Interleukin-1β up-regulates tumor necrosis factor receptors in the mouse airways

Cytokines like interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), released during the inflammatory process, play important roles in the development of airway hyperresponsiveness. The effects of these cytokines are mediated by cell surface receptors, specific for each cytokine. The expression of cytokine receptors is a dynamic process, where receptors can be up- or down-regulated in response to changes in the environment. One such environmental factor is the presence of cytokines per se. The present study was designed to evaluate the effects of IL-1β on the expression of its corresponding receptor IL-1 RI, as well as on the closely related TNFα receptors TNF RI and TNF RII in airways using a mouse organ culture assay and intranasal inoculation model. Immunohistochemical staining was used to quantify expressional differences between fresh and cultured tracheal segments. In the fresh, uncultured, segments, IL-1 RI and TNF RI were seen in the epithelial layer and TNF RI in the smooth muscle layer. After 4 days of culture, the expression of TNF RI decreased in the epithelial layer, whereas the corresponding expression of IL-1 RI and TNF RI in the smooth muscle remained unchanged. When culture was performed in the presence of IL-1β, the expression of IL-1 RI and TNF RI in the epithelial cells and TNF RI in the smooth muscle cells increased. TNF RII was not detected in either fresh or cultured trachea, but after treatment with IL-1β an expression was found in both the epithelial layer and in the smooth muscle cells. The IL-1β-induced increased expression, on TNF RI and TNF RII in the smooth muscle ex vivo and in the lung parenchyma after intranasal challenge in vivo, was verified at the mRNA level using real-time RT PCR. To summarize, presence of IL-1β increases the expression of IL-1 R1 and TNF RI and induces expression of TNF RII in the airway wall. It is not inconceivable that these alterations of the IL-1 and TNF receptors may have important functional implications for the development of hyperresponsiveness in inflammatory airway diseases like asthma.     

Interleukin-6 receptor shedding is enhanced by interleukin-1beta and tumor necrosis factor alpha and is partially mediated by tumor necrosis factor alpha-converting enzyme in osteoblast-like cells

OBJECTIVE: Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process. METHODS: Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding. RESULTS: IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells. This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA. Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels. CONCLUSION: IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation.     

Plasma levels of tumor necrosis factor alpha and soluble tumor necrosis factor receptors in patients with narcolepsy

BACKGROUND: Narcolepsy is a disabling sleep disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis. Recent studies suggest that the immune system might play a pathogenic role pointing to a possible involvement of inflammatory cytokines. METHODS: We investigated a sample of 30 patients with narcolepsy in comparison with 120 sex- and age-matched and 101 sex-, body mass index (BMI)-, and age-matched randomly selected normal controls. In these groups, plasma concentrations of tumor necrosis factor alpha (TNF-alpha) and its soluble receptors p55 and p75 (soluble TNF receptor [sTNF-R] p55 and sTNF-R p75) were measured using commercial enzyme-linked immunosorbent assays. RESULTS: The narcoleptic patients showed a significantly higher BMI compared with controls of the same age. Soluble TNF-R p75 levels were consistently elevated in the narcoleptic patients compared with their sex- and age-matched (P = .001) as well as sex-, BMI-, and age-matched counterparts (P = .003). Female narcoleptic patients exhibited higher sTNF-R p55 levels compared with their sex- and age-matched controls (P = .01), but this difference disappeared when comparing patients with sex-, BMI-, and age-matched normal controls. Tumor necrosis factor alpha levels did not differ significantly between groups. CONCLUSION: Narcoleptic patients show increased plasma levels of sTNF-R p75, suggesting a functional alteration of the TNF-alpha cytokine system, further corroborating a possible pathogenic role of the immune system in this sleep disorder.     

Circulating soluble tumor necrosis factor receptors,interleukin-2 receptors,tumor necrosis factor α, and interleukin-6 levels in rheumatoid arthritis.

Objective. To assess whether circulating concentrations of soluble tumor necrosis factor receptors (sTNFR; p55 and p75), soluble interleukin-2 receptors (sIL-2R), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) reflect clinical response and whether changes are dependent on the drug used in rheumatoid arthritis (RA) patients taking methotrexate (MTX) or azathioprine (AZA). Methods. These cytokines and soluble receptors were assessed in 20 control subjects and serially for up to 48 weeks in 61 RA patients, by bioassay (IL-6) and immunoassays (sTNFR, sIL-2R, TNFα, and IL-6). Results. Concentrations of p55 and p75, sIL-2R, and TNFα (but not IL-6) were significantly higher in RA patients than in controls. Significant decreases in sIL-2R and p55 concentrations were associated with clinical improvement and were observed in patients treated with MTX, but not AZA. Both treatments induced decreases in IL-6 concentrations, but circulating AZA (or its metabolites) appears to interfere with the measurement of IL-6 bioactivity. TNFα and p75 levels did not show significant changes. Conclusion. Measurement of circulating sIL-2R, p55, and IL-6 may be useful in the evaluation of RA disease activity and response to therapy. Interference by circulating levels of drugs must be ruled out when bioassays are used to evaluate cytokine levels.     

