Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages

There is considerable evidence indicating that intracellular Ca²⁺ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca²⁺ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca²⁺ imaging suggested that the LPS-induced increase in [Ca²⁺]_i involves both the TRPV2-mediated intracellular and extracellular Ca²⁺ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca²⁺ mobilization from intracellular Ca²⁺ store and extracellular Ca²⁺. In addition to Ca²⁺ mobilization through the IP₃-receptor, TRPV2-mediated intracellular Ca²⁺ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca²⁺ entry is involved in NFκB-independent IL-6 production.     

LPS suppresses expression of asialoglycoprotein-binding protein through TLR4 in thioglycolate-elicited peritoneal macrophages

Macrophages are known to express various types of endocytosis receptors that mediate the removal of foreign pathogens. Macrophage asialoglycoprotein-binding protein (M-ASGP-BP) is a Gal/GalNAc-specific lectin, which functions as an endocytosis receptor. We found here that LPS is able to down-regulate the mRNA expression of M-ASGP-BP in a time-dependent manner using thioglycolate-elicited rat and mouse peritoneal macrophages. However, LPS does not modulate the mRNA expression of M-ASGP-BP from macrophages of C3H/HeN mice, which have a point mutation of TLR4, the primary LPS receptor. Furthermore, an inhibitor of NF-κB was observed to efficiently block the suppressive effect of LPS on M-ASGP-BP as well as to inhibit the phosphorylated IκB. These results demonstrate that the mRNA expression of M-ASGP-BP is down-regulated by the LPS-mediated TLR4 pathway involving NF-κB activation, suggesting that engagement of M-ASGP-BP by LPS may yield a negative signal that interferes with the LPS-induced positive signals mediated by proinflammatory cytokines.     

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow–derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1β, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.     

Expression of TREM-1 is inhibited by PGD2 and PGJ2 in macrophages

TREM-1 is a superimmunoglobulin receptor present on neutrophils and monocytes, which plays an important role in the amplification of inflammation. The natural ligands for TREM-1 have not been identified; however, Toll-like receptor ligands are known to induce the expression of TREM-1. Blockade of TREM-1 has shown to improve survival in animal models of sepsis. In the present studies, we investigated the role of lipid mediators in the expression of TREM-1. In a macrophage cell line, we show that the expression of TREM-1 in response to LPS and bacteria Pseudomonas aeruginosa is inhibited by PGD₂ and cyclopentanone prostaglandins PGJ₂ and 15-dPGJ₂. The inhibition of TREM-1 by these prostaglandins is independent of the PGD₂ receptors and PPARγ but occurs by activation of Nrf2 and inhibition of NF-κB. Our data suggest a novel mechanism by which these prostaglandins exhibit anti-inflammatory effects and a new therapeutic approach to inhibition of TREM-1.     

Effect of liposomally encapsulated MTX-DMPE conjugates upon TNFα and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages

The ability of liposomally encapsulated preprations of methotrexate (MTX) and three of its lipophilic derivatives (MTX-γ-DMPE, MTX-α-DMPE and MTX-α,γ-diDMPE) to alter mediator release by lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages (PMΘ) was investigated. The viability of these macrophages when incubated with approximately 6.0 nmol/10⁵ cells of the respective liposomal preparations (MTX-LIPO, MTX-γ-LIPO, MTX-α-LIPO and MTX-di-LIPO) for 20 h was greater than 80%. Treatment of macrophages, which had been incubated with MTX-α-LIPO (5.5 nmol/10⁵ cells), MTX-γ-LIPO (6.9 nmol/10⁵ ± 9.6%, 80.6 ± 5.6% and 91 ± 11.4% phagocytosis respectively (mean ± S.E.M.). At similar concentratio MTX-α-LIPO MTX-γ-LIPO and MTX-di-LIPO (6.5 nmol/10⁵ cells), PGE₂ release from LPS-stimulated rat peritoneal macrophages was inhibited by 85.3% ± 3.7%, 68.7 ± 0.6% and 88.8 ± 2.2% respectively (mean ± S.E.M., n = 4). Incubation of these macrophages with 12, 10 and 9.4 nmol/10⁵ cells of the respective liposomal preparations resulted in 89 ± 3.3%, 62 ± 5.5% and 85 ± 3.9% inhibition of TNF_α release (rmmean ± S.E.M., n = 4). However, at this concentration MTX-di-LIPO was toxic. Neither MTX (20?2.5 nmol/10⁵ cells) nor MTX-LIPO (5.6 nmol/10⁵ cells) affected TNF_α release from LPS-stimulated macrophages. Whilst free MTX wasl also ineffective at inhibiting PGE₂ from these cells, incubation with MTX-LIPO at the above concentration resulted in 76.9 ± 2.6% inhibition of the prostaglandins release.     

