IL-1beta, IL-6 and TNF-alpha in synovial fluid of patients with non-gonococcal septic arthritis

Interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are the main proinflammatory cytokines responsible for the inflammatory process and cartilage destruction of inflammatory arthropathies. The present study sequentially measured the concentrations of these cytokines and their proportions of detectable levels in the synovial fluid (SF) of 23 patients with non-gonococcal (GC) septic arthritis before and after treatment. Persistently high concentrations and proportions of IL-6 and TNF-alpha were found up to day 7 of treatment, while SF IL-1beta concentration declined significantly after day 7 (p = 0.036). SF IL-1beta and TNF-alpha correlated with each other significantly and with SF WBC counts (p < 0.01). Positive correlations between SF IL-1beta concentration and joint effusion (p < 0.01) and between SF TNF-alpha concentration and joint tenderness (p < 0.001) were observed. SF IL-1beta and TNF-alpha were significantly higher in patients with local complications of septic arthritis. In conclusion, high levels of IL-1beta, IL-6 and TNF-alpha were detected in SF of patients with non-GC septic arthritis. Only IL-1beta decreased significantly after day 7 of treatment, but IL-6 and TNF-alpha concentrations were persistently high. SF IL-1beta and TNF-alpha may be useful in predicting the outcome and complications of patients with this disease.     

Thalidomide suppressed IL-1beta while enhancing TNF-alpha and IL-10, when cells in whole blood were stimulated with lipopolysaccharide

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomide's ability to inhibit tumor necrosis factor alpha (TNF-alpha) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-alpha. This is dependent on the stimulant used to evoke TNF-alpha; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-alpha, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 beta (IL-1beta) (p = 0.007), and it enhanced TNF-alpha (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1beta, TNF-alpha, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-alpha is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.     

Circulating proinflammatory cytokines (IL-1 beta, TNF-alpha, and IL-6) and IL-1 receptor antagonist (IL-1Ra) in fulminant hepatic failure and acute hepatitis.

Fulminant hepatic failure (FHF) is characterized by massive necroinflammation of the liver tissue and is associated with high mortality. Serum concentrations of IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 receptor antagonist (IL-1Ra) were measured in 30 patients with FHF and in 23 patients with acute hepatitis (AH) before start of treatment and in 23 healthy controls. Levels of all four molecules were increased significantly in FHF compared with AH, in which values were higher than in the healthy controls. High serum levels of IL-1 beta and a significantly reduced ratio of IL-1Ra to IL-1 beta (IL-1Ra/IL-1 beta) were observed in FHF patients who subsequently died compared with subjects who survived. TNF-alpha and IL-6 concentrations were correlated with levels of human hepatocyte growth factor (hHGF), an index of hepatocyte regeneration. Although serum cytokine levels varied considerably between patients within each group studied, it is suggested that the striking elevation in proinflammatory cytokine levels in FHF may reflect both the insufficiency of hepatitis virus elimination and a failure to control a vicious cytokine cascade leading to overwhelming hepatocyte destruction rather than regeneration. The high cytokine levels observed in these patients and the significantly elevated IL-1Ra/IL-1 beta ratio in FHF patients who survived compared with those who did not suggest the possible therapeutic use of cytokine antagonists for the control of this life-threatening disease.     

A multidonor ELISPOT study of IL-1 beta,IL-2, IL-4, IL-6, IL-13, IFN-gamma and TNF-alpha release by cryopreserved human peripheral blood mononuclear cells

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.     

Blood Serum Levels of IL-2, IL-6, IL-8, TNF-α and IL-1β in Patients on Maintenance Hemodialysis

Cytokines are essential mediators of immune response and inflammatory reactions. Patients with chronic renal failure (CRF) commonly present with abnormalities of immune function related with impaired kidney function and the accumulation of uremic toxins in addition to bioincompatibility of dialyzer membranes. During a hemodialysis (HD) session, cytokines are released mainly by monocytes activated by endotoxin-type compounds in dialyzer fluid, complement factors and direct contact with dialyzer membrane. The study included 15 CRF patients, aged 36.4±2.9 years, on regular HD maintenance therapy for mean 68±10 months and 15 healthy controls. It was designed to assess serum levels of a panel of inflammatory cytokines: IL-1β, IL-2, IL-6, IL-8 and TNF-αin CRF patients on regular maintenance HD before, 20, 60 and 240 minutes of a single HD session in parallel with C-reactive protein (CRP) as an additional parameter. CRP concentration was increased in HD patients when compared with healthy controls. The concentrations of IL-1, IL-6, IL-8 and TNF-αwere increased, whereas the serum level of IL-2 was not altered during a single HD session.     

Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.     

Influence of collection and separation of blood samples on plasma IL-1, IL-6 and TNF-alpha concentrations.

The anticoagulant used for the collection of blood was found to influence in vitro cytokine production in whole blood. Lithium heparin in certain collection tubes was found to contain endotoxin and induced cytokine synthesis in a time-dependent manner whereas endotoxin-free lithium heparin did not. No induction of cytokine occurred in the presence of EDTA which was also able to inhibit endotoxin-induced cytokine synthesis. Synthesis or absence of cytokine correlated with the induction of messenger RNA. Investigation of the kinetics of cytokine induction in whole blood revealed that tumour necrosis factor alpha (TNF) was detectable after 2 h of incubation at 37 degrees C and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) after 3 h. In certain samples IL-1 and IL-6 were detectable in plasma separated immediately from blood collected into endotoxin-free lithium heparin, presumably reflecting in vivo synthesis, and similar concentrations were detected after 3 h of incubation of whole blood at 37 degrees C. These data indicate that as long as blood is collected into endotoxin-free anticoagulant then cytokine measurements will reflect the in vivo status.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Proinflammatory cytokine (IL-1beta, IL-6, IL-12, IL-18 and TNF-alpha) levels in sera of patients with subacute cutaneous lupus erythematosus (SCLE)

Subacute cutaneous lupus erythematosus (SCLE) is a subset of lupus erythematosus that identifies patients with clinically recognized erythematous, nonscarring lesions, photosensitivity and serologic abnormalities. Anti-Ro (SS-A) antibodies are considered to be a typical immunopathologic marker of SCLE. Autoimmune diseases have been also characterized by the disturbances in the cytokine network. The aim of this study was to compare the concentrations of proinflammatory cytokines (IL-1beta, IL-6, IL-12, IL-18 and TNF-alpha) in serum of ANA-positive (antibody against nuclear antigen) and ANA-negative patients with SCLE. Sera samples were collected from 15 patients with SCLE (9 ANA-positive and 6 ANA-negative ones). The preliminary identification of autoantibodies as well as their titers was determined on HEp-2 cells using IIF method. Western blotting (EUROIMMUN) was applied to verify the results of IIF. Proinflammatory cytokine concentrations in the patients' sera samples were determined by enzyme-linked immunosorbent assay (ELISA) (Bender MedSystems). The levels of IL-12 were higher in ANA-positive patients than in ANA-negative subgroups [median (interquartile range), 330 pg/ml (128-708 pg/ml) versus 39.4 pg/ml (31.25-80 pg/ml)]. Similar differences were observed in the level of IL-18 [median (interquartile range), 508.4 pg/ml (180-1222 pg/ml) versus 100.5 pg/ml (78.1-154 pg/ml)]. The differences in TNF-alpha levels between the groups of ANA-positive and ANA-negative patients were at the verge of statistical significance, p<0.05. The sera levels of IL-1beta and IL-6 were low and of no significant difference concerning the ANA-positive and ANA-negative subgroups. Since serum levels of IL-12 and IL-18 were higher in ANA-positive patients than in ANA-negative patients, these cytokines might play an important role in the inflammatory process in SCLE.     

A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line.

Intestinal epithelial cells are known to secrete a variety of cytokines and may play a role in the immune response at the intestinal mucosa. However, the regulatory mechanisms that govern the secretion of these cytokines are largely unknown. In this report, we have focused on the cytokine interactions that regulate interleukin (IL)-6 secretion by the non-transformed rat small intestinal epithelial cell line IEC-6. Tumour necrosis factor-alpha (TNF-alpha) was found to enhance both IL-6 mRNA expression and protein secretion by the IEC-6 cells. Furthermore, TNF-alpha acted in synergy with either transforming growth factor-beta 1 (TGF-beta 1) or IL-1 beta to greatly enhance IEC-6 cell IL-6 secretion. Although the IEC-6 cells are known to produce TGF-beta, autocrine-secreted TGF-beta was found to have no effect on the elevated IL-6 secretion induced by both TNF-alpha plus IL-1 beta. However, the addition of activated TGF-beta 1 to IEC-6 cultures stimulated with both TNF-alpha and IL-1 beta resulted in greatly elevated levels of IL-6 secretion. Therefore, activated TGF-beta 1 can augment IL-6 secretion stimulated by TNF-alpha and IL-1 beta, either alone or in combination, suggesting that intestinal epithelial cell IL-6 secretion may be under the control of a cytokine network at the intestinal mucosa.     

