Novel strategies for targeting innate immune responses to influenza.

We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1ß contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

Role of the lipoxygenase pathway in RSV-induced alternatively activated macrophages leading to resolution of lung pathology.

Resolution of severe Respiratory Syncytial Virus (RSV)-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mφ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)(-/-) and 15-LO(-/-) macrophages or mice failed to elicit AA-Mφ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO(-/-) mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mφ in vitro and, conversely, treatment of 5-LO(-/-) macrophages with downstream products, lipoxin A4 and resolvin E1, but not leukotriene B4 or leukotriene D4, partially restored expression of AA-Mφ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also decreased lung pathology in RSV-infected 5-LO(-/-) mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mφ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mφ differentiation by activating the 5-LO pathway.

Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R alpha-, TLR4-, and IFN-beta-dependent.

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed "Th1" and "Th2" cytokine storm. We hypothesized that differentiation of "alternatively activated" macrophages (AA-M phi) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4R alpha/STAT6-dependent AA-M phi differentiation, and significantly enhanced inflammation in the lungs of IL-4R alpha(-/-) mice. Adoptive transfer of wildtype macrophages to IL-4R alpha(-/-) mice restored RSV-inducible AA-M phi phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4(-/-) and interferon (IFN)-beta(-/-) macrophages and mice also failed to express AA-M phi markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptor gamma expression, a DNA-binding protein that induces AA-M phi genes, whereas IFN-beta regulates IL-4, IL-13, IL-4R alpha, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-M phi. These data suggest new treatment strategies for RSV that promote AA-M phi differentiation.

Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo.

Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.

Toll-like receptor 4 signalling: new perspectives on a complex signal-transduction problem.

We previously reported that Toll-like receptor-2 (TLR2) agonists induce expression of a more limited repertoire of pro-inflammatory genes than TLR4 agonists. Murine macrophages stimulated with the TLR4 agonist, Escherichia coli lipopolysaccharide, induced signal transducer and activator of transcription 1 ('STAT1') tyrosine phosphorylation that was secondary to the autocrine/paracrine action of interferon (IFN)-beta, an immediate early gene. In contrast, TLR2 agonists failed to activate IFN-beta gene expression. TLR4-induced IFN-beta mRNA was found to be MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll/interleukin-1 receptor domain-containing adapter protein)/Mal (MyD88-adapter-like)-dependent. In the present paper, we outline the recent controversy over the role of TIRAP/Mal in TLR2 and TLR4 signalling in the context of the current molecular tools used for such studies. Collectively, our findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.

Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1beta, IL-6, and TNF-alpha upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1beta, but not IL-6 and TNF-alpha production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with < or =100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-alpha, while stimulation of THP-1 cells with > or =1 microg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-alpha levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade I-kappaB-alpha or I-kappaB-beta proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade I-kappaB-beta only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.

Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines.

In murine macrophages, the anti-tumor agent, paclitaxel, induces expression of a wide variety of inflammatory and anti-inflammatory genes, and causes cytokine secretion via signaling pathways that overlap with those engaged by lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Using semi-quantitative RT-PCR for detection of gene expression, coupled with ELISA for the detection of secreted gene products, we analyzed the responsiveness of an extensive panel of cytokine and non-cytokine genes to induction by paclitaxel and LPS in the murine DA-3 breast cancer line. A subset of the genes examined (e.g., G-CSF, MIP-2, iNOS, and IL-1 beta, and GM-CSF) was upregulated >3-20-fold by both LPS and paclitaxel in the DA-3 cell line, while IP-10 mRNA was induced by paclitaxel, but not by LPS. In the human MDA-MB-231 breast cancer cell line, LPS also increased mRNA levels for both GM-CSF and IP-10 significantly, while, paclitaxel increased IP-10 mRNA levels with delayed kinetics and failed to induce GM-CSF mRNA. Co-cultures of murine breast cancer cells and macrophages, stimulated with IFN-gamma plus either paclitaxel or LPS, resulted in augmented release of nitric oxide. As both GM-CSF and IP-10 have been implicated in tumor rejection in vivo through either indirect actions on the host immune system or by inhibiting tumor angiogenesis, our data strengthen the hypothesis that tumor cell-derived inflammatory mediators may, in part, underlie the anti-tumor efficacy of paclitaxel in breast cancer.

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4.

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.

Pro- and anti-inflammatory gene expression in the murine small intestine and liver after chronic exposure to alcohol.

