Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Animals ; Cell Line ; Interleukin-6/*biosynthesis ; Leukotriene B4/metabolism ; Ligands ; Lipopolysaccharides/immunology ; Macrophages/immunology/*metabolism ; Macrophages, Peritoneal/immunology/metabolism ; Mice ; Myeloid Differentiation Factor 88/*metabolism ; NADH, NADPH Oxidoreductases/metabolism ; NF-kappa B/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*metabolism ; Signal Transduction

OBJECTIVETo study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA).

METHODWister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated.

RESULTWuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity.

CONCLUSIONThe anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

Animals ; Arthritis, Experimental ; drug therapy ; immunology ; metabolism ; pathology ; Capsules ; Dehydroascorbic Acid ; analogs & derivatives ; blood ; Dinoprostone ; metabolism ; Disease Models, Animal ; Edema ; drug therapy ; Female ; Interleukin-1beta ; metabolism ; Lymphocytes ; immunology ; metabolism ; Macrophages, Peritoneal ; metabolism ; Male ; Medicine, Mongolian Traditional ; Mice ; Nitric Oxide ; metabolism ; Rats ; Spleen ; cytology ; metabolism ; Superoxide Dismutase ; blood ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe the effect of andrographolide on the activation of mitogen-activated protein kinases (MAPKs) and expression of nuclear factor-κB (NF-κB) in macrophage foam cells.

METHODSThe mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL), ox-LDL+andrographolide, or neither (control). The phosphorylation of MAPK molecules (p38MAPK, JNK, ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.

RESULTSAs compared with cells in the control group, the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide (P<0.01), but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL (P<0.05). The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells (P<0.01), but no significant difference existed between ox-LDL and ox-LDL+andrographolide (P>0.05). The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells (P<0.01), but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium (P>0.05).

CONCLUSIONSAndrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-κB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; immunology ; metabolism ; prevention & control ; Cells, Cultured ; Diterpenes ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Foam Cells ; cytology ; drug effects ; enzymology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoproteins, LDL ; metabolism ; MAP Kinase Signaling System ; drug effects ; immunology ; Macrophages, Peritoneal ; cytology ; drug effects ; enzymology ; Mice ; Mice, Inbred Strains ; NF-kappa B ; metabolism ; Vasculitis ; drug therapy ; immunology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo evaluate the hepatoprotective and immunotherapeutic effects of aqueous extract of turmeric rhizome in CCl4 intoxicated Swiss albino mice.

METHODSFirst group of mice (n=5) received CCl4 treatment at a dose of 0.5 mL/kg bw (i.p.) for 7 days. Second group was fed orally the aqueous extract of turmeric at a dose of 50 mg/kg bw for 15 days. The third group was given both the turmeric extract (for 15 days, orally) and CCl4 (for last 7 days, i.p.). The fourth group was kept as a control. To study the liver function, the transaminase enzymes (SGOT and SGPT) and bilirubin level were measured in the serum of respective groups. For assaying the immunotherapeutic action of Curcuma longa (C. longa), non specific host response parameters like morphological alteration, phagocytosis, nitric oxide release, myeloperoxidase release and intracellular killing capacity of peritoneal macrophages were studied from the respective groups.

RESULTSThe result of present study suggested that CCl4 administration increased the level of SGOT and SGPT and bilirubin level in serum. However, the aqueous extract of turmeric reduced the level of SGOT, SGPT and bilirubin in CCl4 intoxicated mice. Apart from damaging the liver system, CCl4 also reduced non specific host response parameters like morphological alteration, phagocytosis, nitric oxide release, myeloperoxidase release and intracellular killing capacity of peritoneal macrophages. Administration of aqueous extract of C. longa offered significant protection from these damaging actions of CCl4 on the non specific host response in the peritoneal macrophages of CCl4 intoxicated mice.

CONCLUSIONSIn conclusion, the present study suggests that C. longa has immunotherapeutic properties along with its ability to ameliorate hepatotoxicity.

Animals ; Aspartate Aminotransferases ; blood ; Bilirubin ; blood ; Carbon Tetrachloride ; toxicity ; Cell Adhesion ; drug effects ; immunology ; Curcuma ; chemistry ; Cytotoxicity, Immunologic ; drug effects ; Immunologic Factors ; pharmacology ; Liver ; drug effects ; metabolism ; pathology ; Macrophages, Peritoneal ; drug effects ; immunology ; metabolism ; pathology ; Male ; Mice ; Nitric Oxide ; metabolism ; Peroxidase ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology

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.
Animals ; Antigens, CD/immunology/metabolism ; Bacterial Outer Membrane Proteins/*pharmacology ; Cells, Cultured ; Female ; Histocompatibility Antigens Class II/immunology/metabolism ; Host-Pathogen Interactions ; Interleukin-12/biosynthesis ; Interleukin-6/biosynthesis ; Legionella pneumophila/*immunology/metabolism ; Legionnaires' Disease/immunology/metabolism ; Lipoproteins/*pharmacology ; Macrophage Activation/drug effects/immunology ; Macrophages, Peritoneal/drug effects/immunology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Toll-Like Receptor 2/*metabolism ; Tumor Necrosis Factor-alpha/biosynthesis

OBJECTIVETo investigate the immunomodulatory effects of Fomes fomentarius polysaccharides (FFP) in mice.

