Soluble Tumor Necrosis Factor P75 Research Articles

Severity of Plasma Leakage Is Associated With High Levels of Interferon γ-Inducible Protein 10, Hepatocyte Growth Factor, Matrix Metalloproteinase 2 (MMP-2), and MMP-9 During Dengue Virus Infection.

Dengue virus infection typically causes mild dengue fever, but, in severe cases, life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur. The pathophysiological hallmark of DHF and DSS is plasma leakage that leads to enhanced vascular permeability, likely due to a cytokine storm. Ninety patients with dengue during 2010-2012 in Singapore were prospectively recruited and stratified according to their disease phase, primary and secondary infection status, and disease severity, measured by plasma leakage. Clinical parameters were recorded throughout the disease progression. The levels of various immune mediators were quantified using comprehensive multiplex microbead-based immunoassays for 46 immune mediators. Associations between clinical parameters and immune mediators were analyzed using various statistical methods. Potential immune markers, including interleukin 1 receptor antagonist, interferon γ-inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor α receptor, vascular cell adhesion molecule 1, and matrix metalloproteinase 2, were significantly associated with significant plasma leakage. Secondary dengue virus infections were also shown to influence disease outcome in terms of disease severity. This study identified several key markers for exacerbated dengue pathogenesis, notably plasma leakage. This will allow a better understanding of the molecular mechanisms of DHF and DSS in patients with dengue.

Construction and Purification of the Murine p75-murine IgG1 Fusion Protein

Etanercept (Amgen Inc, Thousand Oaks, CA) is a human soluble p75 tumor necrosis factor (TNF) receptor-human-IgG1 (hup75 TNFR-huIgG1) fusion protein used in the treatment of chronic inflammatory diseases in humans, including rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. To be able to study the effects of the soluble receptor fusion protein in mouse models, including those that mimic human granulomatous infections, a murine soluble p75-TNF receptor-murine IgG1 (murine p75-murine IgG1) fusion protein had to be constructed. This article discusses the generation, large-scale production, and purification of this molecule.

Use of etanercept in human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) patients

Etanercept (Enbrel, Amgen, Thousand Oaks, CA), a soluble p75 tumor necrosis factor receptor:FC (TNFR:FC) fusion protein for plasma cytokines, specifically tumor necrosis factor-alpha (TNF-alpha), is used in the treatment of immune-mediated rheumatic diseases. To our knowledge, the use of etanercept in patients with human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) is relatively uncommon. The main purpose of this short review is to examine the safety of etanercept in patients with HIV/AIDS. A Medline search was conducted using the keywords etanercept and HIV and/or AIDS for any published articles between 1966 to the present (September 2004). A case report, one case series, and one clinical trial pertained to the use of etanercept in HIV patients. No reports were found on the use of etanercept in AIDS. In addition, two case reports were found documenting the use of infliximab in HIV patients. Preliminary reports indicate that the administration of etanercept does not appear to increase the morbidity or mortality rates in HIV. The inhibition of TNF-alpha may actually improve the symptoms of HIV/AIDS-associated aphthous ulcers, cachexia, dementia, fatigue, and fever, as well as help manage concomitant rheumatic diseases and psoriasis. The use of etanercept shows promise for applications in disease management in patients with HIV/AIDS. Continued research efforts are necessary to establish the long-term safety and efficacy of etanercept and other biologic agents in this patient population.

Protection of human cerebral neurons from neurodegenerative insults by gene delivery of soluble tumor necrosis factor p75 receptor

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurodegenerative diseases, including amyotrophic lateral sclerosis, Huntington's disease, cerebral ischemia, and human immunodeficiency virus (HIV) encephalopathy. We evaluated the ability of the extracellular binding domain of a dimeric tumor necrosis factor receptor (p75TNFR) to prevent neurotoxicity and death of human fetal cerebral neurons that were exposed in vitro to toxic agents known to be implicated in human neurological disorders, including tumor necrosis factor (TNFalpha) and the HIV proteins Tat and gp120. The extracellular domain of p75TNFR is capable of binding and neutralizing both soluble and transmembrane-anchored TNFalpha. We efficiently transduced human neurons using adenoviral vectors expressing p75TNFR (Ad.p75TNFR) or a control gene (lacZ). Treatment of control cultures with the toxic agents TNFalpha, TNFalpha plus actinomycin D, or Tat and gp120, induced neurotoxic alterations and apoptotic death of neurons. By contrast, transduction of neurons with Ad.p75TNFR prevented apoptosis and cell death due to these agents. We conclude that viral vector transfer of the p75TNFR gene efficiently protects human neurons from TNFalpha-, Tat- or gp120-induced apoptosis and cell death. These results suggest that p75TNFR transduction of neurons by viral vectors could be therapeutically useful in the treatment of many human neurodegenerative diseases.