TNF-alpha, TNF-beta, IL-6, and IL-10 polymorphisms in patients with lung cancer

Apart from cigarette smoking, genetic factors seem to be of importance in the development of lung cancer. The present case-control study investigated frequencies of five inflammatory response gene polymorphisms (TNF-alpha-308, TNF-beta-Intron1-252, IL-6-174, IL-10-819 and IL-10-1082) in patients with lung cancer and controls. The study population consisted of 117 patients with lung cancer (77 patients with NSCLC, including 40 Squamous Cell Carcinoma and 26 Adenocarcinoma, and 40 patients with SCLC), 117 matched controls without pulmonary disease and 243 healthy individuals (population control). Genotype analyses revealed no difference in genotype frequencies using matched-pair analysis. However, in comparison to the population control, the IL-10-1082 genotypes carrying the G allele appeared with higher frequency in the SCLC group (p=0.006) [SCLC: 84.6%, population controls: 64.6%]. This yields an odds ratio of 3.01 for SCLC (95% CI = [1.21, 7.48]). No associations were seen for all other polymorphisms analysed. The study raises the possibility of a correlation between the IL-10-1082_G allele and the presence of SCLC in a German population. The functional IL-10-1082 polymorphism correlates with altered IL-10 levels and might influence lung cancer susceptibility by altered inflammatory responses in the airways.     

IL-1, IL-6 and TNF-alpha concentrations in the peritoneal fluid of women with pelvic adhesions.

BACKGROUND: Pelvic adhesions are a significant cause of morbidity and are associated with infertility and pain. The three pro-inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha are involved in adhesion formation/reformation. METHODS: The concentration of these three cytokines was examined in the peritoneal fluid of women undergoing laparoscopy, in order to examine the factors affecting their concentrations, with specific reference to the presence or absence of adhesions. RESULTS: We found that the concentration of TNF-alpha in the peritoneal fluid was significantly correlated to the menstrual cycle day (P < 0.01), with increasing concentration as the menstrual cycle progressed from the follicular to the luteal phase. In contrast, IL-1 and IL-6 levels did not show any variation throughout the menstrual cycle. Increased TNF-alpha was found in patients with pelvic adhesions compared with those with normal pelvis; the concentration of TNF-alpha was highest in mild compared with severe adhesions. In contrast, IL-1 concentration was higher in the presence of severe adhesions. IL-6 levels were significantly correlated with the grade of endometriosis (P < 0.05), but there were no significant correlations of either TNF-alpha or IL-1 concentrations with the various grades of endometriosis. CONCLUSION: The exact role of TNF-alpha and IL-1 in adhesion formation is still unknown, but the results from this study suggest that their concentration in the peritoneal fluid is associated with the degree of adhesions present.     

Induction of IL-1Ra in resistant and responsive hepatitis C patients following treatment with IFN-con1.

Hepatitis C virus (HCV) infection is resistant to interferon-alpha (IFN-alpha) in some patients. The mechanism of this resistance is unknown. Interleukin-1 receptor antagonist (IL-1Ra) is induced by IFN-alpha and is a good indicator of IFN activity. In the current study, we compared IL-1Ra levels in rapid virologic responders and flat responders who showed resistance to IFN. Three groups of patients were examined, including those who received a single dose of consensus IFN (IFN-con1), patients who received daily IFN-con1 for 1 week, and patients who received IFN-con1 daily for 24 weeks. Serum IL-1Ra, IL-6, and HCV RNA were measured serially in all groups. Serum IL-1Ra levels increased rapidly in all patients with hepatitis C after IFN-alpha administration, irrespective of their virologic response. IL-1Ra levels remained elevated at 1 week but were similar to baseline by week 2 of treatment in patients receiving continuous therapy. IL-6 levels also increased acutely but rose more slowly than IL-1Ra levels. The increase in IL-1Ra and IL-6 observed in both flat and rapid virologic responders indicates that IFN receptors are functioning in patients with IFN-resistant hepatitis C and that the lack of response is related to other virologic or immunologic factors.     

