Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

IL-6 and TNF-alpha expression in,and release from,contracting human skeletal muscle

The aim of the present study was to examine whether IL-6 and TNF-alpha are expressed in, and released from, human skeletal muscle during exercise. We hypothesized that the skeletal muscle will release IL-6, but not TNF-alpha, during exercise because of previous observations that TNF-alpha negatively affects glucose uptake in skeletal muscle. Six healthy, male subjects performed 180 min of two-legged knee-extensor exercise. Muscle samples were obtained from the vastus lateralis of one limb. In addition, blood samples were obtained from a femoral artery and vein. Plasma was analyzed for IL-6 and TNF-alpha. We detected both IL-6 and TNF-alpha mRNA in resting muscle samples, and whereas IL-6 increased (P < 0.05) approximately 100-fold throughout exercise, no significant increase in TNF-alpha mRNA was observed. Arterial plasma TNF-alpha did not increase during exercise. Furthermore, there was no net release of TNF-alpha either before or during exercise. In contrast, IL-6 increased throughout exercise in arterial plasma, and a net IL-6 release from the contracting limb was observed after 120 min of exercise (P < 0.05).     

Differential regulation of IL-6 and TNF-alpha via calcineurin in human skeletal muscle cells

Interleukin-6 increases in skeletal muscle during exercise, and evidence points to Ca2+ as an initiator of IL-6 production. However, the signalling pathway whereby this occurs is unknown. One candidate for Ca2+ -mediated IL-6 induction is calcineurin, an activator of NF-AT. Here we investigated whether skeletal myocytes produce IL-6 in a Ca2+/calcineurin-dependent manner, and whether TNF-alpha, an inducer of IL-6, is affected by these stimuli. Human skeletal muscle cell cultures were stimulated with ionomycin time-and dose-dependently to elevate intracellular Ca2+ levels, with or without addition of cyclosporin A (CSA); a calcineurin inhibitor. mRNA was extracted from myocytes and analysed for IL-6 and TNF-alpha gene expression. IL-6 mRNA increased time- and dose-dependently with ionomycin stimulation, an effect that was blunted by approximately 75% in the presence of CSA. In contrast, TNF-alpha gene expression was decreased by approximately 70% in response to ionomycin treatment, but increased in response to addition of CSA. These data demonstrate that IL-6 and TNF-alpha are regulated differentially in skeletal muscle cells in response to a Ca2+ stimulus. Blocking the calcineurin pathway resulted in inhibition of the IL-6 response to ionomycin, whereas TNF-alpha increased by addition of CSA, further indicating a differential regulation of IL-6 and TNF-alpha in human skeletal myocytes.     

Selenium prevents the growth stimulatory effects of cadmium on human prostatic epithelium

Cadmium has been implicated in the increase in prostate cancer incidence in men exposed to high levels. A decrease in zinc and a concomitant increase in cadmium levels in the human prostate has been shown. The role and mechanism of cadmium action in prostate carcinogenesis is not clear. Selenium, on the other hand, has been shown to inhibit carcinogenesis in several animal systems. Results show that cadmium stimulates the growth of human prostatic epithelium in vitro, between 10(-9) M and 10(-7) M concentrations. Selenium, at concentrations between 10(-12) M and 10(-7) M shows no growth stimulatory or inhibitory effects on these cells. However, when present at 10(-8) M level, selenium inhibits the growth stimulation induced by cadmium. These results suggest that selenium may be useful in counteracting the effects of cadmium in the human prostate and offer possibilities for investigations on the protective effects of selenium in cadmium-related carcinogenesis in man.     

Differential effects of hemorrhage and LPS on tissue TNF-alpha, IL-1 and associate neuro-hormonal and opioid alterations