METHODSMTT assay was employed to evaluate the in vitro metabolic activity of the mouse splenocytes treated with FFP at different concentrations, and the secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin 2 (IL-2) from the cells were measured by enzyme-linked immunosorbent assay. The changes in the phagocytotic activity of mouse macrophage in response to FFP treatment were evaluated by phagocytosis percentage of chicken red blood cells (CRBCs). The effect of FFP on the humoral immunity was assessed in mice immunized with sheep red blood cells (SRBCs) by measuring the serum levels of specific antibody (hemolysin) against SRBCs.

RESULTSFFP at the concentrations of 25, 50, and 100 microg/ml all significantly enhanced the metabolic activity of mouse splenocytes in vitro and increased the production of TNF-alpha, IFN-gamma and IL-2. FFP treatment also markedly enhanced the metabolic activity of mouse peritoneal exudate cells and TNF-alpha production by the cells. At the doses of 25, 50, and 100 mg/kg, FFP significantly increased serum hemolysin level in mice immunized with SRBCs, and FFP at 50 and 100 mg/kg obviously increased the capacity of mouse peritoneal macrophages in vivo for CRBC phagocytosis.

CONCLUSIONFFP can promote the secretion of TNF-alpha, IFN-gamma and IL-2 by mouse immunocytes and enhance mouse humoral immune response and the phagocytotic activity of the macrophages.

Adjuvants, Immunologic ; pharmacology ; Animals ; Coriolaceae ; chemistry ; Female ; Immunologic Factors ; immunology ; pharmacology ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Macrophages, Peritoneal ; drug effects ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Phagocytosis ; drug effects ; Polysaccharides ; isolation & purification ; pharmacology ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo explore the influence of bone marrow (BM) mesenchymal stem cells (MSCs) on macrophage activation after lipopolysaccharide (LPS) stimulation.

METHODSMouse BM MSCs were isolated and purified by adherence screening, and mouse peritoneal macrophages (MPM) were collected by sodium thioglycollate peritoneal injection, and the co-culture system was established by planting macrophages on the MSCs monolayer. The grouping of experiments: group A: MPM; group B: MPM + LPS; group C: MPM + LPS + MSC; group D: MPM + LPS + MSC supernatant. Cell culture supernatants were collected to detect the changes of TNF-alpha/TGF-beta and nitrogen monoxide (NO) after stimulating macrophages with LPS for 18 hours. At the same time Escherichia coli standard strain (ATCC25922) was added into the culture system and incubated for another 24 hours, macrophages were stained and phagocytosis were examined.

RESULTSThe concentrations of TNF-alpha and NO in culture supernatants were increased significantly to (147.4 +/- 37.1) pg/ml and (59.9 +/- 8.7) micromol/L respectively after macrophage activation, however, at the present of MSC, the concentration of TNF-alpha was dramatically decreased [(97.6 +/- 30.3) pg/ml, P = 0.032], and the concentration of NO was decreased to (50.9 +/- 29.5) micromol/L (P > 0.05). The concentrations of TNF-alpha and NO were further decreased after addition of MSC supernatants [(58.3 +/- 31.5) pg/ml and (-3.4 +/- 2.3) micromol/L respectively, P < 0.01]. There was no change in the phagocytic rate and phagoindex of macrophages after activation.

CONCLUSIONSMSCs can inhibit the activation of mouse peritoneal macrophages after stimulating with LPS but has no influence on the phagocytosis.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Lipopolysaccharides ; pharmacology ; Macrophage Activation ; drug effects ; Macrophages, Peritoneal ; immunology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.

METHODSMouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).

RESULTSThe average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.

CONCLUSIONPM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.

Animals ; Cells, Cultured ; Lectins, C-Type ; Macrophages, Peritoneal ; cytology ; immunology ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; Penicillium ; immunology ; Toll-Like Receptor 2 ; biosynthesis ; Toll-Like Receptor 4 ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.
Toxoplasmosis/*enzymology/*immunology ; Toxoplasma/*immunology/*metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; Mice, Inbred BALB C ; Mice ; Macrophages, Peritoneal/enzymology/immunology/parasitology ; Humans ; Hela Cells ; Enzyme Activation ; Cyclooxygenase 2/*biosynthesis ; Chemokines/*biosynthesis ; Animals

OBJECTIVETo investigate nuclear factor kappa B (NF-kappaB) activation induced by lipopolysaccharide (LPS) in rat peritoneal macrophages (PMAs) and the inhibitory effect of resveratrol on NF-kappaB activation.

METHODSPMAS from normal SD rats were randomly divided into 7 groups, including a control group, a LPS group and 5 resveratrol groups (I-V). PMAs of the control group were incubated in DMEM, and those in LPS group in DMEM containing LPS (10 microg/ml). PMAS of resveratrol groups I-V were incubated in DMEM containing LPS (10 microg/ml) and different concentrations of resveratrol. After 24 h of incubation, NF-kappaB activation in the PMAs was determined, and the expression levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and nitric oxide (NO) in the culture medium were measured.

RESULTSExposure to LPS resulted in an excessive enhancement of cytokine and NO expressions in the PMAs. Resveratrol at 1.25-10 microg/ml produced a dose- dependent inhibition of cytokine and NO expressions and on NF-kappaB activation in LPS-stimulated PMAs.

CONCLUSIONResveratrol can inhibit LPS-induced NF-kappaB activation in rat PMAs and subsequently suppress the expressions of TNF-alpha, IL-1 and NO.

Animals ; Cytokines ; metabolism ; Lipopolysaccharides ; pharmacology ; Macrophage Activation ; drug effects ; Macrophages, Peritoneal ; drug effects ; immunology ; metabolism ; Male ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology