Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion

Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.     

Oral administration of milk fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 to DBA/1 mice inhibits secretion of proinflammatory cytokines

We have previously shown that oral administration of skimmed milk(SM) fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1/SM) to DBA/1 mice inhibited the development of collagen-induced arthritis (CIA). In this study, our aim was to examine possible mechanisms of inhibiting the development of CIA. We studied the effect of OLL1073R-1/SM on cytokine secretion from cells of popliteal lymph nodes (lymph node cells; LNC) of mice. The results showed that feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines such as interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and the chemokine, monocyte chemoattractant protein 1 (MCP-1). The most prominent effect was inhibition of TNF-α. Secretion of IL-2 and IL-4 were not influenced. Feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines produced by accessory cells, but not T cells. We conclude that CIA may be prevented via down regulation of secretion of proinflammatory cytokines such as IL-6, TNF-α and IFN-γ, and of the chemokine of MCP-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

TNF-α, IFN-γ, and IL−1β modulate hyaluronan synthase expression in human skin fibroblasts: Synergistic effect by concomital treatment with FeSO4 plus ascorbate

Several reports have shown that a number of cytokines such as tumor necrosis-α (TNF-α), interferon-γ (IFN-γ), and interleukin-β (IL-1β) are capable to induce hyaluronan sinthases (HASs) mRNA expression in different cell culture types. The obvious consequence of this stimulation is a marked increment in hyaluronan (HA) production. It has been also reported that oxidative stress, by itself, may increase HA levels. The aim of this study was to evaluate how TNF-α, IFN-γ,IL−1β, and exposition to oxidative stress may modulate HAS activities in normal human skin fibroblasts. Moreover, the effects on HAS mRNA expression of the concomitant treatment with cytokines and oxidants, and the HA concentrations after treatments, were studied. TNF-α, IFN-γ, and IL-1β were added to normal or/and exposed to FeSO₄ plus ascorbate fibroblast cultures and HAS1, HAS2 and HAS3 mRNA content, by PCR-real time, was assayed 3,h later. HA levels were also evaluated after 24,h incubation. The treatment of fibroblasts with cytokines up-regulated HASs gene expression and increased HA production. IL-1β induced HAS mRNA expression and HA production more efficiently than TNF-α and IFN-γ. The exposition of the fibroblasts with the oxidant system markedly increased HAS activities while slightly HA production. The concomitant treatment of cells with the cytokines and the oxidant was able to further enhance, in a dose dependent way, with synergistic effect on HAS mRNA expression. On the contrary HA levels resulted unaffected by the concomitant treatment, and resemble those obtained with the exposure to FeSO₄ plus ascorbate only. This lack in HA production could be due to the deleterious action of free radicals on the HA synthesis.     

Severe hypercalcaemia and extensive osteolytic lesions in an adult patient with T cell acute lymphoblastic leukaemia

Hypercalcaemia is a rare feature of acute lymphoblastic leukaemia (ALL) in adults, particularly of the T cell type. We report on a 24-year-old patient with T-ALL, who presented with symptoms of hypercalcaemia (vomitus, acute renal failure), bone pain, extensive osteolytic lesions and normal white cell count without circulating blasts. An increased serum tumor necrosis factor (TNF-α) concentration of 35 pg/ml was found; it remained elevated at, 52 pg/ml four weeks later, after having achieved haematological remission. Serum concentrations of IL-1β, IL-6 and IL-2 were within the control range. The pathophysiology of hypercalcaemia in malignancy and possible mediators of bone resorption, in particular TNF-α, are discussed.     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.     

Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients afterbacillus Calmette-Guerin immunotherapy

Intravesical immunotherapy for bladder cancer is the most effective form of tumour immunotherapy. Following repeated instillations of bacillus Calmette-Guérin (BCG) organisms into the bladder large 0quantities of several cytokines are detected in the urine. These cytokines include interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor α (TNFα), interferon γ (IFNγ) and also soluble intercellular adhesion molecule ICAM-1. In the work reported here we simultaneously quantified urinary levels of TNFα, TNFβ, TNF receptor I and TNF receptor II by enzyme-linked immunosorbent assay (ELISA) techniques and compared this with bioactive levels of TNF. This was undertaken with a limited number of patients throughout a course of six instillations of immuno therapy. Sequential instillations of BCG induced secretion of TNFα and TNFβ into urine. These cytokines were not always secreted simultaneously, perhaps suggesting differential regulation of their synthesis. Maximal concentrations of TNFα were 675 pg/ml and TNFβ 47 pg/ml. High levels of both species of soluble TNF receptor were readily identified in urine. Maximal levels of sTNF-RI were 6200 pg/ml (range from 0) and for sTNF-RII 7800 pg/ml (range from 0). Contrasting with earlier published observations concerning cytokine levels, the concentration of soluble receptor did not increase with repeated instillation. In apparent contrast with the ELISA data, very low levels of bioactive TNF were identified by the L929 bioassay (maximum concentration 1 U/ml) despite the elevated concen t ration of immunoreactive TNF. The large concentrations of soluble TNF receptor in patients’ urine samples could account for the apparently low bioactivity as determined by the L929 cytotoxicity assay. The precise nature of the role of TNF in BCG immunotherapy remains undetermined; however, it is thought that proinflammatory cytokines are in part responsible for the clinical efficacy of this therapeutic approach. Whether other cytokines are antogonised by soluble binding proteins remains to be determined. Furthermore, whether TNF is bioactive in the bladder wall and only neutralised in the urine also requires investigation. Received: 24 August 1994 / Accepted: 17 October 1994     

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.     

