Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity. (1/9413)

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.  (+info)

Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42. (2/9413)

The c-Jun N-terminal kinases (JNKs) are activated strongly by inflammatory cytokines and environmental stresses, but only weakly by growth factors. Here we show that platelet-derived growth factor (PDGF) strongly potentiates activation of JNK by interleukin 1 (IL-1) in human fibroblasts and a pig aortic endothelial (PAE) cell line. This synergistic activation of JNK by IL-1 and PDGF was unaffected by bacterial toxins that inactivate Rho proteins and Ras. Since Rho proteins have been implicated in JNK activation, their possible involvement was investigated further using stably expressed, inducible N17 or V12 mutants in PAE cell lines. N17 Rac non-selectively reduced JNK activity by 30% in resting or stimulated cells (IL-1 alone, or with PDGF). N17 Cdc42 had no effect. V12 Rac weakly activated JNK and synergized with IL-1, but not with PDGF. V12 Cdc42 weakly activated JNK, but synergized with PDGF and not IL-1. Our results imply that Rho GTPases are not directly involved in mediating IL-1-induced JNK activation, or in the potentiation of this activation by PDGF.  (+info)

Involvement of tumor necrosis factor alpha and interleukin-1beta in enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae. (3/9413)

Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in Shigella-related seizures. Administration of Shigella dysenteriae 60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the Shigella-pretreated group. Preinjection of mice with anti-murine TNF-alpha (anti-mTNF-alpha) or anti-murine IL-1beta (anti-mIL-1beta) 30 min prior to administration of Shigella sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-alpha from 1.2 to 0.8 (P = 0.017) and by anti-mIL-1beta from 1.3 to 0.7 (P = 0.008). Preinjection of anti-mTNF-alpha also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (P = 0.002), and preinjection of anti-mIL-1beta reduced it from 53 to 21% (P = 0. 012). Neutralization of TNF-alpha or IL-1beta did not protect the mice from death due to S. dysenteriae 60R. These findings indicate that TNF-alpha and IL-1beta play a role in the very early sensitization of the central nervous system to convulsive activity after S. dysenteriae administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases.  (+info)

Emergent immunoregulatory properties of combined glucocorticoid and anti-glucocorticoid steroids in a model of tuberculosis. (4/9413)

In Balb/c mice with pulmonary tuberculosis, there is a switch from a protective Th1-dominated cytokine profile to a non-protective profile with a Th2 component. This switch occurs while the adrenals are undergoing marked hyperplasia. Treatment with the anti-glucocorticoid hormones dehydroepiandrosterone or 3 beta, 17 beta-androstenediol, during the period of adrenal hyperplasia, maintains Th1 dominance and is protective. We investigated the effects of these hormones as therapeutic agents by administering them from day 60, when the switch to the non-protective cytokine profile was already well established. Given at this time (day 60), doses that were protective when given early (from day 0) were rapidly fatal. A physiological dose of the glucocorticoid corticosterone was also rapidly fatal. However when the corticosterone and the anti-glucocorticoid (AED or DHEA) were co-administered, there was protection, with restoration of a Th1-dominated cytokine profile, enhanced DTH responses, and enhanced expression of IL-1 alpha and TNF alpha. Therefore this combination of steroids has an emergent property that is quite unlike that of either type of steroid given alone. It may be possible to exploit the ant-inflammatory properties of glucocorticoids while preserving a Th1 bias, by combining glucocorticoids with DHEA or suitable metabolites.  (+info)

Inhibition of transforming growth factor beta production by nitric oxide-treated chondrocytes: implications for matrix synthesis. (5/9413)

