Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Interleukin-4 differentially regulates prostaglandin production in amnion-derived WISH cells stimulated with pro-inflammatory cytokines and epidermal growth factor.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase in human skeletal muscle

Physiological stress associated with muscle damage results in systemic insulin resistance. However, the mechanisms responsible for the insulin resistance are not known; therefore, the present study was conducted to elucidate the molecular mechanisms associated with insulin resistance after muscle damage. Muscle biopsies were obtained before (base) and at 1 h during a hyperinsulinemic-euglycemic clamp (40 mU x kg(-1) x min(-1)) in eight young (age 24+/-1 yr) healthy sedentary (maximal O(2) consumption, 49.7+/-2.4 ml x kg(-1) x min(-1)) males before and 24 h after eccentric exercise (ECC)-induced muscle damage. To determine the role of cytokines in ECC-induced insulin resistance, venous blood samples were obtained before (control) and 24 h after ECC to evaluate ex vivo endotoxin-induced mononuclear cell secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Glucose disposal was 19% lower after ECC (P<0.05). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was 45% lower after ECC (P<0.05). Insulin-stimulated phosphatidylinositol (PI) 3-kinase, Akt (protein kinase B) serine phosphorylation, and Akt activity were reduced 34, 65, and 20%, respectively, after ECC (P < 0.05). TNF-alpha, but not IL-6 or IL-1beta production, increased 2.4-fold 24 h after ECC (P<0.05). TNF-alpha production was positively correlated with reduced insulin action on PI 3-kinase (r = 0.77, P = 0.04). In summary, the physiological stress associated with muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase, presumably leading to decreased insulin-mediated glucose uptake. Although more research is needed on the potential role for TNF-alpha inhibition of insulin action, elevated TNF-alpha production after muscle damage may impair insulin signal transduction.     

Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines.

Macrophage inflammatory protein (MIP)-3alpha is a chemokine involved in the migration of T cells and immature dendritic cells. To study the contribution of proinflammatory cytokines and chemokines to the recruitment of these cells in rheumatoid arthritis (RA) synovium, we looked at the effects of the monocyte-derived cytokines IL-1beta and TNF-alpha and the T cell-derived cytokine IL-17 on MIP-3alpha production by RA synoviocytes. Addition of IL-1beta, IL-17, and TNF-alpha induced MIP-3alpha production in a dose-dependent manner. At optimal concentrations, IL-1beta (100 pg/ml) was much more potent than IL-17 (100 ng/ml) and TNF-alpha (100 ng/ml). When combined at lower concentrations, a synergistic effect was observed. Conversely, the anti-inflammatory cytokines IL-4 and IL-13 inhibited MIP-3alpha production by activated synoviocytes, but IL-10 had no effect. Synovium explants produced higher levels of MIP-3alpha in RA than osteoarthritis synovium. MIP-3alpha-producing cells were located in the lining layer and perivascular infiltrates in close association with CD1a immature dendritic cells. Addition of exogenous IL-17 or IL-1beta to synovium explants increased MIP-3alpha production. Conversely, specific soluble receptors for IL-1beta, IL-17, and TNF-alpha inhibited MIP-3alpha production to various degrees, but 95% inhibition was obtained only when the three receptors were combined. Similar optimal inhibition was also obtained with IL-4, but IL-13 and IL-10 were less active. These findings indicate that interactions between monocyte and Th1 cell-derived cytokines contribute to the recruitment of T cells and dendritic cells by enhancing the production of MIP-3alpha by synoviocytes. The inhibitory effect observed with cytokine-specific inhibitors and Th2 cytokines may have therapeutic applications.     

Effects of interleukin-1-beta,interleukin-6 and tumor necrosis factor-alpha,alone or in association with hexarelin or galanin,on growth hormone gene expression and growth hormone release from pig pituitary cells

OBJECTIVE: We studied the effects of IL-1beta, IL-6 and TNF-alpha on GH gene expression and secretion with or without galanin and hexarelin. METHODS: Pituitary cells from adult pigs were treated with IL-1beta, IL-6 or TNF-alpha (1, 10 and 100 ng/ml), alone or in association with galanin or hexarelin (10(-8) M): GH mRNA was measured by RT-PCR and GH secretion by ELISA. RESULTS: IL-1beta (1, 10 and 100 ng/ml) and IL-6 (1 and 10 ng/ml) significantly (p < 0.05) enhanced GH output. IL-1beta and TNF-alpha (1 and 10 ng/ml) reduced (p < 0.05) the galanin-induced GH secretion and IL-6 (10 ng/ml) potentiated the effect of both GH releasers (p < 0.05). GH gene expression was increased only by IL-6 at the concentrations of 1 and 10 ng/ml, either alone or in association with both galanin and hexarelin. CONCLUSIONS: We hypothesize that cytokines may play a paracrine/autocrine role in GH regulation in the pituitary independently from the intracellular pathways of the GH secretagogues.     

Interleukin-1beta in the functional and structural luteolysis. Relationship with the nitric oxide system

The aim of the present report was to investigate the in vitro effect of interleukin-1beta(IL-1beta) on corpus luteum (CL) function and some aspects of this mechanism involved. Ovarian rat dispersates from mid-luteal phase were exposed to different doses of IL-1beta (1, 10, 20 ng/ml). Meanwhile 1, 10 and 20 ng/ml of IL-1beta decreased progesterone (P4) production, only the highest doses of IL-1beta increased prostaglandin F2alpha (PGF2alpha) levels. To investigate the possible relationship between PGs production and P4 synthesis, we incubated together IL-1beta (20 ng/ml) and indomethacin (0.1 mM) a potent inhibitor of cyclooxygenase pathway. We found that P4 inhibition induced by IL-1beta was completely prevented by addition of indomethacin. On the other hand, when ovarian rat tissue were exposed at 20 ng/ml of IL-1beta (doses that affected both PGF2alpha and P4 production) the nitric oxide synthase (NOS) activity was augmented. Moreover, IL-1beta effects on PGF2alpha and P4 levels were impaired when a NOS inhibitor N(W)-nitro- L -arginine methyl ester (L-NAME, 600 microM) was added to the incubation media. These data demonstrate that: (i) at the tested doses (1-20 ng/ml), IL-1beta is involved in CL function through the diminution of P4 production of whole ovarian dispersate culture; (ii) at the highest doses assayed (20 ng/ml) IL-1beta increased PGF2alpha production; (iii) at these doses, IL-1beta decreased P4 production by means of a cyclooxygenase pathway and (iv) the NO system would be a key intermediary second messenger in the IL-1beta actions.     

Morphological and immunological evidence of a unique selective production and endoplasmic reticular accumulation of interleukin-1alpha in rat peritoneal macrophages induced by Pseudomonas aeruginosa exotoxin A

The immunotoxicity of Pseudomonas aeruginosa exotoxin A (ETA) on macrophages was evaluated by incubating rat peritoneal macrophages (RPM) with 1-100 ng/ml ETA for 3-60 h. Although the overall changes in cell viability and DNA, RNA, and protein synthesis of the ETA-treated RPM (E-RPM) were reduced in a dose- and time-dependent manner, there was a transient but evident rebound in RNA and/or protein synthesis at 24-36 h post-incubation (HPI) at 1-50 ng/ml ETA. However, a more apparent enhancement appeared in RNA and protein synthesis at 36-48 HPI in 10 and 50 ng/ml E-RPM after normalized on the basis of viable cell. Most 50-100 ng/ml E-RPM underwent necrosis/apoptosis before 24 HPI. By 36 HPI, 41% of 10 ng/ml E-RPM remained viable but were full of cytoplasmic granules due to the accumulation of glycoprotein in segmentally dilated endoplasmic reticulum. Immunological staining of the granules revealed strong IL-1alpha but weak or no signals for IL-1beta, IL-1 receptor antagonist, IL-6, and TNF-alpha. A time-dependent increase in IL-1alpha but no IL-1beta was detected in cell lysate of 10 ng/ml E-RPM; however, neither IL-1alpha nor IL-1beta was detected in culture supernatant. Thus, besides cytopathic and functional effects, ETA could induce a unique selective production and endoplasmic reticular accumulation of IL-1alpha in RPM.     

Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-alpha bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-alpha. RA synoviocytes were cultured with TNF-alpha (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-alpha and p55 and p75 soluble receptors were measured using ELISA. TNF-alpha induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 +/- 23.3 ng/ml versus 27.4 +/- 20.9 ng/ml; P = 0.05). This high circulating TNF-alpha bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 +/- 23.7 ng/ml versus 3.4 +/- 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 +/- 6.2 ng/ml versus 0.0 +/- 3.0 ng/ml; P < 0.05). Levels of circulating TNF-alpha bioactivity were predictive of clinical response to TNF-alpha inhibition, confirming a key role for TNF-alpha in these RA patients.     

Influence of carbohydrate ingestion on immune changes after 2 h of intensive resistance training.

Thirty strength-trained subjects were randomized to carbohydrate (CHO) or placebo (Pla) groups and lifted weights for 2 h (10 exercises, 4 sets each, 10 repetitions, with 2- to 3-min rest intervals). Subjects received 10 ml x kg(-1) x h(-1) CHO (6%) or Pla beverages during the weight training bout. Blood, saliva, and vastus lateralis muscle biopsy samples were collected before and after exercise. Blood cell counts were determined, and plasma was analyzed for IL-6, IL-10, IL-1 receptor antagonist (IL-1ra), IL-8, and cortisol. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-gamma, TNF-alpha) by use of real-time quantitative RT-PCR. Significant but modest increases were measured for plasma IL-6, IL-10, IL-1ra, and IL-8, but the pattern of increase did not differ between CHO and Pla groups. The rate of decrease in muscle glycogen content did not differ between CHO and Pla (P = 0.463). Muscle cytokine mRNA was detected preexercise for IL-1beta, IL-6, IL-15, IL-8, and TNF-alpha, and of these, IL-1beta, IL-6, IL-8, and TNF-alpha were significantly increased after the 2-h weight training bout. The increase in mRNA (fold difference from preexercise) did not differ between CHO and Pla groups. In summary, CHO vs. Pla ingestion did not alter modest increases measured for plasma IL-6, IL-10, IL-1ra, and IL-8, and muscle gene expression for IL-1beta, IL-6, IL-8, and TNF-alpha in strength-trained subjects lifting weights intensively for 2 h.     

Changes in airway resistance by simultaneous exposure to TNF-alpha and IL-1beta in perfused rat lungs

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are formed simultaneously under inflammatory conditions such as asthma and acute respiratory distress syndrome. Here we investigated the effects of TNF-alpha (10 ng/ml) and/or IL-1beta (10 ng/ml) in isolated blood-free perfused rat lungs. In lungs precontracted with methacholine, IL-1beta alone and IL-1beta/TNF-alpha decreased airway resistance 10 min after administration, whereas TNF-alpha alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2 mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the steroid dexamethasone. We conclude that IL-1beta has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-alpha. However, at later time points (after 40 min), IL-1beta and TNF-alpha in concert cause a COX-2- and thromboxane-dependent bronchoconstriction. Our findings show that TNF-alpha and IL-1beta exert complex and time-dependent effects on lung functions that cannot be predicted by studying each cytokine alone.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressive effect of TNF-alpha and IL-1 on alveolar fibroblast proliferation in sarcoidosis

The nature of soluble factors that regulate fibroblast proliferation have not been finally characterized. Our aim was to study the role of tumour necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) in the suppressive activity of alveolar macrophages on autologous lung fibroblasts proliferation in sarcoidosis. We found that supernatants recovered from alveolar macrophages suppressed the proliferation of alveolar fibroblast in sarcoidosis by 35.5 +/- 1.13% compared to 3 +/- 16% in controls (p < 0.001 between the two groups). This suppression correlated with high content of TNF-alpha and IL-1 in sarcoidosis patients stage II-III (7.7 +/- 2.9 ng/ml TNF-alpha and 157 +/- 53 U/ml IL-1 compared to 3.4 +/- 2.4 ng/ml TNF-alpha and 43 U/ml IL-1 in controls; p < 0.01 and p < 0.001, respectively). Both cytokines in sarcoidosis stage I were within the normal ranges. Exogenous TNF-alpha (1000-0.5 ng/ml) and IL-1 (500-0.24 ng/ml) had an additive suppressive activity on fibroblast proliferation which was partially reversed by indomethacin.     

Proinflammatory cytokines and the hypermetabolism of children with sickle cell disease

Sickle cell anemia (HbSS) includes chronic inflammation, but the origin is unclear. We hypothesized that in stable HbSS patients the inflammation was associated with hypermetabolism. We compared selected hypermetabolic and key immunomodulator indicators in HbSS versus control children and examined associations between measures of hypermetabolism and inflammation. Twelve fasting asymptomatic HbSS children 6-12 years and 9 controls matched for age, gender and fat mass (FM) were studied. Proportional reticulocyte count (retic%) and resting energy expenditure (REE) represented hypermetabolism, and C-reactive protein (CRP) indicated inflammation. Proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), chemokine monocyte chemoattractant protein-1 (MCP-1), and energy balance cytokine leptin were measured. Methods were indirect calorimetry, enzyme-linked immunosorbent assay, and radioimmunoassay. Statistical analysis included simple correlation and regression analysis. REE (51 +/- 6 vs. 43 +/- 12 kcal/kg per fat-free mass (FFM), mean +/- SD), retic% (12 +/- 4 vs. 0.7 +/- 0.3%), CRP (5 +/- 3 vs. 0.3 +/- 0.4 mg/liter), and IL-6 (71 +/- 40 vs. 20 +/- 7 pg/ml) were significantly higher for HbSS than controls (P < 0.05). Conversely, leptin (0.1 +/- 0.1 vs. 2 +/- 1 microg/liter per kgFM) and MCP-1 (34 +/- 5 vs. 41 +/- 4 pg/ml) were significantly lower for the HbSS subjects (P < 0.01). TNF-alpha was not significantly different. There were no significant associations between REE or retic% and any cytokine measured. However, CRP was significantly associated with REE in HbSS (r = 0.8, P = 0.003) and an important predictor of REE/FFM. We provide new evidence for low circulating levels of inflammatory chemokine MCP-1 in stable HbSS children, confirm mostly low cytokine levels, inflammation, and hypermetabolism and demonstrate association of hypermetabolism with inflammation via CRP but not via cytokines.     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

Brown fat UCP1 is not involved in the febrile and thermogenic responses to IL-1beta in mice

The activity of brown adipose tissue (BAT), a site of nonshivering metabolic thermogenesis, has been reported to increase after interleukin (IL)-1beta/lipopolysaccharide injection. To clarify the possible contribution of BAT thermogenesis to whole body febrile response, we investigated febrile and thermogenic response to IL-1beta using mice deficient in uncoupling protein-1 (UCP1), a key molecule for BAT thermogenesis. In wild-type (WT) mice, IL-1beta injection (5 microg/kg ip) increased body temperature (+1.82 degrees C at 20 min), decreased physical activity (-37% at 1 h), and produced a slight and insignificant rise (+15% at 1 h) in oxygen consumption (Vo(2)). Vo(2) dependent on metabolic thermogenesis (DeltaVO2 thermogenesis) calculated by correcting the effect of physical activity was increased after IL-1beta injection (726 +/- 200 ml x h(-1) x kg(-1) at 1 h). Almost the same responses were observed in UCP1-deficient mice, showing 638 +/- 87 ml x h(-1) x kg(-1) of DeltaVO2 thermogenesis at 1 h. In contrast, CL316,243, a selective activator of BAT thermogenesis, increased body temperature, decreased physical activity, and produced a significant rise in Vo2 in WT mice, showing 1,229 +/- 35 ml x h(-1) x kg(-1) of DeltaVO2 thermogenesis at 1 h. These changes were not observed in UCP1-deficient mice. These results, conflicting with a previously proposed idea of a role of BAT in fever, suggest a minor contribution of BAT thermogenesis to IL-1beta-induced fever. In support of this, we found no effect of IL-1beta on triglyceride content and UCP1 mRNA level in BAT, in contrast with apparent effects of CL316,243.     

Preexposure of macrophages to low doses of lipopolysaccharide inhibits the expression of tumor necrosis factor-alpha mRNA but not of IL-1 beta mRNA

LPS is known to be a potent activator of macrophages and induces the production of TNF-alpha and IL-1. However, the signaling events and regulatory mechanisms required for the activation of macrophages by LPS have not been resolved precisely. We show that LPS modulates its own response in macrophages. Proteose peptone-induced murine peritoneal macrophages (P-PEM) produce significant amount of TNF-alpha and IL-1 after stimulation with LPS. However, preexposure of macrophages to low doses (less than 1 ng/ml) of LPS renders them refractory to stimulation by a second round of LPS, as evaluated by production of TNF-alpha. The loss of sensitivity to a second round of LPS was selective for TNF-alpha production as the LPS-primed macrophages retained the ability to produce IL-1. Northern blot analysis was performed with total RNA obtained from control and LPS- (1 ng/ml) primed P-PEM after 3-h stimulation with a second round of LPS. The expression of TNF-alpha mRNA was inhibited in LPS-primed P-PEM, whereas the expression of IL-1 beta mRNA was the same in control and LPS-primed P-PEM, consistent with the data of biologic activities of these two cytokines. Zymosan-induced TNF-alpha production was the same in control and LPS-primed macrophages, indicating that not all of the pathways required for TNF-alpha production were affected by LPS priming. Monokines such as human (h) rIL-1 alpha, hrTNF-alpha, hrIL-6, and murine rIFN-beta could not substitute for the action of low doses of LPS, and addition of indomethacin could not restore TNF-alpha production. These results suggest that exposure of macrophages to low doses of LPS suppresses the production of TNF-alpha, but not of IL-1, by inhibiting the expression of mRNA through a noncyclooxygenase-dependent mechanism. Thus, LPS-induced production of TNF-alpha and IL-1 in macrophages are differently regulated.