LPS administration and hemorrhage are frequently used models for the in vivo study of the stress response. Both challenges stimulate cytokine production as well as activate opiate and neuro-endocrine pathways; which in turn modulate the inflammatory process. Differences in the magnitude and tissue specificity of the proinflammatory cytokine and neuro-hormonal responses to these stressors are not well established. We contrasted the tissue specificity and magnitude of the increase in circulating and tissue cytokine (TNF-alpha, IL-1alpha and IL-1beta) content in response to either fixed-pressure hemorrhage (approximately 40 mm Hg) followed by fluid resuscitation (HEM) or lipopolysaccharide (LPS; 100 microg/100 g BW) administration. LPS and HEM elevated circulating levels of TNF-alpha, while neither stress altered circulating IL-1-alpha and IL-beta. LPS-induced increases in TNF-alpha content were greater than those elicited by HEM in all tissues studied except for the lung, where both stressors produced similar increases. Tissue (lung, spleen and heart) content of IL-1alpha was increased by HEM but was not affected by LPS. Tissue (lung, spleen, and heart) content of IL-1beta was increased by LPS but was not affected by HEM. HEM produced greater increases than LPS in epinephrine (16- vs. 4-fold) and norepinephrine (4-fold vs. 60%) levels and similar elevations in beta-endorphin. LPS produced greater elevation in corticosterone levels (2-fold) than HEM (50%). These results suggest differential tissue cytokine modulation to HEM and LPS, both with respect to target tissue and cytokine type. The hormonal milieu to HEM is characterized by marked catecholaminergic and moderate glucocorticoid while that of LPS is characterized by marked glucocorticoid with moderate catecholaminergic influence.     

Role of Ca(2+) on TNF-alpha and IL-6 secretion from RBL-2H3 mast cells

Ca2+ acts as an important second messenger in mast cells. However, the mechanisms involved in the secretion of inflammatory cytokines from activated mast cells are unknown. In this study, we examined the signaling pathway involved in calcium-related cytokine secretion in a mast cell line, RBL-2H3 cells. We report that treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a chelator of intracellular calcium, can inhibit IgE-stimulated TNF-alpha and IL-6 secretion in a concentration-dependent manner with IC50 values of 0.41 and 0.014 microM, respectively. Maximal inhibition of TNFalpha- and IL-6 secretion was 58.5 +/- 3% and 87 +/- 8% in BAPTA-AM, respectively. BAPTA-AM also completely inhibited the IgE-induced TNF-alpha and IL-6 mRNA levels. In activated RBL-2H3 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus. However, the level of NF-kappaB/Rel A in nucleus was decreased by treatment of BAPTA-AM. In addition, BAPTA-AM completely inhibited the IgE-induced IkappaB kinase beta (IKKbeta) activation and IkappaBalpha phosphorylation. These observations demonstrate that the intracellular Ca2+ may play an important role in IgE-induced TNF-alpha and IL-6 secretion from mast cells via IKKbeta activation.     

Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2-JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4-NF-kappaB and TLR4-p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.     

Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells

We investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-alpha) and one chemokine (IL-8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-alpha mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius. IL-8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.     

Apoprotein E isoform-dependent expression and secretion of pro-inflammatory cytokines TNF-alpha and IL-6 in macrophages

The anti-atherogenic properties of human apoprotein E-associated lipoproteins have been partially attributed to its anti-inflammatory properties. We studied if endogenously expressed apoprotein E (apoE) elicits isoform-dependent effects on pro-inflammatory cytokine expression and secretion. Mouse J774A.1 peritoneal macrophages without native expression of apoE were used to establish cell lines with stable expression of the three human apoE isoforms, apoE2, apoE3 and apoE4. In the presence of lipopolysaccharide (LPS), expression and secretion of TNF-alpha and IL-6 in cells expressing different apoE isoforms were determined by RT-PCR, immunoblotting and ELISA assays. ApoE3-expressing cells have significantly lower expression and secretion levels of the two cytokines as compared to cells with apoE2 and apoE4 expression. Such observations were accompanied with the lowest ERK1/2 activity in apoE3-expressing cells. Further study shows that the apoE isoform-dependent variations of TNF-alpha and IL-6 expression/secretion in macrophages are diminished in the presence of ERK1/2 inhibitor U0126. In conclusion, apoE elicits isoform-dependent effects on macrophage TNF-alpha and IL-6 expression as well as secretion. The ERK1/2 signaling pathways are involved in mediating such apoE isoform-dependent effects.     

Ex vivo lipopolysaccharide (LPS)-induced TNF-alpha, IL-1beta, IL-6 and PGE2 secretion in whole blood from Type 1 diabetes mellitus patients with or without aggressive periodontitis

Several studies have demonstrated that diabetes is a risk factor for developing periodontal disease, increasing its prevalence and severity. Furthermore, periodontitis may impair the metabolic control and adequate treatment of diabetic patients. LPS from Gram-negative bacteria penetrates the periodontal tissues and subsequently recruits and activates immune cells. Progression to severe periodontitis with loss of supporting structures is mediated by several factors, including secretion of a broad spectrum of inflammatory and destructive (PGE2). mediators such as cytokines (TNF-alpha, IL-1b and IL-6), chemokines (IL-8) and prostaglandin E2. The aim of this work is to investigate differences in the TNF-a, IL-1b and IL-6 expression and prostaglandin E2 (PGE2) release in blood from diabetic patients with and without aggressive periodontitis (AP) stimulated with lipopolysaccharide (LPS). For this purpose we recruited 29 Type 1 diabetes mellitus (DM) patients, 14 with AP and 15 without AP. Fourteen healthy individuals formed the control group. For cytokine expression and PGE2 secretion, an ex vivo whole blood culture system was used. Cytokines and PGE2 were detected by commercial immunometric assays. A wide range of inter-individual variability in spontaneous and LPS-induced TNF-alpha, IL-1b and IL-6 levels in patient groups and controls was found. The mean of spontaneous and LPS-induced TNF-alpha and IL-1b levels did not differ significantly (p > 0.5) when patients were compared to control individuals. Although not significant, the spontaneous TNF-alpha, IL-1b and IL-6 levels in the group of Type 1 DM with AP were higher than in controls, while in diabetic patients without AP, these values were depressed in comparison with controls. In both groups of patients, the means of LPS-induced IL-6 levels were higher than the controls but the differences observed were not significant (p = 0.07). However, the LPS-induced PGE2 levels varied significantly when all groups were compared (p = 0.007). The means of LPS-induced PGE2 levels for Type 1 diabetic patients with AP (p = 0.0009) and without AP (p = 0.024) were significantly higher than the levels observed for healthy controls. Finally, we conclude that Type 1 diabetic patients with or without AP did not express higher LPS-induced TNF-a, IL-1b and IL-6 levels than controls. However, the PGE2 levels released were significantly higher than those detected in controls.     

Immunization of mice with T cell-dependent antigens promotes IL-6 and TNF-alpha production in muscle cells

IL-6 and TNF-alpha are proinflammatory cytokines involved in various inflammatory or non-inflammatory disorders characterized by muscle wasting. While infiltrating leukocytes are known to be the major source of these cytokines, it is unclear whether muscle cells also contribute to local inflammation. In this study, we first showed that cultured muscle cells and naive mouse muscle tissue were capable of producing IL-6 and TNF-alpha. We demonstrated an increased expression of IL-6 and TNF-alpha on muscle sections of C57BL/6J mice immunized with acetylcholine receptor (AChR) in the complete Freund's adjuvant (CFA) or with CFA only. In the presence of IL-6 or TNF-alpha, cultured AChR-expressing mouse (G-8) and human (TE671) skeletal muscle cells showed significantly decreased alpha-bungarotoxin-binding sites as measured by cellular ELISA. Moreover, IL-6- or TNF-alpha-treated muscle cells displayed a considerable increase in apoptotic cell ratios. Collectively, this study suggests a direct role for these two cytokines in muscle cell destruction and a possibility of muscle cell damage via an autocrine fashion.     

A bacteria-induced switch of sympathetic effector mechanisms augments local inhibition of TNF-alpha and IL-6 secretion in the spleen.

It is believed that an inflammation-induced activation of the CNS leads to an inhibition of overshooting immune responses to prevent extensive local cytokine secretion. However, immunosuppression by the sympathetic nervous system may be unfavorable when bacteria are present locally and when TNF-alpha is necessary to overcome infection. We now report in a superfusion model, using mouse spleen slices, that although local Pseudomonas aeruginosa increased splenic TNF-alpha and IL-6 secretion severalfold over basal levels, electrically released neurotransmitters attenuated cytokine secretion to similar basal level as under bacteria-free conditions. Bacteria reversed noradrenergic inhibitory effector mechanisms: Under bacteria-free conditions, TNF-alpha secretion was very low and IL-6 secretion was mainly inhibited by alpha2-adrenoreceptor ligation. In the presence of bacteria, TNF-alpha and IL-6 secretion were high and IL-6 secretion was mainly inhibited by beta-adrenoreceptor ligation. The alpha- to beta-adrenoswitch of IL-6 inhibition in the presence of bacteria was mediated by the prior adrenergic regulation of TNF-alpha. In vivo, chemical abrogation of sympathetic inhibition reduced accumulation of bacteria in the spleen, which depended at least in part on TNF-alpha. This suggests that activation of the sympathetic nervous system may be a forerunner for accumulation of bacteria in tissue and consecutively sepsis due to intensified inhibition of TNF-alpha secretion.     

Natural killer and lymphokine-activated killer cell activities from human marrow precursors. II. The effects of IL-3 and IL-4

Both IL-3 and IL-4 have multi-CSF activity on early marrow progenitors. We have examined the effect of IL-3 and IL-4 on the differentiation of NK cells from their marrow-derived precursors and have further examined the interactions of these cytokines with IL-2 and IL-1. We tested marrow which had been depleted of mature cells and of E rosette-positive cells (including NK cells) by treatment with soybean lectin and SRBC (SBA-E-BM). The cytolytic activities of the SBA-E-BM samples were tested in 51Cr-release assays after 7 days of liquid culture. K562 targets were used as a measure of NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity was detectable in marrow cultured in medium without cytokines, or in medium containing IL-3, or IL-4 alone. Both of these cytokines were shown to be inhibitory to the IL-2-induced generation of NK and LAK activity from SBA-E-BM at concentrations as low as 1 U/ml. The inhibitory activity of both IL-3 and IL-4 was found to occur early in the marrow cultures, with little or no inhibitory effects seen if added 48 h after IL-2. IL-3 appeared to be specifically inhibitory to NK cell precursors since addition of IL-3 to cultures of PBMC did not inhibit IL-2-induced lytic activities. In contrast, IL-4 was equally inhibitory to the activation of marrow and peripheral blood NK cells by IL-2. Mixing experiments demonstrated that the reduced lytic activity in IL-3 or IL-4 containing marrow cultures were not due to suppression of the NK effectors, nor could marrow cultured in IL-3 or IL-4 serve as targets for IL-2-activated NK cells. Phenotype analysis of the lymphoid cells in marrow cultures containing IL-2 combined with IL-3 or IL-4 revealed fewer cells expressing Leu-11 (CD16), or Leu-19 (CD56) and fewer CD16, CD56 coexpressing cells compared with marrow cultured in medium containing IL-2 alone. The inhibitory activity of IL-4, but not IL-3, could be partially reversed if IL-1 was added to the cultures, suggesting that IL-1 and IL-4 have opposing activities on NK cells responsiveness to IL-2. These interactions between cytokines might be important in the regulation of NK cell differentiation and on the functional activity of mature NK cells.     

Differential effects of LPS and TGF-beta on the production of IL-6 and IL-12 by Langerhans cells, splenic dendritic cells, and macrophages

We examined modulatory effects of lipopolysaccharide (LPS) on IL-6 and IL-12 production by mouse Langerhans cells (LC), spleen-derived CD11c+ dendritic cells (DC), and macrophages (Mphi). Low dose LPS (1 ng/ml) increased IL-6 and IL-12 p40 production by Mphi. LPS slightly augmented IL-6 production but showed no effect on IL-12 p40 production by DC. In contrast, only high dose LPS (1 microg/ml) induced IL-6 but not IL-12 p40 production by LC. CD14 expression was the highest on Mphi and then on DC, but not on LC, which may explain the difference in responsiveness to LPS. We also found that TGF-beta inhibited IL-6 and IL-12 p40 production by LPS-stimulated Mphi. However, TGF-beta did not inhibit IL-6 production and even enhanced IL-12 p40 production by anti-CD40/IFN-gamma-stimulated Mphi. Concerning LC, TGF-beta enhanced IL-6 and IL-12 p40 production when stimulated with anti-CD40/IFN-gamma alone or with anti-CD40/IFN-gamma and LPS. Taken together, these findings indicate diverse effects of LPS and TGF-beta on these antigen presenting cells, which probably represents their differential roles in the innate immunity.