OBJECTIVETo study the mechanism of macrophage injury after trauma-hemorrhagic shock.

METHODSWistar male rats underwent trauma (closed bone fracture) and hemorrhage (mean arterial blood pressure of 35 mm Hg+/-5 mm Hg for 60 minutes, following fluid resuscitation). Rats without trauma, hemorrhage or fluid resuscitation served as controls. Peritoneal macrophages were harvested at 6 hours and 1, 2, 3, 7 days after traumatic hemorrhagic shock to determine the effects of pertussis toxin (PTX, as a specific inhibitor to Gi(alpha) and cholera toxin (CTX, as a stimulant to Gs(alpha) on macrophage-Ia expression and TNF-alpha production and levels of Gi(alpha) and Gs(alpha).

RESULTSThe macrophages from the injured rats revealed a significant decrease of Ia positive number and TNF-alpha release in response to LPS. Wi th pretreatment with PTX 10-100 ng/ml Ia positive cells and LPS-induced TNFalpha production in both control and impaired macrophages populations were dos e dependently increased. Both macrophages populations were not responding to CTX treatment (10-100 ng/ml). Western blot analyses showed that the levels of Gi(alpha) protein expression increased as much as 116.5%-148.8% of the control level fro m 6 hours through 7 days after traumatic hemorrhage. The levels of Gs protein expression were reduced at 6 hours and decreased to the lowest degree; 36% o f the control at day 1, began to return at day 2 and returned to the normal level at day 7, following traumatic hemorrhagic shock.

CONCLUSIONSPTX-sensitive G-protein may participate in th e modulation of macrophage-Ia expression and TNF-alpha release following traumatic hemorrhagic shock. Analyses of the alteration of Gi(alpha) and Gs protein express ions further supports the concept that G-protein is involved in trauma-induced macrophage signal transduction pathways.

Analysis of Variance ; Animals ; GTP-Binding Proteins ; immunology ; metabolism ; Histocompatibility Antigens Class II ; immunology ; Immunoblotting ; Lipopolysaccharides ; pharmacology ; Macrophages, Peritoneal ; immunology ; metabolism ; Male ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; blood ; immunology ; Signal Transduction ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo investigate the role of triggering receptors expressed on myeloid-1(TREM-1) in innate response to Porphyromonas gingivalis(Pg) in mice macrophages and its potential role in periodontitis development.

METHODSPeritoneal macrophages from mice were harvested, separated and cultured, then challenged with viable Pg. Transcription and protein expression in macrophages were assessed with real time PCR and flow cytometry respectively.LP-17 peptide (10, 100 and 1000 µg/L) was utilized to block TREM-1, and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme linked absorbent analysis.

RESULTSAt 2 h after Pg challenge, transcription of TREM-1 was significantly up-regulated after Pg challenge[(7.99 ± 1.11) fold vs blank]. At 24 h after bacteria infection, increased TREM-1 expression was demonstrated by flow cytometry, with mean fluorescent intensity increasing from (7.05 ± 1.85) in blank group to (13.17 ± 2.33) in experimental group. Proinflammatory cytokine (TNF-α and IL-6) production was significantly decreased after blocking TREM-1 by LP-17 peptide(100 and 1000 µg/L).

CONCLUSIONSTREM-1 enhanced innate immune response to Pg in macrophages, which may facilitate periodontitis development.

Animals ; Cells, Cultured ; Interleukin-6 ; metabolism ; Macrophages, Peritoneal ; cytology ; immunology ; metabolism ; Membrane Glycoproteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Peptides ; pharmacology ; Porphyromonas gingivalis ; immunology ; Receptors, Immunologic ; metabolism ; Triggering Receptor Expressed on Myeloid Cells-1 ; Tumor Necrosis Factor-alpha ; metabolism ; Up-Regulation

OBJECTIVETo explore the role of toll-like receptor 4 (TLR4) on oxidized low-density/β₂-glycoprotein I/β₂-glycoprotein I (ox-LDL/β₂GPI/anti-β₂GPI) antibodies complex induced macrophage foam cell formation.

METHODSThe peritoneal macrophages were separated from TLR4 intact C3H/HeN mice and TLR4 defective C3H/HeJ mice. The cells were treated with ox-LDL, ox-LDL/β₂GPI, ox-LDL/anti-β₂GPI, anti-β₂GPI/β₂GPI, ox-LDL/β₂GPI/anti-β₂GPI, lipopolysaccharide (LPS) for 48 h and the foam cells were identified by Oil red O staining for intracellular lipids. The total cellular RNA and the protein lysates were collected. The levels of tissue factor (TF) mRNA in two groups were detected by real-time PCR (RT-PCR), and the expression of phosphorylated nuclear factor-κB (NF-κB) p65 was detected by Western blotting. Monocyte chemotactic protein-1 (MCP-1) secretion from peritoneal macrophages was determined by enzyme linked immunosorbent assay (ELISA) kits.

RESULTSCompared with C3H/HeJ mice, lipid droplets in the cytoplasm of peritoneal macrophages from C3H/HeN mice were significantly increased and phosphorylation-NF-κB expression was significantly upregulated after stimulating by ox-LDL/β₂GPI/anti-β₂GPI complex (P < 0.01). TF mRNA and MCP-1 expression were also upregulated post ox-LDL/β₂GPI/anti-β₂GPI complex stimulation [TF mRNA: 0.041 ± 0.023 vs. 0.005 ± 0.003; MCP-1: (6 200.2 ± 6.4) pg/ml vs. (803.3 ± 5.5) pg/ml, P < 0.01].

