Effects of tumor necrosis factor-alpha and interleukin-6 in elderly populations

Aging is associated with low-grade increases in circulating levels of TNF-alpha and IL-6. A wide range of factors, including smoking, obesity, infections, the decline in sex hormones, and the genotype, induce and modify this age-related inflammatory activity, which on the other hand may cause age-related pathology. Several classical risk factors are indeed controlled by TNF-alpha and IL-6. TNF-alpha induces insulin resistance and endothelial dysfunction, IL-6 promotes procoagulant changes and both cytokines cause dyslipidaemia. Moreover, systemic low-grade elevations in both cytokines have been related to cardiovascular diseases and TNF-alpha has been associated with Alzheimer's disease and type 2 diabetes mellitus. TNF-alpha and IL-6 are also differently and independently of each other associated with mortality in elderly populations, indicating points of distinction in the biological effects of the two cytokines. Moreover, the association between cytokines and mortality is independent of co-morbidity, suggesting that low-grade increases in circulating cytokines are strong, independent risk factors of morbidity and mortality in old populations, although life style factors and co-morbidity may modulate levels.     

The effects of peritoneal dialysis and hemodialysis on serum tumor necrosis factor-alpha, interleukin-6, interleukin-10 and C-reactive-protein levels

BACKGROUND: Markers of an acute phase reaction, such as C-reactive protein (CRP) or tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6, are predictive for cardiovascular morbidity and mortality in normal subjects and in chronic renal failure patients. In this study, we aimed to investigate serum TNF-alpha, IL-6, IL-10 and CRP levels in continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) patients. MATERIALS AND METHODS: Serum levels of TNF-alpha, IL-6, IL-10 and CRP levels were measured in 30 patients who were just diagnosed with end-stage renal failure and treated, with 16 CAPD (nine female, seven male) and 14 HD (eight female, six male) patients, before CAPD or HD treatment and after 3 months from the beginning of CAPD or HD in patients with no clinical signs of infection. The control groups were 20 healthy persons of similar age and sex. Serum levels of TNF-alpha, IL-6, IL-10 and CRP were measured by enzyme-linked immunosorbent assay in stable CAPD and HD patients and in healthy persons. RESULTS: The mean serum levels of TNF-alpha, IL-6, IL-10 and CRP showed no significant differences between the CAPD and HD patients for the beginning values and the third month of treatment. However, serum TNF-alpha, IL-6, IL-10 and CRP levels were higher than the control group in the CAPD and HD patients regarding the beginning values and the third month of treatment (p < 0.001). CONCLUSIONS: CAPD and HD of the renal replacement therapy have no effects on serum CRP and cytokines.     

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.     

Mechanisms of tumor necrosis factor-alpha and interleukin-6 induction during human liver transplantation

In human orthotopic liver transplantation (LTX) intraoperative elevations of TNF-alpha (> 100 pg/ml) and IL-6 (>800 pg/ml) have been found to correlate with early post-operative rejections and infections respectively. In this study the possible mechanism responsible for the induction of these cytokines has been investigated during liver allografting in 38 recipients. Intraoperative elevations of TNF-alpha (> 100 pg/ml) were detected in the majority of pre-transplant endotoxin positive recipients (8/12, > 10 endotoxin units/ml), the patients turning endotoxin positive until the end of grafting (3/5), and in a subgroup (6/21 patients), apparently endotoxin negative for the whole operation. Therefore endotoxin (ET) seems to stimulate release of TNF-alpha in approximately 50% of the patients, whereas sensitized Kupffer graft cells or immediate allograft reactivity of the host are likely to account for the remaining TNF-alpha positive cases. Elevations of IL-6 > 800 pg/ml) were found in approximately 50% of the TNF-alpha positive cases, indicating partially independent regulatory pathways for IL-6 induction in the TNF-alpha negative patients. In agreement with a previous study, 11/13 (85%) of the intraoperative TNF-alpha positive recipients rejected their grafts within the first 10 days post-operatively. These data demonstrate that ET/infection associated as well as ET independent/reperfusion associated intraoperative TNF-alpha elevations, promote the initiation of allograft rejection in human liver transplantation. The transient and low endotoxaemia caused by the liver grafting procedure performed without veno-venous bypass seems to be of minor importance in the intraoperative induction of TNF-alpha.     