BACKGROUND: Endotoxin has been proposed to play a primary role in ALD, by initiating an inflammatory cascade within the liver. Although the source of these cytokines has been presumed to be circulating monocytes or tissue macrophages, ethanol-induced, nonhepatic sources of soluble mediators recently have been identified. One potential, but not clearly defined, extrahepatic source of cytokines in ALD is the intestine. In the current study, we hypothesized that alcohol would alter cytokine expression within the small intestine of mice exposed to ethanol and that LPS would alter levels of cytokine expression even more dramatically. METHODS: Mice were fed a modified Lieber-DeCarli liquid ethanol or control diet for up to 14 days prior to injecting either saline or LPS. Plasma alanine aminotransferase (ALT) and cytokine levels, histology, and RT-PCR of pro- and anti-inflammatory cytokine gene expression were determined from distal ileum and liver samples. Translocation of intestinal bacterial flora also was assessed. RESULTS: Ethanol exposure upregulated basal gene expression of IL-1 beta, TNF-alpha, IL-6, and iNOS in the distal ileum, but similar effects of ethanol on the liver were not observed. In contrast, LPS challenge of ethanol-exposed mice increased intestinal gene expression of some cytokines, but decreased expression of others. These effects were not associated with bacterial translocation. Also, ethanol alone induced a modest increase in both ICAM-1 and TLR4 mRNA expression in the intestine, but expression of both molecules was inhibited in mice that received both ethanol and LPS. Finally, whereas basal levels of hepatic IL-11 mRNA were not elevated by exposure to ethanol, intestinal IL-11 mRNA levels were increased more than 100-fold. CONCLUSIONS: These studies are the first to show that ethanol affects cytokine gene expression in the ileum and identifies the ileum as a potential target for ethanol effects. In addition, our results suggest that IL-11 expression may be enhanced in the intestine to help repair or protect this organ from alcohol-induced damage. Collectively, these studies suggest that both pro- and anti-inflammatory soluble mediators in the intestine maintain and exacerbate the local hepatic response to ethanol.

Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses.

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.

Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression.

Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.

Measurement of tumor necrosis factor alpha and beta.

Tumor necrosis factor (TNF) a and b are multifunctional cytokines elaborated primarily by monocytes and macrophages (TNF-a) or T cells (TNF-b). Some cells that bind TNF-a or -b on their surface receptors lyse as a consequence of the binding reaction. This unit presents a protocol that employs TNF-sensitive, actinomycin D-treated murine L929 fibroblasts to quantify TNF activity in supernatants derived from cell cultures, serum samples, or cerebral spinal fluid. While the assay can measure picogram concentrations of human, rat, and murine TNF-ab, it cannot distinguish between the a and b forms of any species. A describes propagation and preparation of L929 fibroblasts.

Measurement of antiviral activity induced by interferons alpha, beta, and gamma.

The Basic Protocol in this unit describes an assay for murine IFN-induced antiviral activity and employs vesicular stomatitis virus (VSV) and IFN-sensitive fibroblasts. Support Protocol describes the preparation of VSV cultures and the calculation of multiplicity of infection (MOI; i.e., concentration of viral particles required to infect cells). Support Protocol describes the antibody neutralization assay, which can be used to identify the bioactive species of IFN in a sample, or to test the potency of an antibody preparation against a particular species of IFN. Alternate Protocol 1 covers measurement of human IFN-induced antiviral activity; while the steps are quite similar to Basic Protocol, different viral and cell cultures are described.

Impaired IFN-gamma production in IFN regulatory factor-1 knockout mice during endotoxemia is secondary to a loss of both IL-12 and IL-12 receptor expression.

Mice with a targeted mutation in the gene that encodes the transcription factor IFN regulatory factor-1 (IRF-1) were used to assess the contribution of IRF-1 to IL-12-dependent and IL-12-independent pathways of IFN-gamma production. In response to LPS, IRF. 1-/- mice produced less IL-12 p40, IL-12 p35, and IFN-gamma mRNA in the liver than IRF-1+/+ mice. While pulmonary IFN-gamma mRNA levels were also mitigated in IRF-1-/- mice, pulmonary IL-12 p40 and IL-12 p35 mRNA were not dysregulated. Circulating IL-12 p70 and IFN-gamma levels were profoundly attenuated in LPS-challenged IRF-1-/- mice. Further analysis revealed a major deficiency in hepatic IL-12Rbeta1 and IL-12Rbeta2 mRNA expression as well as pulmonary IL-12Rbeta1 mRNA expression in LPS-challenged IRF-1-/- mice. In vitro, IFN-gamma up-regulated IL-12Rbeta1 mRNA in macrophages from IRF-1+/+, but not IRF-1-/-, mice. IFN-gamma-induced IL-12Rbeta2 mRNA expression was also diminished in macrophages from IRF-1-/- mice. In contrast to IRF-1+/+ mice, administration of exogenous IL-12 to IRF-1-/- mice resulted in reduced serum IFN-gamma and hepatic and pulmonary IFN-gamma mRNA, demonstrating that loss of IL-12R results in diminished IL-12 responsiveness. While LPS-challenged IRF-1-/- mice also had reduced IL-15 mRNA levels, serum IL-18 responses were intact. Finally, induction of IRF-1 mRNA by LPS in livers of IFN-gamma knockout mice were markedly attenuated, suggesting a feedback amplification loop. These studies indicate that IRF-1 deficiency disrupts both IL-12-dependent and -independent pathways of IFN-gamma production and that IRF-1 is a critical transcription factor involved in the regulation of not only IL-12, but also IL-12R.

