Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.

Differential response of cytokines induced by Leptospira interrogans, serogroup Pomona, serovar Pomona, in mouse and human cell lines.

Leptospira interrogans, the causative agent of leptospirosis, is an important zoonotic bacterium. The mechanisms and roles of cytokine induction in both humans and animals remain unclear. Therefore, the IFN-gamma, IL-6, IL-10 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) in human THP-1 and mouse RAW 264.7 monocyte cell lines following stimulation with heat-killed Leptospira interrogans serogroup Pomona serovar Pomona, L. biflexa, E. coli or Salmonella group B. The production of IL-6 and IL-12 were higher and rose more rapidly in the RAW 264.7 cells with all bacteria. The IL-10 was not detected in the RAW 264.7 cells when induced by leptospires. The IFN-gamma level in human peripheral blood mononuclear cells (PBMCs) induced by leptospires was also significantly lower than with other bacteria. When IL-10 and IL-12 mRNA expressions were detected in hamster's spleen, their patterns were similar to what was observed in THP-1 in that IL-12 was only slightly increased while IL-10 expression was high. Moreover, the IFN-gamma expression could not be detected in hamsters. The more potent cytokine responses in the RAW 264.7 cells may indirectly reflect the disease outcome in mice which render them to be a good reservoir of leptospirosis. Whether these cytokines have contributed to immunoprotection during the L. interrogans infection remains to be further investigated.

BCG-Induced Dendritic Cell Responses and Suppression of Interleukin-5 Production from T Cells in Atopic Asthmatics

Bacille Calmette-Guerin (BCG) induces potent Th1 responses with the help of interleukin (IL)-10 and IL-12 released from dendritic cells (DCs), and suppresses Th2- associated allergic reactions. However, there are still some controversies on therapeutic effects of BCG in asthmatics. This study investigated whether BCG administration to DCs suppresses IL-5 production from T cells in atopic asthmatics. DCs derived from peripheral blood of subjects were cultured with or without BCG and Dermatophagoides farinae extract. Some DCs were co-cultured with T cells in the presence of BCG or the above culture supernatants. In the atopic asthmatics, BCG significantly increased IL-10 and IL-12 production from DCs. In the presence of D. farinae extract, BCG further increased IL-10 production. BCG-induced IL-10 production was significantly higher in the atopics (n=14) than in the non-atopics (n=9). Both BCG and the BCG-treated DCs culture supernatant significantly increased IFN-gamma production from T cells. Both BCG and the supernatant from DCs+BCG+D. farinae co-cultures significantly decreased IL-5 production (all p<0.05), but the supernatant from DCs+BCG co-cultures did not. In conclusion, administration of BCG together with D. farinae extract effectively decreased IL-5 production from T cells, probably through the action of IL-10 and IL-12 released from DCs in D. farinaesensitive asthmatics.

Radioprotective effects of an acidic polysaccharide of Panax ginseng on bone marrow cells

An acidic polysaccharide of Panax ginseng (APG), so called ginsan is known to have important immunomodulatory activities. It was recently reported that APG has radioprotective effects in mice but the detailed mechanism was not fully elucidated. This study examined the effects of APG on bone marrow cells (BMs). The phenotypical and functional changes in APG-treated BMs after gamma radiation were studied. The benefit of APG on BMs damaged by gamma radiation was determined by measuring the cell viability. Using 2 different assays, a pretreatment with APG significantly increased the viability of BMs against gamma radiation. APG-treated BMs had a significantly higher amount of IL-12, which is a major cytokine for immune responses, compared with the medium-treated BMs. The expression of MHC class II molecules of APG-treated BMs was also increased, and APG-treated BMs showed significantly higher levels of allogeneic CD4+ T lymphocyte proliferation. Furthermore, APG-treated mice had a larger number of BMs after gamma radiation than the control mice, and the BMs of APG-treated mice were successfully cultured into dendritic cells, which are the representative antigenpresenting cells. Overall, this study shows that APG alters the phenotype of BMs, increases the viability and alloreactivity of BMs after gamma radiation both in vitro and in vivo. Therefore, APG may be a good candidate radioprotective agent for BMs.