Survival Following Etanercept Therapy for the Treatment of Idiopathic Pneumonia Syndrome Post Allogeneic Stem Cell Transplantation.

Pulmonary complications, both infectious and non-infectious, significantly contribute to mortality following allogeneic bone marrow transplantation (BMT). In the acute setting, a diffuse non-infectious process termed Idiopathic Pneumonia Syndrome (IPS) may occur. Clinically, IPS is associated with a rapid progression to respiratory failure and mortality rates of 50 – 70%. Historically, we have noted a 9.0% incidence of IPS at our center, with a median survival of only 14 days following its onset. Etanercept, a soluble tumor necrosis factor (TNF) receptor, consists of two soluble p75 TNF receptors fused to the Fc portion of a human IgG1. A trial examining etanercept in the treatment of IPS following allogeneic BMT was undertaken. Fifteen patients (median 18 yrs, range 1 – 60 yrs), each meeting the diagnostic criteria for IPS were enrolled. Broncho-alveolar lavage (BAL) specimen were obtained pre and post therapy, undergoing analysis for both infectious pathogens (viral, bacterial, fungal, PCP, AFB) and for a panel of inflammatory cytokine markers, including TNFa and TNFR1. Patients in whom the pre-therapy BAL was positive for a potential pathogen (by specific stain or culture) were ineligible for therapy. Etanercept was administered subcutaneously at a dose of 0.4 mg/kg (maximum dose 25 mg) twice weekly, for a maximum of 8 doses. All patients required supplemental oxygen at therapy onset, with seven patients requiring mechanical ventilation. Etanercept therapy was initiated a median of 17 days (range 11–87 days) post transplant. All patients received corticosteroids (2 mgkg/day) x 7 days, which were then tapered as clinically indicated. RESULTS: Nine of 15 patients had a complete response, defined as the ability to completely withdraw from supplemental oxygen support within 28 days of initiation of etanercept therapy. In responders, the median time to complete response was 7 days (range 3–18 days). The median survival was extended from 14 days (historical controls) to 67 days (range 3–822) in treated patients. Survival at day 28 and day 56 (following the first etanercept dose) was 73% and 60% respectively. The duration of mechanical ventilation required prior to therapy onset had a significant impact on survival. Patients requiring < 48 hours of mechanical ventilation prior to study entry had a median survival of 150 days (range 46 – 822), compared to 17 days (range 3–148) for those requiring mechanical ventilation > 48 hours prior to study entry. Overall, therapy was well tolerated. Two episodes of bacteremia, without sepsis, were noted during therapy, . No infectious pulmonary complications were noted. Three patients died while on therapy, from progressive pulmonary or organ dysfunction. Post therapy BAL fluid analysis noted significant reductions in all inflammatory cytokines tested, including TNFa, TNFR1, sCD14, LBP and MCP-1, when compared to pre-therapy BAL values. CONCLUSION: The addition of etanercept to corticosteroids for the treatment of IPS post allogeneic BMT may improve survival, with minimal toxicity. Moreover, the timing of such intervention may have a significant impact on overall outcome. A phase III trial, investigating the efficacy of etanercept in treating IPS is now being considered. IPS therapy n day 28 day 56 Group A: etanercept + corticosteroids 15 73% 60% Group B: corticosteroids (historical controls with IPS) 49 33% 26%

Soluble tumor necrosis factor receptor: enbrel (etanercept) for the treatment of idiopathic pneumonia syndrome following allogeneic stem cell transplantation

