BACKGROUNDAlthough the onset of anemia during infectious disease is commonly correlated with production of inflammatory cytokines, the mechanisms by which cytokines induce anemia are poorly defined. This study focused on the mechanism research.

METHODSDifferent types of mice were infected perorally with Toxoplasma gondii strain ME49. At the indicated times, samples from each mouse were harvested, processed, and analyzed individually. Blood samples were analyzed using a Coulter Counter and red blood cell (RBC) survival was measured by biotinylation. Levels of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and inducible protein 10 (IP-10) mRNA in liver tissue were measured by real-time polymerase chain reaction.

RESULTST. gondii-infected mice exhibited anemia due to a decrease in both erythropoiesis and survival time of RBC in the circulation (P < 0.02). In addition, infection-stimulated anemia was associated with fecal occult, supporting previous literature that hemorrhage is a consequence of T. gondii infection in mice. Infection-induced anemia was abolished in interferon gamma (IFNγ) and IFNγ receptor deficient mice (P < 0.05) but was still evident in mice lacking TNF-α, iNOS, phagocyte NADPH oxidase or IP-10 (P < 0.02). Neither signal transducer and activator of transcription 1 (STAT1) deficient mice nor 129S6 controls exhibited decreased erythropoiesis, but rather suffered from an anemia resulting solely from increased loss of circulating RBC.

CONCLUSIONSInfection-stimulated decrease in erythropoiesis and losses of RBC have distinct mechanistic bases. These results show that during T. gondii infection, IFNγ is responsible for an anemia that results from both a decrease in erythropoiesis and a STAT1 independent loss of circulating RBC.

Anemia ; genetics ; metabolism ; Animals ; Erythrocytes ; pathology ; Interferon-gamma ; metabolism ; Male ; Mice ; Mice, Knockout ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Receptors, Interferon ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Toxoplasma ; pathogenicity ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed.
Cytokines/genetics ; Cytokines/biosynthesis* ; Hepatitis B Surface Antigens/pharmacology ; Hepatitis B Surface Antigens/immunology* ; Hepatitis B, Chronic/immunology* ; Human ; Interferon Type II/genetics ; Interferon Type II/biosynthesis ; Leukocytes, Mononuclear/immunology ; Liver/cytology ; Recombinant Fusion Proteins/pharmacology ; Recombinant Fusion Proteins/immunology ; Th1 Cells/immunology* ; Th1 Cells/drug effects ; Th2 Cells/immunology* ; Th2 Cells/drug effects ; Trans-Activators/pharmacology ; Trans-Activators/immunology*

OBJECTIVETo investigate the effect of interferon regulatory factors (IRFs) on neointimal formation after vascular injury in the mouse, and its possible mechanism.

METHODSVascular injury was induced by polyethylene cuff placement around the left femoral artery of IRF-1-deficient mice and C57BL/6J mice. The mRNA expressions of IRF-1, IRF-2, angiotensin II type 2 (AT2) receptor, interleukin-1 beta converting enzyme (ICE), inducible nitric oxide synthase (iNOS) were detected by RT-PCR and immunohistochemical staining.

RESULTSNeointimal formation after vascular injury was significantly greater in IRF-1-deficient mice than that in C57BL/6J mice (P<0.05). In contrast, TUNEL-positive nuclei to total nuclei in the neointima and media in vascular smooth muscle cell (VSMC) in the injured artery significantly attenuated in IRF-1-deficient mice compared to C57BL/6J mice (P<0.05). The expressions of AT2 receptor as well as pro-apoptotic genes such as ICE and iNOS in C57BL/6J mice were up-regulated in response to vascular injury, but this upregulation was attenuated in IRF-1-deficient mice.

CONCLUSIONSOur results suggest that IRF-1 induces VSMC apoptosis and inhibits neointimal formation after vascular injury at least partly due to the upregulation of AT2 receptor, ICE and iNOS expressions. These results indicate that IRF-1 exerts an inhibitory effect on neointimal formation through the induction of apoptosis in VSMCs.

Animals ; Apoptosis ; physiology ; Caspase 1 ; genetics ; metabolism ; Femoral Artery ; anatomy & histology ; pathology ; Interferon Regulatory Factor-1 ; genetics ; metabolism ; Interferon Regulatory Factor-2 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Muscle, Smooth, Vascular ; cytology ; metabolism ; pathology ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; Receptor, Angiotensin, Type 2 ; genetics ; metabolism ; Tunica Intima ; pathology ; physiology

TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta TNF-alphaor IFN-gamma TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gammainduced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-reated with IFN-gamma they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.
Tumor Necrosis Factor-alpha/genetics/*metabolism ; TNF-Related Apoptosis-Inducing Ligand ; Membrane Glycoproteins/genetics/*metabolism ; Interferon Type II/*pharmacology ; Humans ; Cells, Cultured ; Astrocytes/*cytology/drug effects/metabolism ; Apoptosis Regulatory Proteins/genetics/*metabolism ; Apoptosis/*drug effects/physiology ; Antineoplastic Agents/*pharmacology

TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta TNF-alphaor IFN-gamma TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gammainduced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-reated with IFN-gamma they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.
Tumor Necrosis Factor-alpha/genetics/*metabolism ; TNF-Related Apoptosis-Inducing Ligand ; Membrane Glycoproteins/genetics/*metabolism ; Interferon Type II/*pharmacology ; Humans ; Cells, Cultured ; Astrocytes/*cytology/drug effects/metabolism ; Apoptosis Regulatory Proteins/genetics/*metabolism ; Apoptosis/*drug effects/physiology ; Antineoplastic Agents/*pharmacology

Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level.
Base Sequence ; Gene Expression Regulation, Neoplastic/drug effects* ; HLA-DR Antigens/genetics* ; Human ; Interferon Type II/pharmacology* ; Melanoma/genetics* ; Molecular Sequence Data ; Polymerase Chain Reaction ; Promoter Regions (Genetics) ; RNA, Messenger/analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of activated greater omental milky spots and peritoneal macrophages in mice on tumoricidal activity against gastric carcinoma SGC-7901, following intraperitoneal (i.p.) injection of INF-gamma, staphylococcin aureus or NDV-L.

METHODSThe quantitative changes of milky spots were determined by activated carbon, the number of the macrophage in milky spots was assessed by nonspecific esterase stain and the number of peritoneal macrophages was counted by trypan blue exclusion. The morphology of peritoneal macrophages was observed by scanning electron microscope, the amount of TNF-alpha and iNOS mRNA expressed by peritoneal macrophages was measured by fluorescence quantitative PCR and the cytotoxicity of peritoneal macrophages supernatant against SGC-7901 was evaluated by MTT assay.

RESULTSIt was found in the treated groups that: 1. The amount of greater omental milky spots and the macrophages in milky spots increased, 2. The number of peritoneal macrophages increased. The peritoneal macrophages were in activated status. The effect TNF-alpha and iNOS mRNA expression increased and 3. The cytotoxicity against in vitro SGC-7901 increased.

CONCLUSIONIntraperitoneal injection of IFN-gamma, staphylococcin aureus or NDV-L could activate the milky spots of the greater omentum and the macrophages in peritoneal cavity in mice, with IFN-gamma being the best. The supernatant of activated peritoneal macrophages has cytotoxicity against SGC-7901. Administration of LPS to macrophages cultured in vitro could amplify the activation and enhance the cytotoxicity of the supernatant against SGC-7901.

Animals ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Female ; Interferon-gamma ; pharmacology ; Macrophages, Peritoneal ; drug effects ; immunology ; ultrastructure ; Mice ; Nitric Oxide Synthase Type II ; genetics ; Omentum ; drug effects ; immunology ; ultrastructure ; RNA, Messenger ; analysis ; Stomach Neoplasms ; immunology ; Tumor Necrosis Factor-alpha ; genetics

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

The kinetics of cytokine mRNA expression was studied in porcine alveolar macrophages using an RT-PCR assay. The expression levels of IFN- gamma, IL-2, IL-4, IL-6, GM-CSF, IL-12 p35, and IL-12 p40 were examined after 2, 4, 14, 24, 48, and 72 h of incubation in unstimulated control and LPS-stimulated cells. The expression contents of IFN-gamma, IL-2, and IL-4 were not detected in both unstimulated and LPS-stimulated cells. On the other hand, the expression levels of IL-6, GM-CSF, and IL-12 in LPS-stimulated cells were almost always higher than those in control cells. Among those cytokines, IL-6 exhibited the predominant expression, and GM-CSF, IL-12 p40, and IL-12 p35 followed in the descending order. The times to reach the peak expression levels for IL-6, and GM-CSF, IL-12 p35, and IL-12 p40 were 14, and 24 h, respectively. After reaching the peak expression point, the expression levels of IL-6, GM-CSF, and IL-12 p40 reduced to the baseline by 72 h after stimulation, however, IL-12 p35 still kept a substantial expression by the same time. This study demonstrates that porcine alveolar macrophages primarily respond to express IL-6, GM-CSF, and IL-12 by LPS-stimulation and have a cytokine-specific expression profile during the stimulation time.
Animals ; Cells, Cultured ; Cytokines/*genetics ; Female ; Gene Expression Profiling ; Gene Expression Regulation/*drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; Interferon Type II/genetics ; Interleukins/genetics ; Kinetics ; Lipopolysaccharides/*pharmacology ; Macrophages, Alveolar/*drug effects/*metabolism ; RNA, Messenger/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Swine/*genetics

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