The effect of Panax ginseng on forced immobility time & immune function in mice.

BACKGROUND & OBJECTIVES: Panax ginseng has been used as a traditional medicine for many years mainly among Asian peoples for developing physical strength. We undertook this study to determine the immune-enhancement effect of P. ginseng using a forced swimming test (FST) and by measuring cytokine production in MOLT-4 cell culture and mouse peritoneal macrophages. METHODS: P. ginseng was orally administered to mice once a day for 7 days. The anti-immobility effect of P. ginseng on the FST and blood biochemical parameters related to fatigue, glucose (Glc); blood urea nitrogen (BUN); latic dehydrogenase (LDH); total protein (TP) and production of cytokines in human T cell line, MOLT-4 cells and mouse peritoneal macrophages were investigated. RESULTS: After two and seven days, the immobility time was decreased in the P. ginsengadministrated mice as compared to the control group; however, this reduction was not significant. In addition, the amount of TP in the blood serum was significantly increased. However, the levels of Glc, BUN, and LDH did not show a significant change. P. ginseng significantly (P<0.05) increased interferon (IFN)-gamma production and expression as compared to control at 48 h in MOLT-4 cells. P. ginseng plus recombinant IFN-gamma instead of P. ginseng alone significantly increased the production of the tumour necrosis factor (TNF)-alpha in the mouse peritoneal macrophages. INTERPRETATION & CONCLUSION: Our results suggest that P. ginseng may be useful for an immune promoter. Further studies are needed to understand the mechanism of its action.

Down regulation of IL-12 production in healthy non-responder neonates to recombinant hepatitis B vaccine

A proportion of healthy neonates and adults fail to develop a protective antibody response to recombinant hepatitis B [HB] vaccine. Unresponsiveness to vaccination could be attributed to defect in a number of immunological regulatory mechanisms. In this study, IL-12 was quantitated in culture supernatant following in vitro stimulation of peripheral blood mononuclear cells isolated from a group of responder and non-responder neonates. Our results indicate significantly decreased production of HBsAg-induced IL-12 in non-responder subjects compared to responders [P<0.01]. Since IL-12 is produced mainly by antigen presenting cells [APC] and is considered to be crucial for initiation and polarization of CD4+ T-cell function, therefore, our findings could be interpreted to imply APC dysfunction in non-responder vaccines

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-g production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8+ T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

Citoquinas inducidas por la inmunización experimental antitetánica. Efecto de la formulación vacunal / [Cytokines induced by experimental anti-tetanus immunization. Vaccine formulation effect]

Several factors are involved in the selective activation of T helper 1 or T helper 2 cells, such as the type of antigen-presenting cells involved in the immune response and the different physical characteristics of antigens. The aim of this work was to evaluate if adding other antigens to tetanus toxoid modifies the original immune response. BALB/c mice were immunized with tetanus and diphtheria toxoids associated with whole-cell Bordetella pertussis (DTPw vaccine), B. pertussis soluble antigens (DTPa vaccine) or Salmonella typhi plus DTPa (DTPaSt vaccine). DTPw and DTPaSt immunization induced a T helper 1/T helper 2 (Th1/Th2) anti-tetanus response with gamma interferon and interleukin 5 production. DTPa immunization induced a Th2 response with production of interleukin 5 and interleukin 6. Only DTPw vaccine induced higher levels of IL-12 in non-immunized mice. Our findings indicate that the co-injection of whole-cell antigens such as B. pertussis or S. typhi, modifies the anti-tetanus response shifting it from Th2 to Th1 type. However, the original Th2 immune response is not modified when the vaccine consists only of soluble antigens.