Non-infectious lung injury is as a frequent and severe complication of allogeneic bone marrow transplantation (BMT). In the acute setting, a diffuse non-infectious process termed Idiopathic Pneumonia Syndrome (IPS) may occur. Despite advances in supportive care and the use of high dose steroids, mortality from IPS remains unacceptably high (> 70%). Etanercept is a soluble tumor necrosis factor (TNF) receptor, consisting of two soluble p75 TNF receptors fused to the Fc portion of a human IgG1. A trial examining etanercept in the treatment of IPS following allogeneic transplant has now been undertaken. Fourteen patients (median 18 yrs, range 1–60 yrs), each meeting the diagnostic criteria for IPS were treated. Broncho-alveolar lavage (BAL) specimen were obtained pre and post therapy, undergoing analysis for both infectious pathogens (viral, bacterial, fungal, PCP, AFB) and for inflammatory cytokine markers (TNF, TNFR1, sCD-14, LBP, and MCP-1). Patients in whom the pre-therapy BAL was positive for a potential pathogen (by specific stain or culture) were ineligible for therapy. Etanercept was administered subcutaneously at a dose of 0.4 mg/kg (maximum dose 25 mg) twice weekly, for a maximum of 8 doses. All patients required supplemental oxygen at therapy onset, with seven patients requiring mechanical ventilation. Etanercept therapy was initiated a median of 17 days (range 11–87 days) post transplant. Results: Therapy was well tolerated, with no infusion related reactions. Eight of 14 patients had a complete response, defined as the ability to withdraw completely from supplemental oxygen support. In those responding, the median time to complete response was 7 days (range 3–18 days), and the median time to normalization of radiographic findings was 6 days (range 2–10 days). Two other patients had a significant reduction in their FiO2 requirement during therapy. Three patients died while on therapy, from progressive organ dysfunction. Post therapy BAL fluid analysis noted a significant reductions in all inflammatory cytokines tested, including TNFa, TNFR1, sCD14, LBP and MCP-1 (Table below). Conclusion: Etanercept therapy was well tolerated, with minimal toxicity and favorable response in patients with IPS post allogeneic transplant. The clinical and biochemical responses seen with therapy support a mechanistic role for TNFa in the pathogenesis of this disorder. Further trials, investigating the responsiveness of IPS to etanercept therapy are warranted.

Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein.

Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. At 2 days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into one selected site in the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3 ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate-to-severe CIA in mice treated with sTNFR:Fc was prevented on a significant level compared with the control mice (P<0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P<0.05). The expression of IL-1beta and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P<0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.

1032. Electro-Gene Therapy of Collagen-Induced Arthritis by Using an Expression Plasmid for the Soluble p75 Tumor Necrosis Factor Receptor-Fc Fusion Protein

1032. Electro-Gene Therapy of Collagen-Induced Arthritis by Using an Expression Plasmid for the Soluble p75 Tumor Necrosis Factor Receptor-Fc Fusion Protein

TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling.

The cytokine tumor necrosis factor (TNF)-alpha has been causally linked to left ventricular (LV) remodeling, but the molecular basis for this effect is unknown. Matrix metalloproteinases (MMPs) have been implicated in cardiac remodeling and can be regulated by TNF-alpha. This study tested the central hypothesis that administration of a TNF-alpha blocking protein would prevent the induction of MMPs and alter the course of myocardial remodeling in developing LV failure. Adult dogs were randomly assigned to the following groups: 1) chronic pacing (250 beats/min, 28 days, n = 12), 2) chronic pacing with concomitant administration of a TNF-alpha blocking protein (TNF block) using a soluble p75 TNF receptor fusion protein (TNFR:Fc; administered at 0.5 mg/kg twice a week subcutaneously, n = 7), and 3) normal controls (n = 10). LV end-diastolic volume increased from control with chronic pacing (83 +/- 12 vs. 118 +/- 10 ml, P < 0.05) and was reduced with TNF block (97 +/- 9 ml, P < 0.05). MMP zymographic levels (92 kDa, pixels) increased from control with chronic pacing (36,848 +/- 9,593 vs. 87,247 +/- 12,912, P < 0.05) and was normalized by TNF block. Myocardial MMP-9 and MMP-13 levels by immunoblot increased with chronic pacing relative to controls (130 +/- 10% and 118 +/- 6%, P < 0.05) and was normalized by TNF block. These results provide evidence to suggest that TNF-alpha contributes to the myocardial remodeling process in evolving heart failure through the local induction of specific MMPs.

Detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in Brazilian patients with dengue fever and dengue hemorrhagic fever.

Pro-inflammatory cytokines are believed to play an important role in the pathogenesis of dengue infection. This study reports cytokine levels in a total of 54 patients examined in Recife, State of Pernambuco, Brazil. Five out of eight patients who had hemorrhagic manifestations presented tumor necrosis factor-alpha (TNF-alpha) levels in sera which were statistically higher than those recorded for controls. In contrast, only one out of 16 patients with mild manifestations had elevated TNF-alpha levels. The levels of interleukin-6 (IL), IL-1beta tested in 24 samples and IL-12 in 30 samples were not significantly increased. Interferon-g was present in 10 out of 30 patients with dengue. The data support the concept that the increased level of TNF-alpha is related to the severity of the disease. Soluble TNF receptor p75 was found in most patients but it is unlikely to be related to severity since it was found with an equivalent frequency and levels in 15 patients with dengue fever and another 15 with dengue hemorrhagic fever.