Differential effect of Cryptococcus neoformans on the production of IL-12p40 and IL-10 by murine macrophages stimulated with lipopolysaccharide and gamma interferon

In the present study, we examined the in vitro effect of Cryptococcus neoformans on the production of interleukin-12 (IL-12) and IL-10 by murine macrophages. At a dose of 1 x 10(5), 1 x 10(6) or 1 x 10(7) ml-1, a highly virulent strain of C. neoformans (strain YC-11) suppressed the production of IL-12p40 by a murine macrophage cell line, J774.1 stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma, while the production of IL-10 was not inhibited, but rather slightly augmented. The suppression of IL-12p40 production did not change by neutralizing anti-IL-10 mAb. A direct contact of C. neoformans with macrophages was largely involved in this inhibitory effect, since placement of a 0.45 micron pore membrane between the organism and macrophages prevented such effect. On the other hand, the culture supernatant of YC-11 did not inhibit macrophage IL-12p40 production when used at a lower dose, which contained an equivalent amount of capsular polysaccharide to that in the supernatant of YC-11 cultured at 1 x 10(5) or 1 x 10(6) ml-1, although it showed a small suppression at higher doses. Our results suggest that C. neoformans may suppress the induction of Th1 responses by inhibiting macrophage IL-12 production predominantly through a direct contact-dependent mechanism and to a lesser extent by a certain soluble factor(s) released from this microorganism.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

A novel α-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. α-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production. Consistent with these findings, α-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. α-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit. Furthermore, α-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel α-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.     

Role of TLR1 and TLR6 in the host defense against disseminated candidiasis

Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers with TLR2. The role of TLR1 and TLR6 for the recognition of the fungal pathogen Candida albicans was investigated. TLR1 is not involved in the recognition of C. albicans, and TLR1 knock-out (TLR1-/-) mice showed a normal susceptibility to disseminated candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between Th1 and Th2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10 and an increased IFN-gamma release. Production of the monocyte-derived cytokines tumor necrosis factor, IL-1, and IL-6 was normal in TLR6-/- mice, and this was accompanied by a normal susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of C. albicans and modulates the Th1/Th2 cytokine balance, but this results in a mild phenotype with a normal susceptibility of TLR6-/- mice to Candida infection.     

Modulation of cytokine production and enhancement of cell viability by TLR7 and TLR9 ligands during anthrax infection of macrophages

Inhalation of Bacillus anthracis, a bioterrorism agent, results in a high mortality rate despite appropriate antibiotic therapy. Macrophages appear to be a key factor in B. anthracis pathogenesis. The burst of pro-inflammatory cytokines from macrophages could be a major cause of death in anthrax. However, preactivation of Toll-like receptors (TLRs) could modify the host response. TLR ligands stimulate the release of activating cytokines but may also down-modulate the subsequent deleterious cytokine response to pathogens. We developed a cell culture model to measure macrophage responses to B. anthracis spores and bacilli. We found that germination from spores to bacilli produced a substantial stimulus for the secretion of the cytokines IL-6, TNF-alpha, IL-10, and IL-12 p40. Our studies showed that pretreatment of mouse macrophages with the TLR9 ligand ISS-1018, or the TLR7 ligands R-848 and IT-37, results in a substantial decrease in the subsequent secretion of IL-6 and TNF-alpha in response to B. anthracis infection of macrophages. Furthermore, the TLR7 and TLR9 ligands significantly decreased anthrax-induced cytotoxicity in the macrophages. These findings suggest that TLR ligands may contribute to the enhancement of innate immunity in B. anthracis infection by suppressing potentially deleterious pro-inflammatory cytokine responses and by improving macrophage viability.     

Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts

Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.     

Neutralization of interleukin (IL)-10 released by monocytes/macrophages enhances the up-regulatory effect of monocyte/macrophage-derived IL-6 on expressions of IL-6 and MUC1, and migration in HT-29 colon cancer cells

The interactions between monocyte-derived IL-6 and IL-10 in colon cancer are unknown. We continued previous work that showed monocyte/macrophage-derived IL-6 induces IL-6 and MUC1 expression in HT-29 cancer cells, and evaluated if IL-10 present in monocyte/macrophage is involved in this IL-6-mediated effect. We treated HT-29 cells with monocyte/macrophage supernatant following neutralization of monocyte/macrophage-released IL-10. Neutralization markedly enhanced monocyte/macrophage-derived IL-6 effects on HT-29 cells including IL-6 and MUC1 production and cell migration. Double blocking of IL-6 and IL-10 in monocyte/macrophage supernatants abolished this enhancement. Western blot analysis of STAT3 phosphorylation showed that this augmented response in HT-29 cells following IL-10 neutralization is probably mediated through enhanced IL-6-induced phosphorylation (Tyr⁷⁰⁵) of STAT3 proteins. Therefore, monocytes/macrophages have the capacity to release the functionally associated cytokines IL-6 and IL-10 whose interactions can account for the pathogenesis and progression of colon cancer.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Prostaglandin E2 production by rat peritoneal macrophages: role of cellular and humoral factors in vivo in transfusion-associated immunosuppression

Abstract The transfusion of blood is associated with long-term immunosuppression, which has been postulated to influence immunosurveillance and cancer cell killing. The mononuclear phagocyte synthesises large quantities of PGE₂, and PGE₂ has been shown to inhibit the activity of a range of immunocompetent cell types. The role of mononuclear phagocyte PGE₂ synthesis in transfusion-associated immunosuppression, and the elements of transfused blood which control this immunosuppression, were investigated using a transfused rat model. A significant increase in macrophage PGE₂ synthesis was detected 7 days after transfusion with blood and serum. The storage of blood for 24 h increased the stimulatory activity of transfused blood. The effects of storage and serum on macrophage PGE₂ synthesis were greater than effects due to genetic differences between blood donor and recipient, and the serum effects indicated that a major factor activating PGE₂-mediated immunosuppression in transfused subjects may be humoral in nature.