The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors

The physiological function of the adaptor protein TRADD remains unclear because of the unavailability of a TRADD-deficient animal model. By generating TRADD-deficient mice, we found here that TRADD serves an important function in tumor necrosis factor receptor 1 (TNFR1) signaling by orchestrating the formation of TNFR1 signaling complexes. TRADD was essential for TNFR1 signaling in mouse embryonic fibroblasts but was partially dispensable in macrophages; abundant expression of the adaptor RIP in macrophages may have allowed some transmission of TNFR1 signals in the absence of TRADD. Although morphologically normal, TRADD-deficient mice were resistant to toxicity induced by TNF, lipopolysaccharide and polyinosinic-polycytidylic acid. TRADD was also required for TRIF-dependent Toll-like receptor signaling in mouse embryonic fibroblasts but not macrophages. Our findings definitively establish the biological function of TRADD in TNF signaling.     

Lipoteichoic acid downregulates FcepsilonRI expression on human mast cells through Toll-like receptor 2

BACKGROUND: FcepsilonRI on the surface of mast cells (MCs) plays a central role in allergic responses. Recent evidence shows that exposure to microbial components corresponds with a significant reduction in the risk for allergic diseases. Although many reports suggest that this is due to changes in T-cell functions, how MC functions are altered by bacterial infection remains unknown. OBJECTIVE: We sought to elucidate the effect of bacterial infection on MC function and expression of Fc receptors, such as FcepsilonRI. METHODS: Isolated human pulmonary MCs and a human MC line (LAD2) were stimulated with bacterial components, and the function and surface expression of Fc receptors were measured. RESULTS: Lipoteichoic acid (LTA) and peptidoglycan, but not LPS, flagellin, or 3CpG-oligodeoxynucleotide, reduced the expression of FcepsilonRI on LAD2 cells. An antibody to Toll-like receptor (TLR) 2 partially blocked the effect of LTA but not peptidoglycan. Both LTA and peptidoglycan reduced MC degranulation caused by an antigen-specific IgE. Furthermore, exposure of pulmonary MCs to LTA reduced both FcepsilonRI expression and IgE-induced degranulation. None of the bacterial components affected the expression of other Fc receptors, such as Fcgamma receptors or Fcalpha receptor I. CONCLUSIONS: Our results indicate that LTA reduces the surface expression of FcepsilonRI through TLR2 and suggests that TLR2 ligands could be used as a novel therapy for controlling allergic disorders. CLINICAL IMPLICATIONS: By knowing how bacterial components modulate MC function, we can expand our possibilities for therapeutic interventions of allergic diseases.     

Induction of tumor necrosis factor alpha by Leishmania infantum in murine macrophages from different inbred mice strains.

The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor alpha (TNF alpha) in murine macrophages. Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNF alpha production with the ability to control parasite growth and replication. Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lshr), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8:1. No significant differences in the percentages of infected peritoneal cells of Lshs versus Lshr mice were observed until 72 h of in vitro culture. On the contrary, Kupffer cells from Lshr mice inhibited Leishmania replication. Peritoneal macrophages of resistant mice produced significantly higher amounts of TNF alpha as compared to susceptible mice. TNF alpha production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite. No TNF alpha was found in supernatants of infected Kupffer cells from all the strains tested. The ability of macrophages from susceptible or resistant mice strains to produce TNF alpha after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro.     

Mycobacterium tuberculosis Rv0652 stimulates production of tumour necrosis factor and monocytes chemoattractant protein-1 in macrophages through the Toll-like receptor 4 pathway