Cytokines and Absence Seizures in a Genetic Rat Model

We investigated the role of two cytokines, IL-1β and TNF-α, in the development of absence seizures using a genetic model of absence epilepsy in WAG/Rij rats. We administered these cytokines to animals systemically and measured the number of spike-wave discharges (SWDs) in the EEG. We also coadministered IL-1β with the GABA reuptake inhibitor tiagabine and measured the levels of IL-1β and TNF-α in the brain and blood plasma of 2-, 4-, and 6-month-old WAG/Rij rats and animals that served as a non-epileptic control (ACI). We found that IL-1β induced a significant increase in SWDs 2-5 h after administration, while TNF-α enhanced SWDs much later. Both cytokines enhanced passive behavior; body temperature was elevated only after TNF-α. The action of tiagabine was potentiated by earlier IL-1β injection, even when IL-1β was no longer active. Young WAG/Rij rats showed higher levels of TNF-α in blood serum than young ACI rats; the effects in the brain tended to be opposite. The marked differences in timing of the increase in SWDs suggest different time scales for the action of both cytokines tested. It is proposed that the results found after TNF-α are due to the de novo synthesis of IL-1β. TNF-α may possess neuroprotective effects. IL-1β might increase GABA-ergic neurotransmission. The changes in the efficacy of antiepileptic drugs related to changes in the cytokine systems may have some clinical relevance.     

<Emphasis Type="Italic">H. pylori</Emphasis> related proinflammatory cytokines contribute to the induction of miR-146a in human gastric epithelial cells

MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a in gastric epithelial cell HGC-27 in NF-κB-dependent manner, while the induction of miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori—induced IL-8, TNF-α, and IL-1β. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators to modulate the inflammation.     

G-Protein-coupled receptor agonists differentially regulate basal or tumor necrosis factor-α-stimulated activation of interleukin-6 and RANTES in human airway smooth muscle cells

Summary Using thapsigargin (Tg), an agent that mobilizes calcium by directly emptying intracellular stores, we previously showed that intracellular calcium may play an important role in the regulation of intercellular adhesion molecule (ICAM)-1 gene expression induced by cytokines in human airway smooth muscle (ASM) cells. In the present study, we extended this previous observation by comparing the effect of Tg and other calcium-mobilizing G-protein-coupled receptor (GPCR) agonists on the expression of different pro-inflammatory genes in response to tumor necrosis factor (TNF)-α in ASM cells. We found that in resting cells, Tg (10–100 nM) or the bradykinin (BK) (1–10 μM) and thrombin (Thr) (1 U/ml) stimulated interleukin (IL)-6 secretion but had no effect on regulated on activation, normal T cells expressed and secreted (RANTES) levels. More importantly, such calcium-mobilizing agents significantly enhanced TNF-α-induced IL-6 secretion while RANTES secretion was abrogated. The use of luciferase-tagged IL-6 and RANTES promoter constructs demonstrated similar effects of Tg on IL-6 and RANTES genes in basal and TNF-α-stimulated conditions. The cyclic adenosine monophosphate (cAMP)-dependent pathway plays a minor role in this differential regulation of IL-6 and RANTES genes expression. 2-Aminoethoxydiphenyl borate (APB), a blocker of store-operated calcium channels (SOCs), and bisindolylmaleimide I (Bis I), a broad-spectrum protein kinase C (PKC) inhibitor, inhibited the basal and synergic effects of IL-6 secretion in response to calcium-mobilizing agents and TNF-α, but did not prevent the abrogated effect of RANTES secretion. We also found that Go-6976, a selective calcium-dependent PKC isozyme inhibitor, did not inhibit IL-6 secretion in response to GPCR agonist and TNF-α; whereas Rottlerlin, a PKC-δ inhibitor, inhibited both Thr- and TNF-α-induced expression of IL-6, while BK-induced IL-6 secretion was not affected. Interestingly, TNF-α-induced interferon regulatory factor (IRF)-1 activation was significantly inhibited by all calcium-mobilizing agents, BK, Thr and Tg. These results show that calcium-mobilizing GPCR agonists functionally interact with TNF-α to differentially regulate pro-inflammatory genes expression in human ASM cells, possibly by involving Tg-sensitive intracellular calcium stores, SOC and PKC.     

Influence of lipopolysaccharides and lipids A from some marine bacteria on spontaneous and Escherichia coli LPS-induced TNF-α release from peripheral human blood cells

Some endotoxic properties of lipopolysaccharides (LPS) and lipids A (LA) from the marine bacteria Marinomonas communis ATCC 27118^T, Marinomonas mediterranea ATCC 700492^T, and Chryseobacterium indoltheticum CIP 103168^T were studied. The preparations tested were shown to have high 50% lethal doses (4 μg per mouse for LPS from M. mediterranea and more than 12 μg per mouse for two other LPS and LA from C. indoltheticum) and were moderate (371 ± 37 pg/ml at 10 μg/ml of C. indoltheticum LPS), weak (148 ± 5 pg/ml at 1 μg/ml of M. mediterranea LPS), and zero (LA and LPS from M. communis and LA from C. indoltheticum) inducers of tumor necrosis factor α (TNF-α) release from peripheral human blood cells. The capacity of the LA and LPS samples from marine bacteria to inhibit TNF-α release induced by LPS from Escherichia coli O55: B5 (10 ng/ml) was also studied. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 936–944.     

Treatment with TNF-α and IFN-γ alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells

The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70^S6k, mitogen-activated protein kinase (MAPK) and p90 ^rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70^S6k, p42^MAPK, p44^MAPK and p90 ^rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42^MAPK (a 2.81-fold increase after 50 min), and the activation of p70^S6k and p90 ^rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70^S6k phosphorylation was significantly diminished, and the increase in p42^MAPK and p90 ^rsk phosphorylation was prevented. The basal p42^MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70^S6k, p42^MAPK and p90 ^rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70^S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.