A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.
Animals ; Cell Cycle ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Fungal Polysaccharides ; pharmacology ; Immunity ; drug effects ; Interferon-gamma ; biosynthesis ; metabolism ; Interleukin-6 ; biosynthesis ; metabolism ; Macrophages ; immunology ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Pleurotus ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism ; Up-Regulation

In the present study, the effects of Pleurotus nebrodensis polysaccharide (PN-S) on the immune functions of immunosuppressed mice were determined. The immunosuppressed mouse model was established by treating the mice with cyclophosphamide (40 mg/kg/2d, CY) through intraperitoneal injection. The results showed that PN-S administration significantly reversed the CY-induced weight loss, increased the thymic and splenic indices, and promoted proliferation of T lymphocyte, B lymphocyte, and macrophages. PN-S also enhanced the activity of natural killer cells and increased the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the serum. In addition, PN-S treatment significantly increased the phagocytic activity of mouse peritoneal macrophages. PN-S also increased the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and nitric oxide (NOS) in splenocytes. qRT-PCR results also indicated that PN-S increased the mRNA expression of IL-6, TNF-α, INF-γ, and nitric oxide synthase (iNOS) in the splenocytes. These results suggest that PN-S treatment enhances the immune function of immunosuppressed mice. This study may provide a basis for the application of this fungus in adjacent immunopotentiating therapy against cancer and in the treatment of chemotherapy-induced immunosuppression.
Animals ; Antineoplastic Agents, Alkylating ; Biological Products ; pharmacology ; therapeutic use ; Cell Line ; Cyclophosphamide ; Immunity ; drug effects ; Immunologic Factors ; pharmacology ; therapeutic use ; Immunosuppression ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Macrophages ; drug effects ; metabolism ; Male ; Mice, Inbred BALB C ; Neoplasms ; drug therapy ; immunology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Phagocytosis ; drug effects ; Pleurotus ; chemistry ; Polysaccharides ; pharmacology ; therapeutic use ; Tumor Necrosis Factor-alpha ; metabolism

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.
Transplantation, Homologous ; Transfection ; Trans-Activators/genetics/*immunology/metabolism ; Trans-Activation (Genetics)/genetics/immunology ; Skin Transplantation ; Nuclear Proteins/genetics/*immunology/metabolism ; Mutation ; Mice, Transgenic ; Mice, Knockout ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Melanoma, Experimental/genetics/immunology/pathology ; Male ; Interferon Type II/pharmacology ; Humans ; Histocompatibility Antigens Class II/genetics/*immunology/metabolism ; Graft Survival/genetics/immunology ; Graft Rejection/genetics/*immunology ; Genes, MHC Class II/genetics/immunology ; Flow Cytometry ; DNA, Complementary/genetics ; Cell Proliferation/drug effects ; Cell Line, Tumor ; Animals

Regulatory T cells, which stimulate or inhibit the effector functions of distinct T cell subsets, are critical in the control of the immune response. We investigated the effect of TGF-beta and IL-10 on T cell subsets according to the Th1/Th2 immune status. Sixty-two patients with asthma and 38 patients with pulmonary tuberculosis were included. Allergy skin tests, tuberculin tests, and chest radiography were performed. The levels of circulating IL-4, IFN-gamma, TGF-beta1, and IL-10 were measured using ELISA. The level of TGF-beta1 was higher in patients with asthma than in those with tuberculosis, but the IL-10 levels were the same between the asthma and tuberculosis groups. Atopy was unrelated to the tuberculin response. The IFN-gamma level was correlated with the IL-10 level, and the level of IL-4 was unrelated to the IL-10 or TGF-beta1 level. The level of IL-10 was higher in the negative tuberculin reactors than in the positive tuberculin reactors among patients with asthma, and TGF-beta1 was higher in the positive tuberculin reactors than in the negative tuberculin reactors among patients with tuberculosis. These results demonstrate that the regulatory effects of circulating TGF-beta and IL-10 on T cell cytokines may be different between Th2-type asthma and Th1 tuberculosis.
Adult ; Asthma/*blood/immunology ; Cytokines/*blood ; Female ; Humans ; Interferon Type II/blood ; Interleukin-10/blood ; Interleukin-4/blood ; Male ; Research Support, Non-U.S. Gov't ; Respiratory Function Tests ; Skin Tests ; Th1 Cells/*metabolism ; Th2 Cells/*metabolism ; Transforming Growth Factor beta/blood ; Tuberculin Test ; Tuberculosis/*blood/immunology