Mycobacterial proteins interact with host macrophages and modulate their functions and cytokine gene expression profile. The protein Rv0652 is abundant in culture filtrates of Mycobacterium tuberculosis K‐strain, which belongs to the Beijing family, compared with levels in the H37Rv and CDC1551 strains. Rv0652 induces strong antibody responses in patients with active tuberculosis. We investigated pro‐inflammatory cytokine production induced by Rv0652 in murine macrophages and the roles of signalling pathways. In RAW264.7 cells and bone marrow‐derived macrophages, recombinant Rv0652 induced predominantly tumour necrosis factor (TNF) and monocyte chemoattractant protein (MCP)‐1 production, which was dependent on mitogen‐activated protein kinases and nuclear factor‐κB. Specific signalling pathway inhibitors revealed that the extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 and phosphatidylinositol 3‐kinase (PI3K) pathways were essential for Rv0652‐induced TNF production, whereas the ERK1/2 and PI3K pathways, but not the p38 pathway, were critical for MCP‐1 production in macrophages. Rv0652‐stimulated TNF and MCP‐1 secretion by macrophages occurred in a Toll‐like receptor 4‐dependent and MyD88‐dependent manner. In addition, Rv0652 significantly up‐regulated the expression of the mannose receptor, CD80, CD86 and MHC class II molecules. These results suggest that Rv0652 can induce a protective immunity against M. tuberculosis through the macrophage activation.     

Polymorphic analysis of the high-affinity tumor necrosis factor receptor 2

The tumor necrosis factor receptor 2 (TNF-RII, CD120b, TNF-R p75/80) gene has recently been characterised. It is located on chromosome 1p362 and consists of 10 exons and 9 introns A number of biallelic polymorphisms have been found in exons 4, 6, 9 and 10 based on differences between published sequences. In this study we have used polymerase chain reaction methodology in association with sequence-specific primers (PCR-SSP) incorporating mismatches at the 3' end to identify these polymorphisms. We were able to confirm the presence of a single biallelic polymorphism in exon 6 corresponding to a (T/G) at nucleotide 676 of TNF-RII mRNA (gb:M32315) which results in an amino acid change and three biallelic polymorphisms in exon 10 (in the3'UTR) corresponding to (A/G) at nucleotide 1663, (T/G) at nucleotide 1668 and a (C/T) at nucleotide 1690 of gb:M32315, whereas no polymorphisms were observed in exons 4 and 9. Here we report that in 192 unrelated UK Caucasian individuals the allele frequencies determined by direct counting were: 676-T (0.77), 1663-G (0.51), 1668-T (0.95), and 1690-T (0.64) and the calculated gene frequencies were; 676-T (0.52), 676-G (0.12); 1663-G (0.30), 1663-A (0.28); 1668-T (0.77), 1668-G (0.025); and 1690-T (0.40), 1690-C (0.20). Furthermore, the presence of an A allele at nucleotide position 1663 was found to be strongly associated with the presence of a C allele at nucleotide position 1690 and a G allele at nucleotide position 1668 whereas the presence of a G allele at position 1663 was associated with the absence of a C allele at nucleotide position 1690.     

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

TRIM21 controls Toll-like receptor 2 responses in bone-marrow-derived macrophages

TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21^−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21^−/− BMDMs had decreased expression of M-CSF signature genes. Although Trim21^−/− BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21^−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.     

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages

Streptococcus gordonii, a Gram-positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram-positive bacteria that are preferentially recognized by Toll-like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat-killed S. gordonii wild-type and an LTA-deficient mutant (ΔltaS) but not a lipoprotein-deficient mutant (Δlgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild-type and ΔltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the Δlgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild-type and ΔltaS induced NF-κB activation, STAT1 phosphorylation, and IFN-β expression, which are important for the induction of iNOS gene expression, with little activation by Δlgt. S. gordonii wild-type and ΔltaS showed an increased adherence and internalization to RAW 264.7 cells compared to Δlgt. In addition, S. gordonii wild-type and ΔltaS, but not Δlgt, substantially increased TLR2 activation while none of these induced NO production in TLR2-deficient macrophages. Triton X-114-extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF-κB and STAT1 activation.     

Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production

The receptor activator of NF-κB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-α)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-α and occurred independently of RANKL, interleukin-1β (IL-1β), and IL-6. CS fluids from P. gingivalis-stimulated TLR2^−/− macrophages failed to express TNF-α, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4^−/− macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-α production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-α-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.     

