Release Of sTNF-R Research Articles

Association between cigarette smoking and release of tumour necrosis factor and its soluble receptors by peripheral blood mononuclear cells in patients with rheumatoid arthritis

To investigate the relationship between cigarette smoking and release of TNF-alpha and its soluble receptors (sTNFRI and sTNFRII) by peripheral blood mononuclear cells (PBMCs) from RA patients. We studied 71 RA patients with established disease (mean duration 10.6 yr). Smoking history was established by questionnaire. T lymphocytes and monocytes were isolated from peripheral blood and incubated with or without stimulation (phytohaemagglutinin and lipopolysaccharide, respectively). Release of TNF-alpha and sTNFR into culture medium was measured by enzyme-linked immunosorbent assay. TNF-alpha release by stimulated T lymphocytes was significantly higher in patients with a history of smoking than in those who had never smoked (1416.0 vs 767.4 pg/ml, P = 0.04), and showed a relationship with smoking duration and intensity (P for trend < or =0.009). Monocyte TNF-alpha release was not associated with smoking status. Release of sTNFR showed no clear relationships with extent of smoking, although release by stimulated T lymphocytes was higher in past smokers than in those who had never smoked (P < or = 0.03). The ratio of TNF-alpha/sTNFR released from T lymphocytes was higher in past and current smokers, and was associated with extent of smoking. No relationship was found between smoking and plasma TNF-alpha levels, but levels of both receptors were higher in past smokers. In RA patients who smoke there is an alteration in the ratio of TNF-alpha/sTNFR released by stimulated T cells that might favour increased TNF-alpha activity. The increased TNF-alpha/sTNFR ratio is associated with extent of smoking, and remains elevated after smoking cessation.

Interferon-gamma regulates apoptosis by releasing soluble tumor necrosis factor receptors in a gastric epithelial cell line.

Interferon (IFN)-gamma and tumor necrosis factor (TNF) are predominant cytokines produced in the gastric mucosa of patients with Helicobacter pylori-infected gastritis. Several studies reported that IFN-gamma and TNF induced the synergistic effect on many cell lines. We attempted to clarify the apoptotic activity and the synergistic effect of IFN-gamma and TNF on the gastric epithelial cell, and whether IFN-gamma relates to soluble TNF receptors (sTNF-R) release from the gastric epithelial cell. On the gastric epithelial cell line MKN45, cytotoxic and apoptotic effects of IFN-gamma and TNF were examined. Next, sTNF-R released in response to IFN-gamma and the protective effect of sTNF-R against the cytotoxic activity of TNF and IFN-gamma were examined by blocking the release of sTNF-R with a serine protease inhibitor such as phenylmethylsulfonyl fluoride. Interferon-gamma significantly decreased cell viability, but TNF decreased it only slightly. Interferon-gamma and TNF did not make a synergistic effect on cell viability and apoptosis. Interferon-gamma and TNF induced sTNF-R release from gastric epithelial cells. Phenylmethylsulfonyl fluoride significantly inhibited shedding of sTNF-R and a synergistic effect of TNF and IFN-gamma on apoptosis was observed. These results suggest that sTNF-R released by IFN-gamma regulate the injury on the gastric epithelial cell line induced by TNF.

Regulation of tumour necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pyloriinfection

BACKGROUNDTumour necrosis factor (TNF) is a predominant cytokine produced in the gastric mucosa of patients with Helicobacter pyloriinfection. TNF induces apoptosis in a variety of cells. The soluble TNF receptors...

Regulation of the release of tumour necrosis factor (TNF)alpha and soluble TNF receptor by gamma irradiation and interferon gamma in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells.

