Extracellular Release of CD11b by TLR9 Stimulation in Macrophages

CpG-DNA upregulates the expression of pro-inflammatory cytokines, chemokines and cell surface markers. Investigators have shown that CD11b (integrin αM) regulates TLR-triggered inflammatory responses in the macrophages and dendritic cells. Therefore, we aimed to identify the effects of CpG-DNA on the expression of CD11b in macrophages. There was no significant change in surface expression of CD11b after CpG-DNA stimulation. However, CD11b was released into culture supernatants after stimulation with phosphorothioate-backbone modified CpG-DNA such as PS-ODN CpG-DNA 1826(S). In contrast, MB-ODN 4531 and non-CpG-DNA control (regardless of backbone type and liposome-encapsulation) failed to induce release of CD11b. Therefore, the context of the CpG-DNA sequence and phosphorothioate backbone modification may regulate the effects of CpG-DNA on CD11b release. Based on inhibitor studies, CD11b release is mediated by p38 MAP kinase activation, but not by the PI3K and NF-κB activation. CD11b release is mediated by lysosomal degradation and by vacuolar acidification in response to CpG-DNA stimulation. The amount of CD11b in the exosome precipitant was significantly increased by CpG-DNA stimulation in vivo and in vitro depending on TLR9. Our observations perhaps give more insight into understanding of the mechanisms involved in CpG-DNA-induced immunomodulation in the innate immunity.     

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.     

Evidence for lipopolysaccharideinduced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells

Effect of lipopolysaccharide (LPS) on RAW264.7 macrophage cell line was studied. LPS-treated RAW264.7 cells increased in cell size and acquired distinct dendritic morphology. At the optimal dose of LPS (1 mg/ml), almost 70% RAW264.7 cells acquired dendritic morphology. Flow cytometric studies indicate that the cell surface markers known to be expressed on dendritic cells and involved in antigen presentation and T cell activation (B 7.1, B 7.2, CD40, MHC class II antigens and CD1d) were also markedly upregulated on LPS-treated RAW 264.7 cells. Our results suggest the possibility that LPS by itself could constitute a sufficient signal for differentiation of macrophages into DC-like cells.     

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion,Phagocytosis, and Cytokine Modulation in Macrophages

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.     

TMAO通过CD147/MMPs通路诱导巨噬细胞活化

肠道菌群代谢产物氧化三甲胺(trimethylamine N-oxide,TMAO)可通过多种途径促进动脉粥样硬化(atherosclerosis,AS)的进展,现研究发现,在临床上其与斑块稳定性存在密切联系,但其分子机制目前尚不明确.金属蛋白酶诱导因子(extracellular matrix metalloprot...     

Cytokine-mediated induction of metallothionein in Hepa-1c1c7 cells by oleanolic acid

Oleanolic acid (OA), a pentacyclic triterpene acid, has been reported to possess inducing activity of hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of OA on the regulation of MT expression in an in vitro model. OA that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the OA-treated conditioned media from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with OA and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in a decrease in the effects of OA on the MT induction. In the OA-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. However, the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the OA-treated conditioned media from the RAW 264.7 cells. These results suggest that the up-regulation of MT expression by OA was mediated by the TNF-alpha and IL-6 released from UA-activated macrophages.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

The glucosamine-mediated induction of CHOP reduces the expression of inflammatory cytokines by modulating JNK and NF-κB in LPS-stimulated RAW264.7 cells

Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles in inflammation in early-stage rheumatoid arthritis (RA). This study investigated whether glucosamine hydrochloride (GlcN), a nonsteroidal anti-inflammatory agent (NSAID) widely used to treat arthritis, affects the expression of inflammatory cytokines via the unfolded protein response (UPR) in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells). Pretreatment with GlcN reduced the expression of inflammatory cytokines and inhibited cell differentiation. Moreover, GlcN treatment increased the expression of CHOP and BiP/Grp78, the UPR target genes, in the presence or absence of LPS. Indeed, knockdown of CHOP using siRNAs prevented the GlcN-mediated reduction of inflammatory cytokines in LPS-stimulated RAW264.7 cells. Finally, we found that GlcN-mediated induction of CHOP reduced the phosphorylation of JNK and NF-?B in LPS-stimulated RAW264.7 cells. Combined, these results suggest that the GlcN-mediated induction of CHOP negatively regulates the inflammatory response by modulating JNK and NF-?B in LPS-stimulated RAW264.7 cells.     