Food allergies affect about 4% of the Korean population, and buckwheat allergy is one of the most severe food allergies in Korea. The purpose of the present study was to develop a murine model of IgE-mediated buckwheat hypersensitivity induced by intragastric sensitization. Young female C3H/HeJ mice were sensitized and challenged intragastricly with fresh buckwheat flour (1, 5, 25 mg/dose of proteins) mixed in cholera toxin, followed by intragastric challenge. Anaphylactic reactions, antigen-specific antibodies, splenocytes proliferation assays and cytokine productions were evaluated. Oral buckwheat challenges of sensitized mice provoked anaphylactic reactions such as severe scratch, perioral/periorbital swellings, or decreased activity. Reactions were associated with elevated levels of buckwheatspecific IgE antibodies. Splenocytes from buckwheat allergic mice exhibited significantly greater proliferative responses to buckwheat than non-allergic mice. Buckwheat-stimulated IL-4, IL-5, and INF-gamma productions were associated with elevated levels of buckwheat-specific IgE in sensitized mice. In this model, 1 mg and 5 mg dose of sensitization produced almost the same degree of Th2-directed immune response, however, a 25 mg dose showed blunted antibody responses. In conclusion, we developed IgE-mediated buckwheat allergy by intragastric sensitization and challenge, and this model could provide a good tool for future studies.
Anaphylaxis/blood/immunology ; Animals ; Cell Proliferation/drug effects ; Comparative Study ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Fagopyrum/*immunology ; Female ; *Flour ; Food Hypersensitivity/blood/*immunology ; Immunoglobulin E/blood/immunology ; Immunoglobulin G/blood/immunology ; Interferon Type II/biosynthesis ; Interleukin-4/biosynthesis ; Interleukin-5/biosynthesis ; Mice ; Mice, Inbred C3H ; Plant Extracts/administration & dosage/immunology ; Research Support, Non-U.S. Gov't ; Spleen/cytology/drug effects/metabolism ; Stomach/drug effects/*immunology ; T-Lymphocytes/cytology/drug effects/metabolism ; Time Factors

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals ; Antibodies, Monoclonal/pharmacology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation ; Female ; Glucocorticoids/*pharmacology ; Herpes Simplex/*immunology ; Herpesvirus 1, Human/pathogenicity ; *Immunity, Cellular ; Interferon Type II/secretion ; *Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/metabolism ; Receptors, Interleukin-2/metabolism ; Receptors, Nerve Growth Factor/genetics/immunology/*metabolism ; Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/*immunology/metabolism/virology

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.
Animals ; Antigen-Presenting Cells/cytology/*drug effects/immunology/*metabolism ; Antigens, CD86/metabolism ; Carrier Proteins/*pharmacology ; Cell Death/drug effects ; Cell Survival/drug effects ; Cells, Cultured ; Cytokines/metabolism ; Flow Cytometry ; Histocompatibility Antigens Class II/metabolism ; Inflammation Mediators ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/cytology/*drug effects/immunology/*metabolism ; Membrane Glycoproteins/*pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Nitric Oxide Synthase Type II/metabolism ; Phagocytosis/drug effects ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/immunology/metabolism ; Up-Regulation/drug effects/genetics

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.
Binding, Competitive ; Cell Survival ; Cells, Cultured ; Corneal Diseases/metabolism ; Electrophoretic Mobility Shift Assay ; Epithelium, Corneal/drug effects/*immunology/metabolism ; Gene Expression ; Humans ; Interferon Type II/metabolism/pharmacology ; Interleukin-1/*pharmacology ; Lipopolysaccharides/metabolism/pharmacology ; NF-kappa B/metabolism ; Promoter Regions (Genetics)/drug effects/genetics ; Research Support, Non-U.S. Gov't ; Tumor Necrosis Factor-alpha/metabolism/pharmacology ; beta-Defensins/*biosynthesis/genetics/metabolism

