Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors

Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.     

Chemokine production and E-selectin expression in activated endothelial cells are inhibited by p38 MAPK (mitogen activated protein kinase) inhibitor RWJ 67657

Endothelial cells play an important role in inflammatory diseases like rheumatoid arthritis by recruitment of inflammatory cells. The cytokines TNF-alpha and IL-1beta are major inducers of endothelial cell activation and are stimulators of inflammatory signal transduction pathway involving p38 MAPK (mitogen-activated protein kinase). The present study investigated the effects of p38 MAPK inhibition on cell adhesion molecule (CAM) expression and chemokine production by endothelial cells both on mRNA and protein level. Pre-treatment of endothelial cells with the pharmacologically relevant concentration of 1 microM of the p38 MAPK inhibitor RWJ 67657 reduced TNF-alpha and IL-1beta induced mRNA and membrane expression of E-selectin. Moderate inhibitory effects on ICAM-1 and VCAM-1 expression were found. Significant reduction of mRNA expression and protein production of the inflammatory cytokine IL-6 and the chemokines IL-8 and MCP-1 was demonstrated. Treatment with RWJ 67657 could lead to reduced leukocyte infiltration by the reduction of E-selectin expression and chemokine production.     

Caffeine suppresses TNF-alpha production via activation of the cyclic AMP/protein kinase A pathway

This study investigated the effect of in vitro exposure to caffeine, and its major metabolite paraxanthine, at concentrations relevant to typical caffeine consumption in humans, on lipopolysaccharide (LPS)-stimulated cytokine production in human whole blood. In addition, a role for the cyclic AMP/protein kinase A (PKA) pathway in the immunomodulatory effect of caffeine was investigated. Diluted whole blood (taken following >/=15 h abstinence from caffeine-containing food and beverages) was preincubated with caffeine or paraxanthine (10-100 microM) and stimulated with LPS (1 proportional, variant g/ml) for 24 h. The proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-12, and the antiinflammatory cytokine IL-10 were measured in cell-free supernatants. Whilst caffeine and paraxanthine had little or no effect on IL-10, IL-1beta, or IL-12 production, TNF-alpha production was suppressed in all individuals studied. The effect was statistically significant at 100 microM and consistent across seven experiments performed. Although not statistically significant, a similar effect was observed with paraxanthine. Caffeine (100 microM) also increased intracellular cyclic AMP concentrations in LPS-stimulated monocytes isolated from whole blood. Moreover, the effect of caffeine on TNF-alpha production was abolished by pretreatment with the protein kinase A inhibitor Rp-8-Br-cAMPS (10(-4) and 10(-5)M). To conclude, this study demonstrates that concentrations of caffeine that are relevant to human consumption consistently suppress production of the proinflammatory cytokine TNF-alpha in human blood and that this effect is mediated by the cyclic AMP/protein kinase A pathway.     

Protein kinase calpha but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1beta: involvement of sequential activation of tyrosine kinase, nuclear factor-kappaB-inducing kinase, and IkappaB kinase 2

IL-1beta induced an increase in ICAM-1 expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C inhibitor (D609) attenuated IL-1beta-induced ICAM-1 expression. IL-1beta produced an increase in PKC activity and this effect was abolished by D609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited IL-1beta-induced response. TPA, a PKC activator, stimulated ICAM-1 expression as well, this effect being inhibited by tyrosine kinase inhibitors. Treatment of cells with IL-1beta resulted in stimulation of p44/42 MAPK, p38, and JNK. However, neither the mitogen activated protein kinase kinase inhibitor PD 98059 nor the p38 inhibitor SB 203580 affected IL-1beta-induced ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by IL-1beta and these effects were inhibited by tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity as well, these effects being inhibited by tyrosine kinase inhibitors. Dominant-negative PKCalpha, NIK, or IKK2, but not IKK1 mutant, inhibited IL-1beta- or TPA-induced ICAM-1 promoter activity. IKK activity was stimulated by either IL-1beta or TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. Taken together, IL-1beta activates phosphatidylcholine-specific phospholipase C and induces activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of NIK, IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression. However, activation of p44/42 MAPK, p38, and JNK is not involved.     

Cytokine-mediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide.