Exercise-induced downregulation of serum interleukin-6 and tumor necrosis factor-alpha in Egyptian handball players

Muscles of candidates work at various grades of intensity during handball exercises according to the pace of exercise. The movement pattern involves large number of contractions, feints, dodges and numerous changes in movements, all of which are highly responsible for changes in trainer's organs, including the immune system. In this study, inflammatory mediators involving interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in serum of 18 Egyptian male handball players, selected from Tanta club handball under 21 year’s old team, were analyzed. The analysis was established on samples collected just before and immediately after intermediate reasonable exercise via enzyme linked immunosorbent assay (ELISA). Moreover, white blood cells (WBCs) count and other hematological markers including hemoglobin %, hematocrit value, and platelet count were assessed. Our results demonstrated a significant decrease in the levels of IL-6 and TNF-α after exercise compared to those before exercise. This was coupled with an increase in WBCs and platelets count. It is also noteworthy that there was a significant positive correlation between serum levels of IL-6 and TNF-α in the study subjects coupled with a significant negative correlation between IL-6 and WBCs after the exercise. Therefore, it is concluded that intermediate reasonable exercises result in decreased levels of IL-6 and TNF-α, which result in decreasing of the inflammation and help in healing and rapid recovery of muscles of the candidates.     

Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.     

Prolactin enhances production of interferon-gamma,interleukin-12, and interleukin-10, but not of tumor necrosis factor-alpha,in a stimulus-specific manner

Prolactin, an anterior pituitary hormone, has been shown to have a role in immunomodulation. Some reports have shown the importance of prolactin in activating lymphocytes and macrophages, while in hyperprolactinemia patients, prolactin was found to decrease lymphocyte activation and natural killer function. In the present work, at physiological (15ng/ml) and stress-induced levels (30ng/ml) of prolactin, interferon-gamma (IFN-gamma) and interleukin (IL)-12 p70 levels, but not of IL-10 and tumor necrosis factor-alpha (TNF-alpha), increased significantly (p<0.05-0.006) in phytohemeagglutinin (PHA)+lipopolysaccharide (LPS)-stimulated whole blood. However, no such effect was observed at high concentrations of prolactin (100-300ng/ml). In addition, 15ng/ml of prolactin reversed hydrocortisone suppressive effect on IFN-gamma, IL-12 p70, and IL-10 production in PHA+LPS-stimulated whole blood. On the other hand, in LPS-stimulated whole blood, prolactin enhanced significantly (p=0.027) the production levels of IL-10, but not of IFN-gamma, IL-12 p70, and TNF-alpha, in non-concentration-dependent manner. These results suggest that prolactin modulates cytokine response during antigenic response, and this modulation is stimulus specific.     

Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue

High circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are found in patients with hyperinsulinemia. Insulin stimulates release of IL-6 from adipocyte cultures, and it stimulates IL-6 gene expression in insulin-resistant, but not control, rat skeletal muscle. In addition, TNF-alpha may be involved in the pathogenesis of insulin resistance. Therefore, we studied the effect of insulin on IL-6 and TNF-alpha gene expression in human skeletal muscle and adipose tissue. Nine healthy young volunteers participated in the study. They underwent a 6-h hyperinsulinemic euglycemic clamp at a fixed insulin infusion rate, with blood glucose clamped at fasting level. Blood samples drawn at 0, 1, 2, 3, 4, 5, and 6 h were analyzed for IL-6 and TNF-alpha. Muscle and fat biopsies, obtained at 0, 2, 4, and 6 h, were analyzed for IL-6 and TNF-alpha mRNA with real-time PCR. IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, in adipose tissue (ANOVA P = 0.027), whereas there was no significant effect of insulin on skeletal muscles. Plasma IL-6 increased during insulin stimulation. TNF-alpha mRNA increased 2.4-, 1.4-, and 2.2-fold in adipose tissue (ANOVA P = 0.001) and decreased 0.74-, 0.64-, and 0.68-fold in muscle tissue (ANOVA P = 0.04). Plasma levels of TNF-alpha were constant. In conclusion, the finding that insulin stimulates IL-6 and TNF-alpha gene expression in adipose tissue only and inhibits the TNF-alpha production in skeletal muscles suggests a differential regulation of muscle- and adipose tissue-derived IL-6 and TNF-alpha.     

Role of FAN in tumor necrosis factor-alpha and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice

Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.     