Clinical experience with soluble TNF p75 receptor in rheumatoid arthritis

Objectives: Managing rheumatoid arthritis (RA) can be difficult: the disease may follow an unpredictable course, and therapies are often ineffective or toxic. Etanercept, a bioengineered fusion protein derived from the human soluble tumor necrosis factor p75 receptor, recently has been approved for use in patients with refractory RA. Methods: Published data on clinical experience with etanercept in conjunctionwith case illustrations are presented. Results: Reports from clinical trials of patients with refractory RA indicate that etanercept significantly improves measures of disease activity, including swollen and tender joint counts, morning stiffness, pain, and erythrocyte sedimentation rate, compared with placebo. In the phase 3 trial, swollen joint counts improved by 47% in patients receiving etanercept 25 mg compared with a 7% worsening in patients receiving placebo. The drug is well tolerated; injection site reaction, the most frequent adverse event, was minor and manageable. In long-term studies, etanercept remains well tolerated and effective. Our clinical experience indicates that patients with refractory RA experience dramatic symptomatic relief, along with reduced fatigue and improved quality of life. One of 18 patients discontinued treatment; the rest have remained on therapy for up to 18 months. Conclusions: Etanercept diminishes disease activity in patients with refractoryRA. Its favorable safety profile provides symptom control without major toxicity. Etanercept is an important addition to RA therapeutic agents.

Etanercept, a novel drug for the treatment of patients with severe, active rheumatoid arthritis

The US adult rheumatoid arthritis (RA) population numbers approximately 2.1 million, with a greater proportion of cases in women. RA is a disease of the immune system that has no known cure, and current drugs do not affect the underlying cause. The side effects such drugs produce limit their usefulness, and many patients stop responding to these treatments over time. Etanercept, a biologic inflammation modulator, is a novel human recombinant version of the soluble p75 tumor necrosis factor (TNF) receptor that is linked to the Fc receptor of human immunoglobulin G subclass 1. It acts as a competitive inhibitor of the binding of TNF-alpha to cell-surface TNF receptors and thereby inhibits TNF-alpha-induced proinflammatory activity in the joints of RA patients. Etanercept acts as a cytokine “carrier” and TNF-alpha antagonist, rendering TNF-alpha biologically inactive, even though prolonging its half-life. In Phase I, II, and III clinical studies in patients with active, severe RA who had not responded to disease-modifying antirheumatic drug (DMARD) therapy, etanercept treatment decreased disease activity, increased functional activity, and improved health-related quality of life. In a recent 12-month continuation of an earlier 6-month study, 105 patients who received etanercept 25 mg subcutaneously twice weekly demonstrated rapid and sustained improvements in disease activity. The US Food and Drug Administration has approved etanercept for marketing for the treatment of moderately to severely active RA in patients who have not responded adequately to other DMARDs.

Circadian rhythm of the soluble p75 tumor necrosis factor (sTNF-R75) receptor in humans--a possible explanation for the circadian kinetics of TNR-alpha effects.

Circadian alterations of several immune functions in vivo are well established, and may have important physiological and clinical implications. In line with this, tumor necrosis factor (TNF)-alpha has been implicated in the circadian regulation of body temperature. As soluble TNF receptors (TNF-R) act as naturally occurring competitive inhibitors of this cytokine, we investigated plasma levels of the soluble sTNF-R55 and sTNF-R75 in comparison with plasma cortisol and body temperature in nine healthy male volunteers during a defined 16 h light/8 h dark cycle. It was found that sTNF-R75, but not sTNF-R55, exhibited a clear-cut circadian rhythm with a significant (P < 0.01) peak at 7:51 a.m. +/- 58 min. The phase of the sTNF-R75 rhythm preceded that of cortisol by approximately 1 h and inversely corresponded to the circadian rhythm of body temperature. Moreover, the individual amplitudes of sTNF-R75 and body temperature exhibited a significant (P < 0.01) positive correlation. These results suggest that (i) the two sTNF-R are regulated independently, (ii) the sTNF-R75 rhythm is not primarily due to the cortisol rhythm and (iii) the fluctuation of the sTNF-R may contribute to the regulation of body temperature by modulating the availability of free TNF.