OBJECTIVE: Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1beta (IL-1beta). If NO production is blocked, much of the IL-1beta inhibition of proteoglycan synthesis is prevented. We tested the hypothesis that this inhibitory effect of NO on proteoglycan synthesis is secondary to changes in chondrocyte transforming growth factor beta (TGFbeta). METHODS: Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFbeta bioactivity in chondrocyte- and cartilage-conditioned medium (CM) was measured with the mink lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35S-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns. RESULTS: IL-1beta increased active TGFbeta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFbeta were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFbeta without affecting the early TGFbeta activation. IL-1beta stimulated a NO-independent, transient increase in TGFbeta3 at 24 hours; however, TGFbeta1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1beta increased CM concentrations of TGFbeta1 from 24-72 hours of culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGFbeta1. Anti-TGFbeta1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1beta + L-NMA, confirming that NO inhibition of TGFbeta1 in IL-1beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFbeta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillamide. Similar results were seen with cartilage slices in organ culture. The autocrine increase in CM TGFbeta1 levels following prior exposure to TGFbeta1 was also blocked by NO. CONCLUSION: NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFbeta1 by chondrocytes exposed to IL-1beta. It prevents autocrine-stimulated increases in TGFbeta1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.  (+info)

Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release. (6/9413)

OBJECTIVE: To assess the expression and function of purine receptors in articular chondrocytes. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to screen human chondrocyte RNA for expression of P1 and P2 purine receptor subtypes. Purine-stimulated prostaglandin E2 (PGE2) release from chondrocytes, untreated or treated with recombinant human interleukin-1alpha (rHuIL-1alpha), was assessed by radioimmunoassay. RESULTS: RT-PCR demonstrated that human articular chondrocytes transcribe messenger RNA for the P1 receptor subtypes A2a and A2b and the P2 receptor subtype P2Y2, but not for the P1 receptor subtypes A1 and A3. The P1 receptor agonists adenosine and 5'-N-ethylcarboxamidoadenosine did not change PGE2 release from chondrocytes. The P2Y2 agonists ATP and UTP stimulated a small release of PGE2 that was potentiated after pretreatment with rHuIL-1alpha. PGE2 release in response to ATP and UTP cotreatment was not additive, but release in response to coaddition of ATP and bradykinin (BK) or UTP and BK was additive, consistent with ATP and UTP competition for the same receptor site. The potentiation of PGE2 release in response to ATP and UTP after rHuIL-1alpha pretreatment was mimicked by phorbol myristate acetate. CONCLUSION: Human chondrocytes express both P1 and P2 purine receptor subtypes. The function of the P1 receptor subtype is not yet known, but stimulation of the P2Y2 receptor increases IL-1-mediated PGE2 release.  (+info)

Effects of lipopolysaccharide on production of interleukin-1 and interleukin-6 by bovine mammary epithelial cells in vitro. (7/9413)

This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 micrograms/ml) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.  (+info)

Mechanisms of prostaglandin E2 release by intact cells expressing cyclooxygenase-2: evidence for a 'two-component' model. (8/9413)

Prostaglandin (PG) release in cells expressing constitutive cyclooxygenase-1 is known to be regulated by liberation of arachidonic acid by phospholipase A2 followed by metabolism by cyclooxygenase. However, the relative contribution of phospholipase A2 to the release of PGs in cells expressing cyclooxygenase-2 is not clear. We addressed this question by using radioimmunoassay to measure PGE2 release by human cells (A549) induced to express cyclooxygenase-2 (measured by Western blot analysis) by interleukin-1beta. Cells were either unstimulated or stimulated with agents known to activate phospholipase A2 (bradykinin, Des-Arg10-kallidin, or the calcium ionophore A23187) or treated with exogenous arachidonic acid. When cells were treated to express cyclooxygenase-2, the levels of PGE2 released over 15 min were undetectable; however, in the same cells stimulated with bradykinin, A23187, or arachidonic acid, large amounts of prostanoid were produced. Using selective inhibitors/antagonists, we found that the effects of bradykinin were mediated by B2 receptor activation and that prostanoid release was due to cyclooxygenase-2, and not cyclooxygenase-1, activity. In addition, we show that the release of PGE2 stimulated by either bradykinin, A23187, or arachidonic acid was inhibited by the phospholipase A2 inhibitor arachidonate trifluoromethyl ketone. Hence, we have demonstrated that PGE2 is released by two components: induction of cyclooxygenase-2 and supply of substrate, probably via activation of phospholipase A2. This is illustrated in A549 cells by a clear synergy between the cytokine interleukin-1beta and the kinin bradykinin.  (+info)