Differential role of tumor necrosis factor receptors in TNBS colitis

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathology of T helper 1-mediated colitis such as Crohn's disease; however, the role of its 2 receptors in mediating pathology has not been fully explored. METHODS: Trinitrobenzene sulfonic acid colitis was used to induce colitis in mice lacking each of the TNF receptors (TNFRs) and in wild-type mice. TNFR1-/- mice lost more weight, became hypothermic, and had increased mortality compared with wild-type C57Bl/6 mice. TNFR2-/- mice, however, lost less weight, had normal temperatures, and had improved survival. RESULTS: Despite the improved clinical outcomes in TNFR2-/- mice, TNFalpha levels were increased in these mice. CONCLUSIONS: TNFalpha signaling through TNFR1 is protective in the trinitrobenzene sulfonic acid mouse model of inflammatory bowel disease.     

Soluble tumor necrosis factor receptors in human inflammatory synovial fluids

Objective. To test synovial fluid (SF) for the presence of soluble fragments originating from distinct tumor necrosis factor receptors (TNF-sR55 and TNF-sR75) which bind to TNF and inhibit its biologic activity. Methods. TNF-sR55 and TNF-sR75 were measured in 62 SF samples by specific immunoassays using monoclonal antibodies. Results. Both TNF-sR were present in all of the SF tested. Their concentrations were higher in SF from patients with seropositive rheumatoid arthritis than in patients with other inflammatory arthritides. The relative amount of TNF-sR75, as compared with TNF-sR55, was higher in seropositive RA SF than in other SF. Conclusion. The balance between TNF and its specific inhibitors may be critical to the biologic outcome mediated by this cytokine.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

Interleukin-1 induced impairment in pancreatic islet oxidative metabolism of glucose is potentiated by tumor necrosis factor

Recent observations suggest that human interleukin-1 (IL-1) causes functional impairment and death of pancreatic B-cells. This action seems to be potentiated by another cytokine, tumor necrosis factor (TNF). In the present investigation, the effects of recombinant human (r)IL-1 (10, 30 and 150 pmol/l), and a combination of rIL-1 and human rTNF (25 micrograms/l), on islet glucose metabolism were examined in the presence of D-[5-3H] and D-[6-14C) glucose. The utilization of glucose was not affected by rIL-1 or rIL-1 plus rTNF. However, rIL-1 induced a 40% decrease in glucose oxidation, which was further potentiated by the addition of rTNF. rTNF alone did not impair islet glucose utilization or oxidation. It is concluded that rIL-1 induces a perturbation of islet glucose handling, mainly at the mitochondrial level. This impairment in the oxidative metabolism of glucose is further increased by the addition of rTNF.     

Interleukin-18 enhances monocyte tumor necrosis factor alpha and interleukin-1beta production induced by direct contact with T lymphocytes: implications in rheumatoid arthritis

OBJECTIVE: At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up-regulation of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha). We examined the regulatory effects of IL-18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA). METHODS: Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)-stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFalpha, IL-1beta, IL-10, and IL-18 were measured by enzyme-linked immunosorbent assay. Expression of adhesion molecules, IL-18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF-kappaB p65, phosphorylated IkappaBalpha, and phosphatidylinositol 3-kinase (PI 3-kinase) p110 was analyzed by Western blotting. RESULTS: IL-18 dose-dependently enhanced the production of IL-1beta and TNFalpha, but not IL-10, by monocytes following contact with RA synovial T cells or PHA-prestimulated T cells. NF-kappaB inhibitors N-acetyl-L-cysteine and Bay 11-7085 and PI 3-kinase inhibitor LY294002 inhibited the enhancing effects of IL-18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF-kappaB in the nucleus, phosphorylated IkappaB, and PI 3-kinase were confirmed in monocytes cocultured with PHA-prestimulated T cells, and the levels were further increased by stimulation with IL-18. Neutralizing antibody to IL-18 inhibited monocyte activation induced by direct contact with PHA-prestimulated T cells. Via cell-cell contact, PHA-prestimulated T cells increased autocrine production of IL-18 by monocytes, which was mediated by activation of the NF-kappaB and PI 3-kinase pathways, and up-regulated the expression of the IL-18 receptor in monocytes. IL-18 up-regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on monocytes. Blocking the binding of the TNF receptors VCAM-1 or ICAM-1 on monocytes to their ligands on stimulated T cells suppressed the IL-18-enhanced production of TNFalpha and IL-1beta in monocytes induced by contact with PHA-prestimulated T cells. CONCLUSION: IL-18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF-kappaB and PI 3-kinase pathways. IL-18 up-regulates the expression of the TNF receptors VCAM-1 and ICAM-1 on monocytes, which mediate the enhancing effects of IL-18 on T cell-monocyte contact.     