This study analyses the production of tumour necrosis factor (TNF)alpha and soluble TNF receptor (sTNF-R) before and after exposure to gamma irradiation and interferon gamma (IFN gamma) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF alpha-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-Rp55 and sTNF-Rp75 ELISA. The tumour cell lines released minimal amounts of TNF alpha, prominent amounts of sTNF-Rp55 (7/12 cell lines) and no sTNF-Rp75. Exposure to gamma irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF alpha release without changing sTNF-Rp55 and sTNF-Rp75 levels. Priming of cultures with recombinant human IFN gamma (rhIFN gamma) markedly enhanced TNF alpha secretion in the radiation-responsive cell lines and had no influence on sTNF-Rp55 and sTNF-Rp75 levels. rhIFN gamma affected the magnitude rather than the sensitivity of the radiation response. The TNF alpha secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF alpha monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AA-COCF3 (a specific inhibitor of phospholipase A2) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated gamma-irradiation-stimulated TNF alpha release. The antioxidants N-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited gamma-irradiation-mediated TNF alpha production. Collectively our findings indicate that IFN gamma priming potentiates the secretion of bioactive TNF alpha by ES/pPNET cells in response to gamma irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the gamma-irradiation-mediated intracellular signalling pathway leading to TNF alpha production.

IL-4, IL-10 and IFN-γ have distinct, but interacting, effects on differentiation-induced changes in TNF-α and TNF receptor release by cultured human monocytes

Monocytes cultured in vitro differentiate to a macrophage-like phenotype and undergo functional changes, including reduced capacity for release of TNF-α and the soluble p55 receptor for TNF (sTNF-R55) but enhanced capacity for release of the soluble p75 receptor (sTNF-R75). The cytokines IL-4 and IL-10 act on monocytes to suppress the release of pro-inflammatory cytokines, including TNF-α, and to influence the release of sTNF-R. We therefore investigated the influence of differentiation over 15 days in vitro on the spontaneous and LPS- and IFN-γ-induced release of TNF- α and sTNF-R from human monocytes and examined the actions of IL-4 and IL-10 on these. Unstimulated monocytes did not release TNF-α at any stage but released progressively larger amounts of sTNF-R75 with time. LPS-stimulated release of TNF-α declined substantially after the first day and was consistently suppressed by IL-10 and IL-4 but increased by IFN-γ. Monocytes cultured with IL-10 released more sTNF-R75 at all times and expressed more mRNA for TNF-R75 at day 8. LPS stimulation consistently enhanced both spontaneous and IL-10-augmented release of sTNF-R75, whilst IFN-γ co-stimulation consistently suppressed them. The influence of IL-4 on sTNF-R75 release, however, depended qualitatively on both the length of time in culture and on conditions of stimulation. The effects of LPS and IFN-γ on TNF-α and sTNF-R75 release were progressively lost with increasing time in culture in the presence of IL-4. sTNF-R55 was not detectable after the first day of culture under any of these conditions. IL-4, IL-10 and IFN-γ therefore have distinct, but interacting, effects on the balance between TNF-α and sTNF-R75 release by maturing monocytes. These interactions may be relevant to the pathogenesis or treatment of TNF-α-mediated diseases, where sTNF-R may act to neutralize or stabilise TNF, thereby modifying biological activity.

Tumor Necrosis Factor α and Interleukin-1α Stimulate Late Shedding of p75 TNF Receptors but Not p55 TNF Receptors from Human Monocytes

Soluble receptors for TNF (sTNF-R) are present at elevated concentrations in the synovial fluid of patients with rheumatoid arthritis. They are presumably released by cells of the synovial membrane, including the monocyte-derived synovial macrophages. Cytokines from the synovium, including IL-1 and TNF-alpha, may stimulate release. We therefore examined the release of sTNF-R from monocytes exposed to IL-1 and TNF-alpha. Elutriator-purified human blood monocytes spontaneously released both the p75 and the p55 sTNF-R (1011 +/- 199 and 177 +/- 20 pg/10(6) cells, respectively, mean +/- SEM) during 48 h of in vitro culture. TNF-alpha and IL-1 alpha induced time- and concentration-dependent increases in the release of sTNF-R75 from monocytes, but neither had a measurable effect on the release of sTNF-R55. The release of sTNF-R75 was inhibited by cycloheximide. Neither lymphocytes nor polymorphonuclear leukocytes (PMN) released measurable sTNF-R spontaneously or in response to stimulation with IL-1 alpha, but TNF-alpha stimulated the release of small amounts of sTNF-R75 by PMN. The timing, cycloheximide sensitivity, and selectivity of stimulated release of TNF-R75 by monocytes are consistent with previous observations on other cell types of late (8-20 h) increased synthesis and turnover of cell surface TNF-R75, but not TNF-R55, after stimulation with TNF-alpha or IL-1. These observations help to explain why elevated levels of sTNF-R in synovial fluid coexist with enhanced expression of cell surface TNF-R on synovial macrophages in rheumatoid arthritis.

Polymorphonuclear granulocytes enhance lipopolysaccharide-induced soluble p75 tumor necrosis factor receptor release from mononuclear cells.

Lipopolysaccharide (LPS), a part of the Gram-negative bacteria cell wall, is a potent inducer of tumor necrosis factor (TNF). TNF is an important mediator in Gram-negative infections such as meningococcal septic shock, but its harmful action can be prevented by the natural occurring soluble (s) TNF receptors (sTNFR) sp55 and sp75. In this study, the effect of LPS on release of sTNFR was investigated. First, we found a selective increase in human whole-blood sp75 TNFR levels following LPS stimulation, accompanied by no increase in sp55. Separating the different blood cell populations, mononuclear cells (PBMC) selectively released sp75 upon LPS stimulation, while LPS induced a minor increase in sp75 release from polymorphonuclear granulocytes. Interestingly, in co-cultures of PBMC and granulocytes, the release of LPS-induced sp75 TNFR was enhanced. Second, adherent monocytes were also found to selectively release sp75 TNFR upon LPS stimulation, where Neisseria meningitidis LPS was found to be 100-1000 times more potent in inducing sp75 release than Escherichia coli LPS. Using flow cytometry, the monocyte membrane distribution of both TNFR were found to be increased after LPS stimulation. Third, human umbilical vein endothelial cells selectively released sp55 TNFR after stimulation with LPS. We conclude that mononuclear and endothelial cells might be the main sources of soluble p75 and p55 TNFR, respectively, observed in Gram-negative sepsis, although these receptors are released in vivo more rapidly than they are in vitro.

Differences in the shedding of soluble TNF receptors between endotoxin-sensitive and endotoxin-resistant mice in response to lipopolysaccharide or live bacterial challenge.

TNF-alpha plays a pivotal role in the pathogenesis of septic shock. It exerts its effects by binding two cell surface receptors, designated TNF-R I and II, also referred to as the p55 and p75 receptors, respectively. TNF-Rs are transmembrane proteins, which on cleavage of their extracellular domains, result in the release of soluble fragments (sTNF-R). sTNF-R levels increase markedly during infection, and may serve to modulate TNF-alpha bioactivity. The mechanisms regulating this process are uncertain. To investigate this, we measured sTNF-R release in endotoxin-sensitive C3H/HeN and endotoxin-resistant C3H/HeJ mice given LPS or live Gram-negative bacteria. In C3H/HeN mice, there was a rapid early response during the first 4 h, and a second peak at 8 h, particularly noticeable in the case of the p75 receptor. Prior administration of neutralizing Abs to TNF-alpha or IFN-gamma had no effect on receptor shedding. Surprisingly, C3H/HeJ mice also responded to both bacterial challenge and to LPS by shedding sTNF-R; the magnitude and duration of the early response was not substantially different from C3H/HeN mice, although the second peak was absent. Peritoneal macrophages from C3H/HeN mice responded promptly (5 h) when stimulated with LPS in vitro, and by 22 h levels had increased five- to 10-fold. In contrast, cells from C3H/HeJ mice demonstrated only a very modest response at 22 h following maximal stimulation. The data suggest that there may be at least two separately regulated pathways that control sTNF-R shedding in these mice.