1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of c-fos and c-jun gene expression by lipopolysaccharide and cytokines in primary cultured astrocytes: effect of PKA and PKC pathways

The effects of lipopolysaccharide (LPS) and several cytokines on the c-fos and c-jun mRNA expression were examined in primary cultured astrocytes. Either LPS (500 ng/mL) or interferon-gamma (IFN-gamma; 5 ng/mL) alone increased the level of c-fos mRNA (1 h). However, tumor necrosis factor-alpha (TNF-alpha; 10 ng/mL) or interleukin-1beta (IL-1beta; 5 ng/mL) alone showed no significant induction of the level of c-fos mRNA. TNF-alpha showed a potentiating effect in the regulation of LPS-induced c-fos mRNA expression, whereas LPS showed an inhibitory action against IFN-gamma-induced c-fos mRNA expression. LPS, but not TNF-alpha, IL-1beta and IFN-gamma, increased the level of c-jun mRNA (1 h). TNF-alpha and IFN-gamma showed an inhibitory action against LPS-induced c-jun mRNA expression. Both phorbol 12-myristate 13-acetate (PMA; 2.5 mM) and forskolin (FSK; 5 mM) increased the c-fos and c-jun mRNA expressions. In addition, the level of c-fos mRNA was expressed in an antagonistic manner when LPS was combined with PMA. When LPS was co-treated with either PMA or FSK, it showed an additive interaction for the induction of c-jun mRNA expression. Our results suggest that LPS and cytokines may be actively involved in the regulation of c-fos and c-jun mRNA expressions in primary cultured astrocytes. Moreover, both the PKA and PKC pathways may regulate the LPS-induced c-fos and c-jun mRNA expressions in different ways.     

Induction of COX-2 and PGE(2) biosynthesis by IL-1beta is mediated by PKC and mitogen-activated protein kinases in murine astrocytes

Interleukin-1 (IL-1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E(2) (PGE(2)) production after IL-1beta stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL-1beta or the phorbol ester, PMA significantly increased PGE(2) secretion. The stimulatory action of IL-1beta on PGE(2) production was totally abolished by NS-398, a specific inhibitor of cyclo-oxygenase-2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL-1beta induced the expression of COX-2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL-1 beta treatment also induced the expression of COX-2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H-7, bisindolylmaleimide and calphostin C) inhibited IL-1beta stimulation of PGE(2). In addition, PKC-depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL-1. The ablation of the effects of PMA and IL-1beta on PGE(2) production, likely results from down-regulation of phorbol ester sensitive-PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC-alpha from cytosol to membrane following treatment with IL-1beta. In addition, IL-1beta treatment led to activation of extracellular signal-regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL-1beta-induced PGE(2) release. ERK1/2 activation by IL-1beta was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE(2), likely by regulating the induction of cyclo-oxygenase-2, in IL-1beta-stimulated astroglial cells.     

Tumour necrosis factor-alpha- and interleukin-1beta-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells

The elevated levels of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF-alpha and IL-1beta on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. TNF-alpha and IL-1beta induced [(3)H]-thymidine incorporation in a time- and concentration-dependent manner. The maximal stimulation of [(3)H]-thymidine incorporation induced by TNF-alpha and IL-1beta was seen 12 h after incubation with these cytokines. In response to TNF-alpha and IL-1beta, p42/p44 MAPK was activated with a concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF-alpha and IL-1beta. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)-phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)-PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. TNF-alpha- and IL-1beta-induced [(3)H]-thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF-alpha and IL-1beta were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF-alpha- and IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in TSMCs.     

Anti-inflammatory activity of fisetin in human mast cells (HMC-1).

Mast cells play an important role in the pathogenesis of allergic diseases through the release of inflammatory mediators such as histamine, cysteinyl leukotrienes, cytokines, and chemokines. Flavonoids, like fisetin are naturally occurring molecules with antioxidant, cytoprotective, and anti-inflammatory actions. The aim of our study was to examine whether fisetin modulates inflammatory reaction in stimulated human mast cells (HMC-1). Fisetin decreased phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated gene expression and production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-4, IL-6, and IL-8 in HMC-1 cells. Fisetin inhibited PMACI-induced phosphorylation of p38 mitogen-activated protein kinase, extracellular-regulated kinase, and c-Jun N-terminal kinase. In addition, fisetin suppressed nuclear factor (NF)-kappaB activation induced by PMACI, leading to expression of IkappaB-alpha phosphorylation and degradation. Fisetin suppressed powerful induction of NF-kappaB promoter-mediated luciferase activity. These pharmacological actions of fisetin produce new suggestion that fisetin is a potential medicine for treatment of inflammatory diseases through the down-regulation of mast cell activation.     