Inhibition of LPS-induced cytokines by Bcl-xL in a murine macrophage cell line.

The antiapoptotic molecule Bcl-xL has been implicated in the differentiation and survival of activated macrophages in inflammatory conditions. In this report, the role of Bcl-xL in LPS-induced cytokine gene expression and secretion was studied. Bcl-xL-transfected RAW 264 macrophages were protected from gliotoxin-induced apoptosis, indicating the presence of functional Bcl-xL. Overexpression of Bcl-xL in this macrophage cell line was also associated with a marked inhibition of LPS-induced TNF-alpha, JE/monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 secretion. Inhibition of LPS-induced cytokine secretion was paralleled by a decrease in levels of steady-state mRNA for the above cytokines and for IL-1beta. Decreased production of TNF-alpha in Bcl-xL transfectants was not due to increased mRNA degradation, as the mRNA half-lives were the same in Bcl-xL transfectants and control macrophages. Although the composition of NF-kappaB complexes detected by EMSA and supershift analysis in nuclear lysates derived from Bcl-xL transfectants and control cells was indistinguishable, LPS-induced inhibitory kappaBalpha degradation, as well as NF-kappaB binding and AP-1 activation, were slightly decreased by ectopic expression of Bcl-xL. More strikingly, LPS-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase was strongly repressed by Bcl-xL overexpression, offering a possible mechanism for the inhibition of LPS-induced cytokine production. These data provide the first evidence for a novel role for Bcl-xL as an anti-inflammatory mediator in macrophages.

Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2.

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as "endotoxin protein," suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Interferon regulatory factor (IRF)-1 and IRF-2 regulate interferon gamma-dependent cyclooxygenase 2 expression.

Cyclooxygenases (Cox) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. Cox-2 is the inducible isoform that is upregulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. In this study, we demonstrate that interferon (IFN)-gamma alone or in synergy with lipopolysaccharide (LPS) or interleukin 1alpha induces Cox-2 expression in mouse peritoneal macrophages, which is paralleled by changes in Cox-2 protein levels and prostaglandin E(2) (PGE(2)) release. Induction of Cox-2 was abrogated in macrophages that lack IFN regulatory factor (IRF)-1, consistent with an attenuated hepatic mRNA response in IRF-1(-/-) mice injected with LPS. Conversely, the absence of IRF-2 in macrophages resulted in a significant increase in both basal and inducible Cox-2 gene and protein expression as well as IFN-gamma-stimulated PGE(2) release, identifying IRF-2 as negative regulator of this promoter. Two IFN stimulation response elements were identified in the mouse Cox-2 promoter that were highly conserved in the human Cox-2 gene. Both bind endogenous IRF-1 and IRF-2 and regulate transcription in an IRF-1/2-dependent manner. Our data demonstrate conclusively the importance of IFN-gamma as a direct activator and coactivator of the Cox-2 gene, and the central role of IRF-1/2 family members in this process.

Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression.

In this study, the effect of in vitro endotoxin tolerance on LPS-induced mitogen-activated protein kinase activation, transcription factor induction, and cytokine, chemokine, and Toll-like receptor (TLR) 2 and 4 gene expression, as well as the involvement of TNF and IL-1 signaling pathways in tolerance, were examined. Pretreatment of mouse macrophages with LPS inhibited phosphorylation of the extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 kinase; degradation of I-kappaBalpha (inhibitory protein that dissociates from NF-kappaB) and I-kappaBbeta; and activation of the transcription factors NF-kappaB and AP-1 in response to subsequent LPS stimulation. These changes were accompanied by suppression of LPS-induced expression of mRNA for GM-CSF, IFN-gamma-inducible protein-10, KC, JE/monocyte chemoattractant protein-1, macrophage-inflammatory protein-1beta, and macrophage-inflammatory protein-2, with concurrent inhibition of chemokine secretion. In contrast to control cells, endotoxin-tolerant macrophages exhibited an increased basal level of TLR2 mRNA, and failed to increase levels of TLR2 mRNA or to down-regulate TLR4 gene expression upon restimulation with LPS. As judged by transcription factor activation, LPS and IL-1 were found to induce a state of cross-tolerance against each other, while no such reciprocal effect was seen for LPS and TNF-alpha. In addition, macrophages from TNFR I/II double knockout mice were LPS tolerizable, and blocking of endogenous TNF-alpha with TNFR-Fc fusion protein did not affect the capacity of LPS to tolerize macrophages. These data extend our understanding of LPS-signaling mechanisms that are inhibited in endotoxin-tolerized macrophages and suggest that endotoxin tolerance might result from impaired expression and/or functions of common signaling intermediates involved in LPS and IL-1 signaling.