Soluble tumor necrosis factor receptors are released independent of TNF in endotoxemia

The pathway by which the in vivo application of LPS induces the release of soluble TNF receptor molecules (sTNF-R) is not yet understood. Earlier findings have demonstrated that the injection of LPS induces the release of TNFα, and injections of TNFα induce sTNF-R I and II. Thus one would assume that sTNF-R are released after LPS application as a result of endogenous production of TNFα. In this study, i.v. bolus injections of Salmonella abortus equi endotoxin (4 ng/kg body weight) once daily for 5 consecutive days were administered to cancer patients. The time course of serum levels of TNFα and sTNF-R I and II were determined. sTNF-R I and II were released after every LPS injection with peak levels being comparable on days 1 through 5. In contrast, the TNFα peak level on day 1 was about 50 times higher than on day 2. On days 3 through 5, no TNFα could be detected, a phenomenon referred to as endotoxin tolerance. Since LPS was able to provoke a comparable release of sTNF-R despite inducing widely differing levels of TNF-α, we conclude that the release of sTNF-R I and II during endotoxemia is not induced via TNF-α.

Tissue distribution and clearance of soluble murine TNF receptors in mice

In this study, clearance of sTNFR was investigated. The data show that bilateral nephrectomy results in an increase of the levels of both sTNFR after which a new steady state situation develops, suggesting that other organs, apart from the kidneys, are involved in clearing of sTNFR. Bilateral nephrectomy also leads to an increase in circulating TNF. This TNF was detected by ELISA and appeared to be not biologically active. To investigate whether the endotoxin induced increase in sTNFR is dependent of renal function, endotoxin was injected in nephrectomized mice. The data show that nephrectomy followed by endotoxin injection resulted in a further increase of the levels of both sTNFR. However, the endotoxin induced increase in nephrectomized mice was similar to the situation in normal mice after LPS indicating that the endtoxin induced increase is kidney independent in these mice. To investigate the relative participation of various organs in sTNFR clearance, 125I labelled sTNFR-P75 was injected. The data reveal that the majority of the sTNFR is removed from the circulation by the kidneys although indications for involvement of the liver and the lungs were also obtained. Calculation of the parametric clearance revealed that nephrectomy resulted in a 50% reduction of sTNFR-P75 clearance. Furthermore, the data presented strongly suggest that sTNFR release seems to be a continuous process, which is in balance with clearance of the sTNFR by the kidney, although other organs such as the liver and the lungs are involved.

Induction of soluble tumor necrosis factor receptor (sTNF‐R75) release by HIV adsorption on cultured human monocytes

Soluble tumor necrosis factor (TNF) receptors were recently detected in the circulation of patients with early HIV-induced disease, at significantly higher levels than in control subjects. They were proposed as markers of disease progression and of the degree of immunodeficiency. We report that adsorption of heat-inactivated HIV-1 LAI to isolated human monocytes triggers the release of both TNF-alpha and its natural specific inhibitor, the soluble TNF receptor (sTNF-R)75, but not that of sTNF-R55. Only limited inhibition of sTNF-R release was obtained in the presence of a fully neutralizing anti-TNF-alpha monoclonal antibody, suggesting that stimulation by TNF-alpha was only partially responsible for sTNF-R release. HIV-1 LAI induced a higher sTNF-R/TNF ratio than lipopolysaccharide, a well-known monocyte activator. Monocytes thus represent a cellular source of sTNF-R that can be detected in the circulation of HIV-infected patients from seroconversion onwards. The release of sTNF-R could be of great significance in the control of HIV infection via the cytokine network and especially TNF-alpha.

Lipopolysaccharide LPS-mediated soluble TNF receptor release and TNF receptor expression by monocytes. Role of CD14, LPS binding protein, and bactericidal/permeability-increasing protein.