CONCLUSIONTLR4 can enhance ox-LDL/β₂GPI/anti-β₂GPI complex induced peritoneal macrophage foam cell formation via upregulating phosphorylation-NF-κB, TF and MCP-1 expression.

Animals ; Antigen-Antibody Complex ; immunology ; Atherosclerosis ; immunology ; metabolism ; Cells, Cultured ; Foam Cells ; immunology ; metabolism ; Lipoproteins, LDL ; immunology ; Macrophages, Peritoneal ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C3H ; NF-kappa B ; metabolism ; Thromboplastin ; metabolism ; Toll-Like Receptor 4 ; immunology ; metabolism ; beta 2-Glycoprotein I ; immunology

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

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 investigate the influence of activated complement on the secretory function of peritoneal macrophage (PMphi) in the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), especially in the role of different G-protein subtypes in this process after burns.

METHODSThe mice inflicted by 18% TBSA full-thickness scald was established and employed as the model. And the mice were divided into A (the complements were preserved and activated) and B (with intraperitoneal injection of CVF to deplete complement before scald) groups. The plasma of the mice in the two groups was collected at 6 postburn hour (PBH) and cultured with PMphi from normal mice. The PMphi were pretreated with pertussis toxin (PT) and with cholera toxin (CT). The NO and TNF-alpha levels in the supernatant of normal PMphi culture with different pretreatment were measured by Greiss assay.

RESULTSThe NO and TNF-alpha contents in group A [(80 +/- 12) micromol/L, (46 +/- 6)%] were obviously higher than those in group B [(34 +/- 5) micromol/L, (26 +/- 5)%, P < 0.01]. The NO content produced by PMphi (45 +/- 10 micromol/L) in A group decreased (P < 0.01), while the TNF-alpha activity (58 +/- 10)% increased by PT pretreatment (P < 0.05). On the contrary, the NO content produced by PMphi (105 +/- 18 micromol/L) in group A increased (P < 0.01), while the TNF-alpha activity (27 +/- 6)% decreased by CT pretreatment (P < 0.01).

CONCLUSIONThese results indicates that the secretory function of normal PMphi can be enhanced by complement activation after thermal injury, which might partly be due to the effect of activated complement components through complement receptor coupled G-protein. In the secretory function of complement stimulated Mphi, Gi protein has a major role in the production of NO, Gs protein is mainly involved in the secretion of TNF-alpha.

Animals ; Burns ; immunology ; metabolism ; Complement Activation ; Complement System Proteins ; metabolism ; Female ; GTP-Binding Proteins ; metabolism ; Macrophage Activation ; immunology ; Macrophages, Peritoneal ; secretion ; Male ; Mice ; Mice, Inbred Strains ; Nitric Oxide ; biosynthesis ; Signal Transduction ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo study the effect of Angelica sinensis, Salvia miltiorrhiza and Ligustrazine on function of peritoneal macrophages during peritoneal dialysis.

METHODSPeritoneal macrophages of mice were cultured in culture medium (control), peritoneal dialysate (PD), drugs contained PD containing Angelica, Salvia and Ligustrazine combined (PD-ASL) or separated (PD-A, PD-S, PD-L) with concentration of 2 micrograms/ml, 10 micrograms/ml and 100 micrograms/ml, separately for 24 hrs. The nitric oxide (NO) content, methyl thiazolyl tetrazolium (MTT) reducing capacity (MTT-RC) and phagocytosis capacity of macrophages were determined and compared.

RESULTSNO content and MTT-RC of macrophages cultured in PD group were significantly lower than those of the control (P < 0.01), as compared with those in drug contained PD groups, the NO content in the PD-L group and the MTT-RC in the PD-ASL group were higher significantly (P < 0.01). The phagocytosis capacity and NO content in the PD-ASL group were raised along with the increased concentration of drug in PD.

CONCLUSIONAdministering Chinese herbal medicine during peritoneal dialysis has important significance in improving the defense function of peritoneal macrophages, reducing the incidence of peritonitis and enhancing the therapeutic effect of peritoneal dialysis.

Angelica sinensis ; Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Macrophages, Peritoneal ; cytology ; immunology ; Male ; Mice ; Nitric Oxide ; metabolism ; Peritoneal Dialysis ; adverse effects ; Phagocytosis ; drug effects ; Phytotherapy ; Pyrazines ; pharmacology ; Salvia miltiorrhiza

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

OBJECTIVETo study the adjustment of Xianggui pill on the cytokine of endometriosis model rat, and investigate the mechanism of Xianggui pill on the treatment of endometriosis.

METHODTo set up endometriosis model by rat self-endometria transplantation, drench sodium chloride, Xianggui pill elixation or Danazol after grouping, and to detect the contents of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) by ELISA.

RESULTThe contents of IL-8, TNF-alpha in the peripheral blood and peritoneal fluid of model group were higher than that of the blank group; The quality of allotopia growth intima tissue, the quantity of macrophage in peritoneal fluid and the contents of IL-8, TNF-alpha in the Xianggui pill group and Danazol group were all lower than those of the model group; but there was no significant difference of each target between the Xianggui pill group and Danazol group.

CONCLUSIONXianggui pill can restrain significantly the growth of allotopia intima tissue, and has apparently adjustment to the cytokine.

Animals ; Cell Count ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endometriosis ; blood ; immunology ; metabolism ; Female ; Interleukin-8 ; blood ; metabolism ; Macrophages, Peritoneal ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

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