Induction of tumor necrosis factor alpha in murine macrophages with various strains of Coxiella burnetii and their lipopolysaccharides

The ability of various strains of Coxiella burnetii (C.b.) and their phase I and II lipopolysaccharides (LPSs) to induce tumor necrosis factor alpha (TNF-alpha) in peritoneal Balb/c mouse macrophages in vitro was investigated. Considerable differences in the induction ability were observed in dependence on the strain applied. In a TNF-alpha bioassay, the most effective inducers were both corpuscles and LPSs of the strains Priscilla and Scurry, followed by Nine Mile, Luga, and Henzerling I. In contrast, in ELISA, the most effective inducers were LPSs of the strains Luga and Henzerling, followed by Nine Mile, Priscilla, and Scurry. The role of toll-like receptor 4 (TLR4) in the induction was confirmed by the use of C3H/HeJ mouse macrophages. Thus, the induction of TNF-alpha was much higher in Balb/c mouse macrophages than that in TLR4-deficient C3H/HeJ mouse macrophages. Differences in the results of the bioassay and those of ELISA suggest a role of another secreted factor(s) induced with C.b. in murine macrophages that could act synergically with TNF-alpha in L929 cells in the bioassay. The observed differences in TNF-alpha induction might play a role in the pathobiology of Q fever.     

Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2^−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Role of tumor necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor 1-deficient mice

Increased gastric production of interleukin 8 and tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of Helicobacter pylori-associated gastroduodenal disease. In the present study we used a mouse model to demonstrate whether loss of the tumor necrosis factor receptor 1 (TNF-R1) function leads to differences in gastric inflammation or the systemic immune response in H. pylori infection. Six different clinical isolates of H. pylori (three cytotoxin-positive and three cytotoxin-negative strains) were adapted to C57BL/6 mice. TNF-R1-deficient (TNF-R1(-/-)) mice (n = 19) and isogenetic controls (n = 24) were infected and sacrificed after 4 weeks of infection. Inflammation of the stomach and the humoral immune response to H. pylori were evaluated by histological, immunohistochemical, and serological methods. There was no detectable difference in the grade or activity of gastritis in TNF-R1(-/-) mice when they were compared with wild-type mice, but the number of lymphoid aggregates was slightly reduced in the gastric mucosa of TNF-R1(-/-) mice. Interestingly, total immunoglobulin G (IgG), as well as IgG1, IgG2b, and IgG3, H. pylori-specific antibody titers were significantly higher in wild-type mice. As revealed by immunoblot analysis, the difference in reactivity against H. pylori antigens was not based on a failure to recognize single H. pylori antigens in TNF-R1(-/-) mice. We therefore suggest that TNF-R1-mediated TNF-alpha signals might support a systemic humoral immune response against H. pylori and that the gastric inflammatory response to H. pylori infection seems to be independent of TNF-R1-mediated signals.     

Microglia and macrophages express tumor necrosis factor receptor p75 following middle cerebral artery occlusion in mice

The proinflammatory and potential neurotoxic cytokine tumor necrosis factor (TNF) is produced by activated CNS resident microglia and infiltrating blood-borne macrophages in infarct and peri-infarct areas following induction of focal cerebral ischemia. Here, we investigated the expression of the TNF receptors, TNF-p55R and TNF-p75R, from 1 to 10 days following permanent occlusion of the middle cerebral artery in mice. Using quantitative polymerase chain reaction (PCR), we observed that the relative level of TNF-p55R mRNA was significantly increased at 1-2 days and TNF-p75R mRNA was significantly increased at 1-10 days following arterial occlusion, reaching peak values at 5 days, when microglial-macrophage CD11b mRNA expression was also increased. In comparison, the relative level of TNF mRNA was significantly increased from 1 to 5 days, with peak levels 1 day after arterial occlusion. In situ hybridization revealed mRNA expression of both receptors in predominantly microglial- and macrophage-like cells in the peri-infarct and subsequently in the infarct, and being most marked from 1 to 5 days. Using green fluorescent protein-bone marrow chimeric mice, we confirmed that TNF-p75R was expressed in resident microglia and blood-borne macrophages located in the peri-infarct and infarct 1 and 5 days after arterial occlusion, which was supported by Western blotting. The data show that increased expression of the TNF-p75 receptor following induction of focal cerebral ischemia in mice can be attributed to expression in activated microglial cells and blood-borne macrophages.     