Soluble tumor necrosis factor receptor (p75) does not attenuate the sleep changes induced by lipopolysaccharide in the rat during the dark period

Sleep is generally enhanced during the early phase of infection. The cytokine tumor necrosis factor (TNF) has been postulated to play an important role in the acute phase sleep response. After demonstrating the ability of a soluble p75 TNF receptor (TNFR) to inhibit TNF activity in vitro, we assessed the influence of TNFR on the sleep changes evoked by lipopolysaccharide (LPS). In this vehicle-controlled experiment, 24 rats received either an intracerebroventricular injection of 10 μg TNFR, an intraperitoneal injection of 30 μg/kg LPS, or both, at the beginning of the dark period. EEG, EMG and brain temperature ( T br) were recorded during the first 12 h post injection. Compared with vehicle, LPS had minimal effects on T br, but promoted non-rapid eye movement sleep (non-REMS), suppressed REMS, shortened the sleep episodes and decreased high-frequency (≥8 Hz) EEG activity during non-REMS. TNFR alone had no significant effects and did not attenuate any of the LPS-induced sleep changes. These results may either indicate that TNF is not critically involved in the sleep response to a low level LPS challenge during the activity phase or that the soluble p75 TNFR does not effectively antagonize the sleep changes evoked by TNF.

Randomized placebo-controlled clinical trial of high-dose interleukin-2 in combination with a soluble p75 tumor necrosis factor receptor immunoglobulin G chimera in patients with advanced melanoma and renal cell carcinoma.

A randomized, double-blind, placebo-controlled trial was performed to compare the toxicity and biologic effects of treatment with high-dose intravenous (IV) bolus interleukin-2 (IL-2) plus the recombinant human soluble p75 tumor necrosis factor (TNF) receptor immunoglobulin G (IgG) chimera (rhuTNFR:Fc) with high-dose IL-2 alone in patients with advanced melanoma and renal cell carcinoma. Twenty patients with advanced melanoma or renal cell carcinoma were randomized to receive IL-2 (Chiron, Emeryville, CA) 600,000 IU/kg every 8 hours on days 1 to 5 and 15 to 19 (maximum, 28 doses) combined with placebo or the rhuTNFR:Fc fusion protein (Immunex, Seattle, WA) 10 mg/m2 on days 1 and 15 and 5 mg/m2 on days 3, 5, 17, and 19. The impact of rhuTNFR:Fc on IL-2 toxicity and biologic effects was evaluated. No clinically significant difference in toxicity was observed in the two treatment arms. The adjusted median number of IL-2 doses administered during cycle 1 was 24.5 (range, seven to 28) and 21.5 (range, five to 27) for the placebo and rhuTNFR:Fc arms, respectively (P = .544). IL-2-induced TNF bioactivity, neutrophil chemotactic defect, and serum IL-6, IL-8, and IL-1 receptor antagonist (IL-1RA) induction were suppressed by rhuTNFR:Fc. Two of nine assessable patients (22%) on IL-2/placebo and three of 10 patients (30%) on IL-2/rhuTNFR:Fc responded. Despite evidence of in vitro neutralization of TNF functional activity and partial inhibition of other secondary biologic effects of IL-2, rhuTNFR:Fc does not reduce the clinical toxicity associated with high-dose IL-2 therapy. These results suggest that the toxicity and antitumor effects of IL-2 treatment are independent of circulating TNF.

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.

Two inhibitors of pro-inflammatory cytokine release, interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes.

The biological activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha depends on the level of TNF-alpha itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF-alpha, the soluble p55 and p75 TNF receptors (TNF-R). Interleukin (IL)-10 and IL-4 are known to inhibit TNF-alpha production by monocytes. We, therefore, investigated the effects of IL-10 and IL-4 on the cell surface expression and release of TNF-R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF-alpha. Exposure to IL-10 (1-10 U/ml) for 24 or 48 h increased soluble p75 TNF-R expression and concomitantly reduced surface expression of p75 TNF-R. Further, IL-1 alpha-stimulated production of TNF-alpha was diminished by IL-10 and only a small proportion of this TNF-alpha was bioactive, consistent with increased production of inhibitory soluble TNF-R. IL-10 also induced down-regulation of surface p55 TNF-R on monocytes, and increased release of soluble p55 TNF-R. However, the expression of soluble p55 TNF-R was much lower than soluble p75 TNF-R, indicating that it contributed less importantly to neutralization of TNF-alpha under these conditions. Like IL-10, IL-4 supressed the release of TNF-alpha by monocytes. In contrast to IL-10, however, IL-4 (0.1-10 ng/ml) supressed the release of soluble p75 TNF-R from monocytes in a dose-dependent manner. Release of soluble p55 TNF-R was also supressed by IL-4. IL-10, therefore, reduces the pro-inflammatory potential of TNF in three ways: by down-regulating surface TNF-R expression whilst increasing production of soluble TNF-R and inhibiting the release of TNF-alpha itself. This suggests that IL-10 may be useful in the treatment of diseases where overexpression of TNF-alpha occurs.