Previously we demonstrated that two soluble(s) tumor necrosis factor receptors, TNF-R55 as well as sTNF-R75, are constitutively released in vitro by monocytes, and that this release was markedly enhanced after activation. Because LPS is an important activator of monocytes, we investigated the effect of LPS on sTNF-R release by monocytes. It was found that release of sTNF-R75, but not (or minimally) release of sTNF-R55, was enhanced after activation with LPS, reaching plateau levels after approximately 2 days. CD14, one of the membrane receptors for LPS, is an intermediate in this process, as shown in experiments using mAb directed against CD14. Under serum-free conditions, LPS-induced sTNF-R75 release was less as compared with release in the presence of serum, suggesting involvement of serum proteins. Addition of LPS binding protein (LBP) enhanced the LPS-induced sTNF-R75 release under serum-free conditions, but had no effect in the presence of serum. On the other hand, bactericidal/permeability-increasing protein (BPI), known to possess LPS neutralizing activity, inhibited LPS-induced sTNF-R75 release. Furthermore, cell surface expression of both types of TNF-R was shown to be controlled by LPS, LBP, and BPI. LPS caused, within 1 h, a complete reduction of TNF-R55 as well as TNF-R75 expression, followed by enhanced re-expression of both receptors after 24 h. The down-modulation of expression was increased by LBP, whereas BPI counteracted the LPS-induced down-regulation. The LPS-enhanced release of sTNF-R75, capable of inactivation of TNF, as well as LPS-induced initial down-modulation of TNF-R expression leading to postulated temporary unresponsiveness to TNF may share in a physiological mechanism to carefully control the effects of TNF.

Slow release of soluble TNF receptors by monocytes in vitro.

In this study, we investigated the release of soluble(s) TNF-R by PBMC in vitro. T cell activation by mAb anti-CD3, as well as activation with phorbol esters (PMA), enhanced the release of both sTNF-R55 and sTNF-R75 by PBMC. In contrast to shedding of TNF-R by neutrophils upon activation, release of sTNF-R by PBMC proved to be a relatively slow process, reaching a plateau after 2 days of culture. Monocytes appeared to be the main source of the released sTNF-R, whereas activation of purified T cells induced only a minor release of sTNF-R as compared with the whole cell population. To unravel the mechanism, a number of cytokines were added during a 2-day culture of cells. IL-10 enhanced sTNF-R levels with similar kinetics as mAb anti-CD3 and PMA, whereas the other cytokines tested did not affect the release of sTNF-R by PBMC, pure T lymphocytes, or purified monocytes, either activated or not. Conversely, inhibitors of cytokines were added during the activation period to study the effect of endogenously produced cytokines on sTNF-R release. mAb anti-IL-10 and IL-1ra partly sTNF-R release, whereas other inhibitors did not affect the release. The results obtained in vitro may extend our insight in the mechanism via which sTNF-R are enhanced in vivo during inflammatory reactions.

LPS-induced sTNF-receptor release in vivo in a murine model. Investigation of the role of tumor necrosis factor, IL-1, leukemia inhibiting factor, and IFN-gamma.

TNF, a primary mediator of the response to infection, can be injurious to the organism when present in excessive quantities. Circulating soluble TNF receptors (sTNFR) appear to represent a natural mechanism that protects against circulating TNF. Two soluble TNF receptors (sTNFR-P55 and sTNFR-P75) circulate in vivo and are up-regulated in response to endotoxin. In this study, we investigated the kinetics of LPS-induced sTNFR release and the role of the cytokines TNF, leukemia inhibiting factor, IFN-gamma, and IL-1 in this process. The results show that LPS injection results in a rapid increase in levels of both sTNFR. Although sTNFR-P55 decreases after a peak at 30 min, sTNFR-P75 levels show a peak after 4 to 8 h, after which they slowly diminish. Both human TNF and murine TNF are capable of increasing levels of both sTNFR. Blocking circulating TNF by administration of 3 different anti-TNF agents before LPS injection (mAb to murine TNF, sTNFR55-Fc or sTNFR75-Fc) results in a significant increase of sTNFR-P55 levels, whereas only both sTNFR-Fc constructs also significantly increase sTNFR-P75 levels. Although IL-1 receptor antagonist pretreatment before LPS has no effect on TNF or sTNFR levels, leukemia inhibiting factor pretreatment significantly increases sTNFR-P55 levels. Pretreatment with anti IFN-gamma mAb before LPS results in a significant reduction in TNF and sTNFR-P55 levels, but sTNFR-P75 levels are significantly increased. Our data show that both sTNFR can be up-regulated by LPS and TNF. The influence of TNF, leukemia inhibiting factor, IL-1, and IFN-gamma on the kinetics of LPS-induced circulating sTNFR is discussed in the context of the pathophysiology of LPS-induced disease.