Two soluble antigens of Plasmodium falciparum induce tumor necrosis factor release from macrophages.

The production of cytokines such as tumor necrosis factor (TNF) may contribute to the pathology of malaria. We showed previously that crude preparations of heat-stable exoantigens from parasite cultures induce the release of TNF in vitro and in vivo. When separated from the culture medium by affinity chromatography, in which immune immunoglobulin G was used as ligand, the mixture of exoantigens of Plasmodium falciparum retained the capacity to induce the secretion of TNF, both by human monocytes from Gambian children and by mouse macrophages. Two individual antigens, Ag1 and Ag7, further purified by affinity chromatography and identified by crossed immunoelectrophoresis, also stimulated TNF production by both types of cell but differed in other functional properties. Thus, the activity of Ag7, but not that of Ag1, was inhibited by polymyxin B, and antisera made against boiled exoantigens of the rodent parasite Plasmodium yoelii which blocked the ability of these antigens to induce the production of TNF also inhibited the activity of Ag7 without affecting Ag1. Since the prevalence of antibody against Ag7 in sera from children in endemic areas appears to correlate with the development of immunity against the manifestations of the disease, this antigen may be one cause of pathology, perhaps through its ability to induce the production of TNF. Its serological relationship with rodent exoantigens suggests that it might be a candidate for an anti-disease vaccine which has the advantage that its active moiety is not subject to significant antigen polymorphism.     

Interleukin-6 antagonizes tumor necrosis factor-mediated mycobacteriostatic and mycobactericidal activities in macrophages.

Interleukin-6 (IL-6) is a cytokine produced by a number of cells, including macrophages, and is directly involved in the inflammatory response. The production of IL-6 can be stimulated by monokines such as IL-1 and tumor necrosis factor (TNF). Mycobacterium avium complex organisms frequently cause disseminated disease in patients with AIDS. M. avium is an intracellular bacterium that that mainly infects macrophages. Treatment of M. avium-infected macrophage monolayers with recombinant IL-6 decreased the ability of TNF to activate cultured macrophages to inhibit growth of or kill intracellular M. avium (68% +/- 14% decrease in intracellular killing compared with that in monolayers not treated with IL-6). To further evaluate whether this effect was dependent on the down regulation of membrane receptors to TNF, we examined 125I-TNF binding to macrophages previously exposed to IL-6: the expression of TNF receptors was decreased by 78% +/- 9%. The effect of IL-6 on TNF receptors was observed after 4 h and was reversible. Infection of macrophages with different M. avium serovars was associated with release of IL-6, and IL-6 production peaked at 48 h after infection in concentrations ranging from 328 +/- 87 ng/10(5) cells to 907 +/- 224 ng/10(5) cells. IL-6 did not have any influence on the rate of growth of the tested strains of M. avium within or outside macrophages. These results suggest that release of IL-6 by M. avium-infected macrophages may influence the host's immune response and the outcome of the disease.     

Substance P augments tumor necrosis factor release in human monocyte-derived macrophages.

Substance P (SP) is an undecapeptide that has the amino sequence Arg-Pro-Lys-Pro-Gin-Gln-Phe-Phe-Gly-Leu-Met-NH2 and that belongs to a family of structurally related peptides known as tachykinins, the latter are widely distributed in the central nervous system. SP is involved in the biological activities of cells in the immune system, including the induction of cytokines in immune cells. We have investigated the effects of SP on constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF) in cultured blood monocyte-derived macrophages (MDM). Cells cultured in vitro for 14 days were treated with SP at various concentrations (10(-10) to 10(-6M) in the presence of LPS before culture supernatants were harvested. TNF bioactivity in culture supernatants was measured with L929 cell line MDM from 10 of 12 donors treated with a SP alone showed increased TNF production. SP and LPS also interacted in a synergistic fashion in upregulating TNF production in MDM from responders. The stimulatory effect of SP was inhibited by two SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-7-D-Trp-9-leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). In addition, an anti SP polyclonal antibody blocked the SP effect on TNF production in cultured MDM, further indicating the specificity of these effects. These results demonstrate that SP is an important regulator of monokine production by human monocytes/macrophages.