Interleukin-1beta and tumor necrosis factor-alpha induce chemokine and matrix metalloproteinase gene expression in human colonic subepithelial myofibroblasts

BACKGROUND: Colonic subepithelial myofibroblasts may play a role in the inflammatory responses and in extracellular matrix (ECM) metabolism. In this study, we investigated the effects of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and ECM turnover (proliferation of subepithelial myofibroblasts, and secretion of ECM and matrix metalloproteinases (MMPs)) in colonic subepithelial myofibroblasts. METHODS: Human colonic subepithelial myofibroblasts were isolated using the method described by Mahida et al. Chemokine and MMP expressions were determined by ELISA and Northern blotting. Nuclear factor (NF)-kappaB and NF-IL6 DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: IL-1beta and TNF-alpha did not affect the proliferation of subepithelial myofibroblasts, but stimulated the secretion of types I and IV collagens weakly. Unstimulated subepithelial myofibroblasts secreted a large amount of MMP-2, but a small amount of IL-8, MCP-1 and MMP-1. IL-1beta and TNF-alpha both induced a dose- and time-dependent increase in IL-8, MCP-1 and MMP-1 secretion, and weakly stimulated MMP-2 secretion. IL-1beta and TNF-alpha both rapidly evoked NF-kappaB DNA-binding activity. The inhibition of NF-kappaB activation markedly blocked both IL-1beta- and TNF-alpha-induced IL-8 and MCP-1 mRNA expression, but did not affect MMP-1 mRNA expression. CONCLUSIONS: These observations indicate that chemokine secretion and ECM metabolism are collectively regulated by the proinflammatory cytokines, IL-1beta and TNF-alpha, in colonic subepithelial myofibroblasts. Thus, colonic subepithelial myofibroblasts may play an important role in the pathophysiology of inflammation in the colon.     

High concentrations of soluble tumor necrosis factor receptors in ascites.

Ascites and plasma concentrations of soluble tumor necrosis factor receptors p55 and p75 were measured in a prospective study in 34 patients (35 occasions of ascites) with hepatic (5 infected and 21 uninfected) and malignancy-related (9) ascites. All patients had high concentrations of both soluble tumor necrosis factor receptors in ascites and plasma; these were about 500 times higher than the corresponding tumor necrosis factor-alpha concentrations. Ascites levels of soluble tumor necrosis factor receptors p55 and soluble tumor necrosis factor receptors p75 were significantly elevated in patients with malignancy-related (p55: 26.0 +/- 8.6 ng/ml; p75: 20.5 +/- 17.4 ng/ml; mean +/- S.D.) and infected ascites (p55: 25.1 +/- 10.9 ng/ml, p75: 22.6 +/- 11.0 ng/ml) compared with patients with uncomplicated hepatic ascites (p55: 10.1 +/- 4.4 ng/ml; p75: 6.0 +/- 2.6 ng/ml). Patients with infected or malignancy-related ascites also showed higher soluble tumor necrosis factor receptor concentrations in plasma than did patients with plain hepatic ascites. Successful antibiotic treatment of peritonitis reduced soluble tumor necrosis factor receptor p55 and p75 ascites levels in three patients from 24.2 +/- 15.2 ng/ml to 10.7 +/- 1.9 ng/ml and from 20.2 +/- 14.4 ng/ml to 7.5 +/- 1.8 ng/ml, respectively. Soluble tumor necrosis factor receptors p55 and p75 at cutoff levels of 16.5 ng/ml and 9.5 ng/ml, respectively, differentiated between infected or malignant and plain hepatic ascites with diagnostic accuracies of 94% and 89%, respectively. They did not differentiate between infected and malignant ascites. The concentrations of soluble tumor necrosis factor receptor p55 were usually higher in ascites than in plasma in all subgroups of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFα stimulation. In cells from TRADD-deficient mice, TNFα-mediated apoptosis and TNFα-stimulated NF-κB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFα-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNγ-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.     

Interleukin-4 (IL-4) inhibits secretion of IL-1 beta, tumor necrosis factor alpha, and IL-6 by human monocytes

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin-4 (IL-4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL-1, tumor necrosis factor alpha (TNF alpha), and IL-6. To elucidate which cytokines produced by monocytes are controlled by IL-4, we tested the effect of IL-4 on the secretion of IL-1 alpha, IL-1 beta, TNF alpha, and IL-6 induced by LPS or IFN gamma. IL-4 was found to inhibit the secretion of IL-1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL-6 was found to be reduced 70% to 85% in the presence of IL-4, whereas there was no effect on the secretion of IL-1 alpha (IL-1 alpha is mainly cell-associated). Time-course experiments demonstrate that IL-4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL-1 beta and TNF alpha was specifically induced by IL-4 because anti-IL-4 antiserum completely restored normal monokine production. These data suggest that IL-4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.     

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.