Prolactin induced expression of interleukin-1 alpha,tumor necrosis factor-alpha,and transforming growth factor-alpha in cultured astrocytes

Prolactin (PRL) is a potent mitogen in cultured astrocytes. Because one of the major effects of astrocyte proliferation is the expression of inflammatory cytokines, we examined the effect of PRL-induced mitogenesis on the expression of interleukin-1 (IL-1α), tumor necrosis factor-α (TNF-α), and transforming growth factor-α (TGF-α) in cultured astrocytes. Astrocytes were stimulated with PRL or growth hormone (GH), and the expression of cytokines was determined by immunohistochemistry and Western blot analysis. Following incubation of astrocytes with 1 nM PRL for 6 h, strong positive staining of IL-1α and TNF-α, but not TGF-α, was found. No detectable staining for the above cytokines was found in vehicle, or GH treated astrocytes. When astrocytes were incubated in the presence of 1 nM PRL for 18 h, strong positive staining for IL-1α and TGF-α was found. Immunocytochemical analysis of the expression of TNF-α and IL-1α in PRL stimulated astrocytes suggested that the expression of IL-1α preceded the expression of TNF-α. To confirm this observation, Western blot analyses were performed on extracts from astrocytes incubated with 1 nM PRL. In unstimulated astrocytes, IL-1α levels were not detectable. In astrocytes stimulated with 1 nM PRL, expression of IL-1α was clearly detected after 1 h of incubation, and IL-1α levels continued to increase during the course of the experiment (6 h). In contrast, in astrocytes stimulated with 1 nM PRL, an increase in the expression of TNF-α was first apparent after 2 h of incubation. TNF-α levels peaked 3 to 4 h after the addition of PRL, and returned to near control levels after 6 h. Finally, injection of PRL into a wound site in female rats increased the expression of glial fibrillary acid protein (GFAP), an astrocyte specific protein. These data suggest that PRL can stimulate astrogliosis at the wound site in vivo. These data clearly indicate that PRL can stimulate the expression of TNF-α and IL-1α in cultured astrocytes and suggest that PRL may play a role in the regulation of the neuroimmune response in vivo.     

Role of the pro-inflammatory cytokines tumor necrosis factor-alpha,interleukin-1 beta,interleukin-6 and interleukin-8 in the pathogenesis of the otitis media with effusion

Inflammation in the middle ear mucosa, caused usually by bacterial and viral pathogens, is the primary event in the middle ear predisposing the development of otitis media with effusion (OME). Numerous inflammatory mediators have been identified in OME. However, cytokines play a central role as initiators, mediators and regulators of middle ear inflammation and subsequent molecular-pathological processes in middle ear tissues, leading to histopathological changes in the middle ear cavity and the pathogenesis of OME. In this article, we aim to present an overview of current research developments in the pro-inflammatory cytokine involvement in the aetiology of otitis media with effusion.     

Spontaneous and stimulated interleukin-6 and tumor necrosis factor-alpha production at delivery and three months after birth

OBJECTIVE: To study the production and interrelations of maternal and neonatal cytokines (IL-6 and TNF-alpha) during labor, after vaginal delivery and at three months after delivery. METHOD: The unstimulated concentrations of cytokines in the supernatants of whole-blood cultures and concentrations after PMA (phorbol 12-myristate 13-acetate) and concanavalin (conA) stimulation were determined by enzyme-linked immunosorbent assays (ELISAs). The blood samples were from the peripheral veins of 27 healthy women during term labor and immediately after delivery and three months after delivery. Neonatal samples were taken at birth (cord blood) and three months after delivery. RESULTS: IL-6 responses to stimulation were increased in the parturients and in umbilical cord blood at delivery compared with maternal and neonatal samples obtained 3 months postpartum. In contrast, the production of maternal TNF-alpha in peripheral blood was down-regulated at delivery compared with values 3 months postpartum. After an IL-6 and TNF-alpha burst in umbilical cord samples, neonatal cytokine production was at a low level three months after delivery. IL-6 production tended to be higher in both umbilical cord blood as well as in maternal samples after delivery in women who were younger. In addition, TNF-alpha production in umbilical cord blood was significantly higher in those women who were younger. CONCLUSIONS: The production of IL-6 was up-regulated in both the maternal and in umbilical cord blood at delivery. The production of TNF-alpha was up-regulated in umbilical cord blood compared with neonatal values 3 months after birth. Maternal age had effects on IL-6 and TNF-alpha production at delivery.     

Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-alpha

IL-6 and TNF-alpha have been associated with insulin resistance and type 2 diabetes. Furthermore, abnormalities in muscle fatty acid (FA) metabolism are strongly associated with the development of insulin resistance. However, few studies have directly examined the effects of either IL-6 or TNF-alpha on skeletal muscle FA metabolism. Here, we used a pulse-chase technique to determine the effect of IL-6 (50-5,000 pg/ml) and TNF-alpha (50-5,000 pg/ml) on FA metabolism in isolated rat soleus muscle. IL-6 (5,000 pg/ml) increased exogenous and endogenous FA oxidation by approximately 50% (P < 0.05) but had no effect on FA uptake or incorporation of FA into endogenous lipid pools. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into diacylglycerol (DAG) by 45% (P < 0.05). When both IL-6 (5,000 pg/ml) and insulin (10 mU/ml) were present, IL-6 attenuated insulin's suppressive effect on FA oxidation, increasing exogenous FA oxidation (+37%, P < 0.05). Furthermore, in the presence of insulin, IL-6 reduced the esterification of FA to triacylglycerol by 22% (P < 0.05). When added in combination with IL-6 or leptin (10 microg/ml), the TNF-alpha-induced increase in DAG synthesis was inhibited. In conclusion, the results demonstrate that IL-6 plays an important role in regulating fat metabolism in muscle, increasing rates of FA oxidation, and attenuating insulin's lipogenic effects. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into DAG, which may be involved in the development of TNF-alpha-induced insulin resistance in skeletal muscle.     

Tumor necrosis factor-alpha/interleukin-10 balance in normal and cystic fibrosis children

BACKGROUND: The balance between tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) is important for immune homeostasis maintenance. Exuberant production of TNF-alpha contributes to overwhelming inflammatory response and tissue damage. But, commonly, increase in TNF-alpha is counterbalanced by simultaneous synthesis of an anti-inflammatory cytokine IL-10, which suppresses production of many activating and regulatory mediators. AIMS: In the present study, the relationships between TNF-alpha and IL-10 in the plasma of healthy school-children and cystic fibrosis (CF) patients have been investigated. METHODS: Blood samples were obtained from 12 CF patients with chronic pulmonary disease and 18 healthy schoolchildren vaccinated with live attenuated rubella vaccine. IL-10 and TNF-alpha were determined in the plasma samples using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Before vaccination, most healthy children (13 of 18) demonstrated superiority of pro-inflammatory TNF-alpha over anti-inflammatory IL-10 (TNF-alpha/IL-10 > 1). In these subjects, a significant positive linear association between the cytokine values has been found. Vaccine challenge resulted in a marked reduction of TNF-alpha/IL-10 ratios. In addition, a disappearance of correlation between the cytokine values was observed. Such disturbance was related to exuberant elevation of the IL-10 levels after inoculation. On the contrary, in CF individuals, plasma cytokine values remained in strong linear association independently of TNF-alpha or IL-10 predominance. No spikes in the plasma levels of IL-10 in CF patients during a 6-month observation period have been revealed. CONCLUSIONS: There were no fundamental differences between CF and healthy children in the regulation of TNF-alpha and IL-10 secretion. Thus, immune quiescence seemed to be associated with the predominance of TNF-alpha, whereas immune disturbance was characterized by IL-10 superiority. The only abnormality that was found in CF patients consisted of their inability to produce unlimitedly IL-10 in response to antigen stimuli.     

Complement factor I is upregulated in rat hepatocytes by interleukin-6 but not by interferon-gamma, interleukin-1beta, or tumor necrosis factor-alpha.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1beta, TNF-alpha and IFN-gamma for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FI-specific RT-PCR signal in isolated hepatocytes, in the two rat hepatoma-derived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-specific mRNA by about ten-fold. These data are in accord with Northern blot analyses in which the FI-mRNA was upregulated by IL-6 between five- and seven-fold. IL-6, but not IL-1beta, TNF-alpha or IFN-gamma also increased FI-protein levels in cell culture supernatants by about five-fold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.     

Lactate induces tumour necrosis factor-alpha, interleukin-6 and interleukin-1beta release in microglial- and astroglial-enriched primary cultures

Hyperammonaemia has deleterious effects on the CNS in patients with liver dysfunction. Cellular mechanisms underlying the effects of hyperammonaemia are largely unknown, although astrocytes have been the main target of interest. This study investigated how treatment with NH4Cl and lactate, which increase in the brain as a consequence of hyperammonaemia, affects cells in primary rat cultures enriched in either astrocytes or microglia. Morphological changes were studied over time using light microscopy. Release of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta was measured using ELISA. NH4Cl was found to induce vacuole formation in both culture systems. Lactate treatment altered astrocytic appearance, resulting in increased space between individual cells. Microglia adopted a round morphology with either NH4Cl or lactate treatment. Lactate, but not NH4Cl, induced release of TNF-alpha and IL-6 in both astroglial- and microglial-enriched cultures, while IL-1beta was released only in microglial cultures. Cytokine release was higher in the microglial- than in the astroglial-enriched cultures. Additionally, the astroglial-enriched cultures containing approximately 10% microglial cells released more cytokines than cultures containing about 5% microglial cells. Taken together, our data suggest that most TNF-alpha, IL-6 and IL-1beta release comes from microglia. Thus, microglia could play an important role in the pathological process of hyperammonaemia.     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.