Inhibition of IL-1, IL-6, and TNF-alpha in immune-mediated inflammatory diseases

Blockade of cytokines, particularly of tumour necrosis factor alpha (TNF-α), in immuno-inflammatory diseases, has led to the greatest advances in medicine of recent years. We did a thorough review of the literature with a focus on inflammation models in rodents on modified gene expression or bioactivity for IL-1, IL-6, and TNF-α, and we summarized the results of randomized controlled clinical trials in human disease. What we have learned herewith is that important information can be achieved by the use of animal models in complex, immune-mediated diseases. However, a clear ranking for putative therapeutic targets appears difficult to obtain from an experimental approach alone. This is primarily due to the fact that none of the disease models has proven to cover more than one crucial pathogenetic aspect of the complex cascade of events leading to characteristic clinical disease signs and symptoms. This supports the notion that the addressed human immune-mediated diseases are polygenic and the summation of genetic, perhaps epigenetic, and environmental factors. Nevertheless, it has become apparent, so far, that TNF-α is of crucial importance in the development of antigen-dependent and antigen-independent models of inflammation, and that these results correlate well with clinical success. With some delay, clinical trials in conditions having some relationship with rheumatoid arthritis (RA) indicate new opportunities for blocking IL-1 or IL-6 therapeutically. It appears, therefore, that a translational approach with critical, mutual reflection of simultaneously performed experiments and clinical trials is important for rapid identification of new targets and development of novel treatment options in complex, immune-mediated, inflammatory diseases.     

Perioperative serum levels of tumour-necrosis-factor alpha (TNF-alpha), IL-1 beta, IL-6, IL-10 and soluble IL-2 receptor in patients undergoing cardiac surgery with cardiopulmonary bypass without and with correction for haemodilution.

Cardiac surgery with cardiopulmonary bypass (CPB) leads to a systemic inflammatory response with secretion of cytokines. Alterations in the serum concentrations of cytokines have important prognostic significance. Reports on cytokine release during cardiac surgery with CPB have yielded conflicting results. Haemodilution occurs with the onset of CPB resulting in large fluid shifts during the perioperative course of cardiac procedures. In the present study we compare the perioperative course of serum concentrations of TNF-alpha, IL-1beta, IL-6, IL-10 and sIL-2R with and without correction for haemodilution in patients undergoing coronary artery bypass grafting (CABG) surgery. Twenty male patients undergoing elective CABG surgery with CPB and general anaesthesia using a balanced technique with sufentanil, isoflurane and midazolam were enrolled into the study. Serum levels of TNF-alpha, IL-1beta, IL-6, IL-10 and sIL-2R were measured using commercially available ELISA kits. Simultaneous haematocrit values were obtained at all sample times. Statistical analysis was performed by non-parametric analysis of variance and t-tests for data corrected for haemodilution and data that were not corrected for haemodilution. Adjusted significance level was P < 0.01. Intra-operatively, up to the second post-operative day PCV values were significantly decreased compared with preoperative values. Cytokine measurements not corrected for haemodilution were significantly lower than the corrected values. The perioperative haemodilution and decrease in PCV may lead to an underestimation of the cytokine secretion in post-operative patients. We conclude that cytokine measurements were significantly influenced by the perioperative haemodilution and the subsequent decrease in PCV and may in part account for the varying results reported in the literature regarding cytokine release in patients undergoing CABG surgery.     

Production of IL-1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE.

Depositions of immune-complexes are responsible for many of the pathological features of systemic lupus erythematosus (SLE). For example, immune-complex-induced tissue damage in glomerulonephritis has been shown to be mediated, at least in part, by interleukin (IL)-1. Inappropriate production or function of IL-1 may therefore contribute to disease manifestations in SLE. We investigated lipopolysaccharide (LPS)- and adherent IgG-stimulated release of IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-10, a potent modulator of IL-1, by blood mononuclear cells from patients with SLE. Mediator production was measured as ng cytokines/10(6) monocytes and compared with clinical parameters. Release of IL-1beta was only detectable in LPS-stimulated cultures and substantially reduced in patients with both active and inactive disease (P < 0.001). LPS-stimulated IL-1ra release was normal and the IL-1ra/IL-1beta ratio was therefore increased (P < 0.05) and correlated inversely to prednisolone dosage (P = 0.009). IgG-stimulated release of IL-1ra was reduced in patients with active disease compared to those with inactive disease and controls (P = 0.002). IL-10 release was similar in patients and controls. We conclude that monocytes from patients with active SLE are deficient in Fc gamma-R-mediated production of IL-1ra, whereas LPS-stimulated IL-1beta release by SLE monocytes is reduced regardless of disease activity. The former may contribute to immune-complex-mediated tissue damage in SLE.