Phagocytosis of serum- and IgG-opsonized zymosan (SOZ and IOZ, respectively) particles into J774A.1 macrophages induced apoptosis of the cells, accompanied by the expression of p21(WAF1), one of cyclin-dependent protein kinase (CDK) inhibitors. Furthermore, phagocytosis of SOZ and IOZ particles into macophages induced superoxide formation. Tat-superoxide dismutase (SOD), which is readily transduced into the cells using Tat-domain, protected the cells from the apoptosis induced by phagocytosis of SOZ and IOZ particles. lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) also caused the apoptosis of the cells. However, Tat-SOD could not protect the cells from LPS/IFN-gamma induced apoptosis, suggesting that apoptosis mechanisms involved are different from each other. In the present study, we determined the amounts of nitric oxide (NO) produced by SOZ, IOZ, and LPS/IFN-gamma, and found that SOZ and IOZ did not induce the generation of NO in macrophages, whereas LPS/ IFN-gamma did. The apoptosis due to phagocytosis was accompanied with the release of cytochrome c from mitochondrial membrane to cytosolic fraction. Furthermore, SOZ and IOZ induced the cleavage of procasapase-3 (35 kDa) to give rise to an active caspase-3 (20 kDa), which was blocked by Tat- SOD but not by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. On the other hand, LPS/IFN-gamma caused the activation of procaspase-3, which was blocked by PTIO but not by Tat-SOD. Taken together, phagocytosis of SOZ and IOZ particles induced apoptosis through superoxide but not NO in macrophages, accompanied with the release of cytochrome c and the activation of caspase-3.
Apoptosis/*immunology ; Caspases/metabolism ; Cell Line ; Cyclins/biosynthesis ; Cytochromes c/metabolism ; Immunoglobulin G/*immunology ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/*immunology/metabolism ; Nitric Oxide/*metabolism ; Opsonins/immunology ; Phagocytosis/*physiology ; Superoxide Dismutase/metabolism ; Superoxides/*metabolism ; Zymosan

Host immune response has been considered as an important disease-modifying factor of periodontitis, however, which immune cell(s) or factor(s) are involved in the destruction of periodontium remains unclear. Previously, we reported that osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+)T cells. We present new data that B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis. Purified murine B cells were activated with anti-IgD mAb, IL-4, and anti-CD40 mAb, in the absence (B(Th2)) or presence of Th1 cytokines, either IL-2 (B(IL-2)) or IFN-gamma (B(IFN-gamma)). Each activated B cell population was co-cultured with RAW264.7 cells in the presence of soluble receptor activator of NF-kappaB ligand (sRANKL), and the effect on osteoclastic differentiation was evaluated. While B(Th2)increased osteoclastogenesis, B(IL-2)and B(IFN-gamma)suppressed it profoundly. To verify the mediating molecule(s), we analyzed cytokine profiles of the activated B cells. Compared to B(Th2), B(IL-2)expressed increased amount of IFN-gamma and B(IFN-gamma)expressed decreased amounts of IL-4, IL-5, and IL-10. IFN-gamma was a key negative regulator of osteoclastic differentiation, and mediated the inhibition by B(IL-2). These results suggest that Th1 cytokines may have new important roles in resistance to periodontitis, acting directly on osteoclasts or indirectly through B cells.
Animals ; B-Lymphocytes/cytology/*drug effects/immunology ; Base Sequence ; Cell Differentiation/*drug effects ; Cytokines/*pharmacology ; Female ; Giant Cells/cytology/drug effects ; Interferon Type II/immunology/metabolism ; Lymphocyte Activation/*drug effects ; Mice ; Molecular Sequence Data ; Osteoclasts/*cytology/*drug effects ; Phenotype ; Support, Non-U.S. Gov't ; Th1 Cells/*immunology ; Tumor Necrosis Factor/pharmacology