The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

Inflammatory response plays an important role in ischaemia reperfusion injury (IRI) through a variety of inflammatory cells. Apart from neutrophils, macrophages and lymphocytes, the role of dendritic cells (DCs) in IRI has been noticed. The study was aimed at investigating whether the high‐mobility group protein box‐1/toll like receptor 4 (HMGB1/TLR4) signalling pathway regulate the migration, adhesion and aggregation of DCs to the myocardium, induce DCs activation and maturation, stimulate the expression of surface costimulatory molecules and participate in myocardial IRI. In vivo, migration, adhesion, and aggregation of DCs was enhanced; the expression of peripheral blood DCs CD80 and CD86, myocardial adhesion molecules were increased; and the infarct size was increased during myocardial ischaemia reperfusion injury myocardial ischemic/reperfusion injury (MI/RI). These responses induced by MI/RI were significantly inhibited by HMGB1 specific neutralizing antibody treatment. Cellular experiments confirmed that HMGB1 promoted the release of inflammatory cytokines through TLR4/MyD88/NF‐κB, upregulated CD80 and CD86 expression, mediated the damage of cardiomyocytes and accelerated the apoptosis. Our results indicate that DCs activation and maturation, stimulate the expression of surface costimulatory molecules by promoting the release of inflammatory factors through NF‐κB pathway and participate in myocardial IRI.     

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.     

Histone deacetylase inhibition by sodium valproate regulates polarization of macrophage subsets

Recent studies suggest that change of macrophage phenotype (M1/M2) is associated with autoimmune diseases. Sodium valproate (VPA) is a class I histone deacetylase (HDAC) inhibitor, which has immunomodulatory function in graft-versus-host disease. However, its impact on macrophage polarization has not been defined. We evaluated the effects of VPA on both mouse macrophage cell line RAW264.7 and primary mouse bone marrow macrophages (BMMs). Exposure to VPA significantly repressed the production of interleukin 12 (IL-12), and tumor necrosis factor α by lipopolysaccharide (LPS)-induced macrophage activation, in contrast, promoted IL-10 expression. VPA also affected the costimulatory molecule expression on LPS-stimulated RAW264.7 and BMMs (downregulation of CD40 and CD80, and upregulation of CD86). Specifically, VPA inhibited macrophage-mediated T helper 1 (Th1) effector but enhanced Th2 effector cell activation. Together, our preclinical study demonstrates that VPA significantly affects the phenotype and function of macrophage, indicating an important role of HDAC activity in immune regulation and inflammation. It also provides a rationale to evaluate VPA activity for the treatment of macrophage dysfunction-associated diseases.     

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.     

Curcumin enhances non-inflammatory phagocytic activity of RAW264.7 cells

Present study was performed to assess the effect of curcumin treatment on macrophage functions using RAW264.7 cells, a murine macrophage cell line. Phagocytic activity of RAW264.7 cells was enhanced by the treatment with curcumin for 48 h while the nitric oxide synthesis from RAW264.7 cells following lipopolysaccharide exposure was blocked. The incubation of RAW264.7 cells with curcumin dose-dependently inhibited the stimulatory responses of macrophage triggered by lipopolysaccharide; the enhanced secretion of inflammatory cytokines such as TNF-α and IL-1β and the up-regulated expression of surface antigens like CD14 and CD40. Curcumin alone, however, was able to increase the basal level of TNF-α secretion and elevated markedly the expression of CD14 and slightly CD40. The marked enhancement of both phagocytic activity and CD14 was detectable as early as 75 min after curcumin treatment which is the minimum time period required for the phagocytosis and CD14 measurement, suggesting a signaling pathway distinct from that triggered by apoptotic cells. In conclusion, this study elucidates that curcumin treatment enhances the phagocytic activity with blocking nitric oxide synthesis, a scavenger function of macrophages in non-inflammatory condition. In addition, this enhancement of phagocytic activity is triggered directly by the signals from curcumin itself not by apoptotic cells.     

Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.