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.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Hydrogen sulfide attenuates lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated protein kinase in microglia

The present study attempts to investigate the effect of H(2)S on lipopolysaccharide (LPS)-induced inflammation in both primary cultured microglia and immortalized murine BV-2 microglial cells. We found that exogenous application of sodium hydrosulfide (NaHS) (a H(2)S donor, 10-300 micro mol/L) attenuated LPS-stimulated nitric oxide (NO) in a concentration-dependent manner. Stimulating endogenous H(2)S production decreased LPS-stimulated NO production, whereas lowering endogenous H(2)S level increased basal NO production. Western blot analysis showed that both exogenous and endogenous H(2)S significantly attenuated the stimulatory effect of LPS on inducible nitric oxide synthase expression, which is mimicked by SB 203580, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. Exogenously applied NaHS significantly attenuated LPS-induced p38 MAPK phosphorylation in BV-2 microglial cells. Moreover, both NaHS (300 micro mol/L) and SB 203580 (1 micro mol/L) significantly attenuated LPS-induced tumor necrosis factor-alpha secretion, another inflammatory indicator. In addition, NaHS (10-300 micro mol/L) dose-dependently decreased LPS-stimulated NO production in primary cultured astrocytes, suggesting that the anti-neuroinflammatory effect of H(2)S is not specific to microglial cells alone. Taken together, H(2)S produced an anti-inflammatory effect in LPS-stimulated microglia and astrocytes, which may be due to inhibition of inducible nitric oxide synthase and p38 MAPK signaling pathways. These findings may have important implications in the treatment of neuroinflammation-related diseases.     

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Aims

Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Main methods

RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently.

Key findings

All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.

Significance

Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.     

Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.     

Anti-inflammatory effects of recombinant arginine deiminase originating from Lactococcus lactis ssp. lactis ATCC 7962

Arginine deiminase (ADI, E.C. 3.5.3.6), one of the arginine deprivation enzymes, exhibits anticarcinogenic activities. The present study investigated the anti-inflammatory activities of the purified recombinant ADI originating from Lactococcus lactis ssp. lactis ATCC7962 (LADI). LADI dose-dependently inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase and the production of nitric oxide in RAW 264.7 murine macrophages. The induction of cyclooxygenase-2 expression and subsequent production of prostaglandin E2 by LPS was also attenuated by LADI treatment. Moreover, LADI inhibited the production of interleukin-6 in LPS-stimulated RAW 264.7 macrophages. These results indicate that LADI exerts anti-inflammatory effects, which may in part explain its chemopreventive potential.     

Anethum graveloens flower extracts inhibited a lipopolysaccharide-induced inflammatory response by blocking iNOS expression and NF-κB activity in macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.     

Anethum graveloens Flower Extracts Inhibited a Lipopolysaccharide-Induced Inflammatory Response by Blocking iNOS Expression and NF-κB Activity in Macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages

Prostaglandin E2 (PGE2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE2--either endogenous or exogenous--on NO formation in J774 cells. Supporting these findings, exogenous PGE2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE2 levels.     

IFN-gamma + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line

Nitric oxide (NO.) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO. by interferon-gamma (IFN-gamma) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gammaNO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38(mapk) inhibited IFN-gamma + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma + LPS, also implicating the c-Jun NH(2)-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38(mapk) appears more complex and may be due to the ability of p38(mapk) to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO. expression by IFN-gamma + LPS.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Murine TOLL-like receptor 4 confers lipopolysaccharide responsiveness as determined by activation of NF kappa B and expression of the inducible cyclooxygenase

Genetic evidence indicating that TOLL-like receptor 4 (Tlr4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine Tlr4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through the activation of NF kappa B. Thus, we determined whether murine Tlr4 confers LPS responsiveness as evaluated by the activation of NF kappa B and COX-2 expression. Transfection of a murine macrophage-like cell line (RAW264.7) with the constitutively active form (delta Tlr4) of Tlr4 is sufficient to activate NF kappa B and COX-2 expression. However, the truncated form (delta Tlr4(P712H)) of the missense mutant Tlr4(P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NF kappa B activation and COX-2 expression. The inability of delta Tlr4(P712H) to activate NF kappa B and induce COX-2 expression is rescued by a constitutively active adapter protein myeloid differentiation factor 88 (MyD88), which interacts directly with the cytoplasmic domain of Tlr proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild-type delta Tlr4 but not with the delta Tlr4(P712H) mutant. Together, these results indicate that Tlr4 confers LPS responsiveness in RAW264.7 cells and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is attributed to the disruption of Tlr4-mediated signaling pathways that results from the inability of the mutant Tlr4(P712H) to interact with MyD88.