM control and showed a significant negative correlation to HbA1c, an indicator of diabetic control. The period for negative conversion of culture finding in TB + DM patients under poor control was prolonged when compared with those in TB patients. These results demonstrated the difference in cytokines secretion profile between TB patients and TB + DM patients, and suggest that the immunological mechanism underlying pathogenesis of tuberculosis might work differently between these two patients groups.