Inhibitory action of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) on some mononuclear phagocyte functions

Peripheral benzodiazepine receptors (PBRs) are widely distributed throughout the body, but their functions are unknown. They are found on mononuclear phagocytes, and they are up-regulated in a number of neurological and other disease states. We explored the functional consequences of PBR ligand binding to mononuclear-derived cells using the high-affinity ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) and 4'-chlorodiazepam (7-chloro-5-(4'-chlorophenyl)-1, 3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; Ro 5-4864). The functions were the following: respiratory burst; secretion of glutamate, interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha); toxicity of culture supernatants towards SH-SY5Y human neuroblastoma cells; and expression of the inflammatory surface markers HLA-DR and Fcgamma RII (CDw32). PK 11195 inhibited the respiratory burst response, reduced release of glutamate and IL-1beta, and suppressed secretion of products cytotoxic to neuronal cells. Selectivity was suggested by the failure of PK 11195 to influence TNF-alpha secretion or expression of HLA-DR and CDw32. Powerful ligands of PBRs, such as PK 11195, may be useful inhibitors of selective macrophage functions, retarding both local and systemic inflammation. Since PK 11195 readily enters the brain, it may be beneficial in treating central as well as peripheral inflammatory diseases.     

Interleukin-1beta-induced mucin production in human airway epithelium is mediated by cyclooxygenase-2, prostaglandin E2 receptors, and cyclic AMP-protein kinase A signaling

We reported recently that interleukin (IL)-1beta exposure resulted in a prolonged increase in MUC5AC mucin production in normal, well differentiated, human tracheobronchial epithelial (NHTBE) cell cultures, without significantly increasing MUC5AC mRNA (Am J Physiol 286:L320-L330, 2004). The goal of the present study was to elucidate the signaling pathways involved in IL-1beta-induced MUC5AC production. We found that IL-1beta increased cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin (PG) E(2) production and that the COX-2 inhibitor celecoxib suppressed IL-1beta-induced MUC5AC production. Addition of exogenous PGE(2) to NHTBE cultures also increased MUC5AC production and IL-1beta-induced Muc5ac hypersecretion in tracheas from wild-type but not from COX-2-/- mice. NHTBE cells expressed all four E-prostanoid (EP) receptor subtypes and misoprostol, an EP2 and EP4 agonist, increased MUC5AC production, whereas sulprostone, an EP1 and EP3 agonist, did not. Furthermore, specific protein kinase A (PKA) inhibitors blocked IL-1beta and PGE(2)-induced MUC5AC production. However, neither inhibition of epidermal growth factor receptor (EGFR) activation with the tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline HCl (AG-1478) or EGFR blocking antibody nor inhibition of extracellular signal-regulated kinase/P-38 mitogen activated protein kinases with specific inhibitors blocked IL-1beta stimulation of MUC5AC mucin production. We also observed that tumor necrosis factor (TNF)-alpha, platelet activating factor (PAF), and lipopolysaccharide (LPS) induced COX-2 and increased MUC5AC production that was blocked by celecoxib, suggesting a common signaling pathway of inflammatory mediator-induced MUC5AC production in NHTBE cells. We conclude that the induction of MUC5AC by IL-1beta, TNF-alpha, PAF, and LPS involves COX-2- generated PGE(2), activation of EP2 and/or EP4 receptor(s), and cAMP-PKA-mediated signaling.     

Regulation of tumor necrosis factor-alpha in glioma cells by lead and lipopolysaccharide: involvement of common signaling pathway

Both lead (Pb) and lipopolysaccharide (LPS) damage nervous system, partly, by the induction of tumor necrosis factor-alpha (TNF-alpha) in glia origin. In this study, we examined the Pb- and LPS-triggered signal leading to TNF-alpha expression in a glioma cell line, U-373MG. Both Pb and LPS increased the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), which depended on the activation of MAPK kinase (MEK) and protein kinase C (PKC). Selective p42/44 MAPK inhibitor could reduce the Pb- and LPS-triggered TNF-alpha expression in U-373MG cells. Suppressing PKC by chelerythrine chloride completely diminished the Pb- and LPS-induced TNF-alpha expression in glial cells in the mouse brain. Thus, our results indicated that PKC-MEK-p42/44 MAPK is a common signaling pathway for Pb- and LPS-induced TNF-alpha expression in glial cells.     

Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages.

Curcumin, a dietary pigment responsible for the yellow colour of curry, has been used for the treatment of inflammatory diseases and exhibits a variety of pharmacological effects such as anti-inflammatory activity. The mechanism in anti-inflammatory activity of curcumin has been investigated; however, little is known about the effect of curcumin on cytokine production by human peripheral blood monocytes and alveolar macrophages. In the present study, we shed light on the effect of curcumin on inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. To this end, we determined the concentrations of interleukin-8 (IL-8), monocyte inflammatory protein-1 (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-1beta (IL-1beta), and tumour necrosis factor-alpha (TNF-alpha) in the culture supernatants from phorbor ester, 4beta phorbor 12beta-myristate-13alpha acetate (PMA)- or lipo-polysaccharide (LPS)-stimulated monocytes and alveolar macrophages in the presence or absence of curcumin. Curcumin inhibited the production of IL-8, MIP-1alpha, MCP-1, IL-1beta, and TNF-alpha by PMA- or LPS-stimulated monocytes and alveolar macrophages in a concentration- and a time-dependent manner. These results show that curcumin exhibits an inhibitory effect on the production of IL-8, MIP-1alpha, MCP-1, IL-1beta, and TNF-alpha by PMA- or LPS-stimulated monocytes and alveolar macrophages.     

Responses of exposure to cigarette smoke at three dosage levels on soleus muscle fibers in Wistar-Kyoto and spontaneously hypertensive rats

Overproduction of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) is importantly involved in the pathogenesis of endotoxemia and atherosclerosis. Calcium antagonists are commonly used as cardiovascular drugs and have a beneficial effect on prolonging survival in various models of endotoxin shock. The present study was to investigate the effect of a calcium antagonist amlodipine on nitrite, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) formation and iNOS induction both in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and in a rat model of endotoxemia. Incubation with amlodipine (0.1 - 10 microM) for 24 h resulted in a significant and dose-dependent attenuation in medium nitrite, TNF-alpha and IL-1beta formation as well as iNOS protein expression in LPS/IFN-gamma-treated RASMC. In addition, amlodipine inhibited leucigenin-induced superoxide formation in RASMC. In the rat endotoxic model, the serum nitrite/nitrate, TNF-alpha and IL-1beta levels as well as iNOS protein expression of lungs were also suppressed by administration of amlodipine (50 microg/kg, i.v.). These results suggest that amlodipine may exert vascular beneficial effects by suppressing pro-inflammatory cytokines and free radical generation as well as iNOS induction in smooth muscle cells during activation of inflammatory mechanism.     

FK506 potently inhibits T cell activation induced TNF-alpha and IL-1beta production in vitro by human peripheral blood mononuclear cells

The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, with a view to assessing this immunosuppressive agent as a potential anti-rheumatic drug. We employed an in vitro model which produces TNF-alpha and IL-1beta through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti-CD3/CD28 monoclonal antibody in this model. FK506 inhibited anti-CD3/CD28 induced TNF-alpha and IL-1beta production at concentrations less than 1 ng ml(-1). Flow cytometric analysis of intracellular TNF-alpha and IL-1beta positive cells showed that FK506 potently suppresses inflammatory cytokine production from CD14+ monocytes as well as from T cells. Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti-CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, specifically inhibited anti-CD3/CD28 induced inflammatory cytokine production without affecting the lipopolysaccaride (LPS) induced effect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC. The inhibitory potency of agents on TNF-alpha and IL-1beta production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition. These results strongly suggest that FK506 may be most effective to specifically prevent T cell activation mediated inflammatory cytokine production in a clinical setting.     

Molecular mechanisms of topical anti-inflammatory effects of lipoxin A(4) in endotoxin-induced uveitis

Lipoxin A(4) (LXA(4)) is a lipid mediator that plays an important role in inflammation resolution. We assessed the anti-inflammatory effect of LXA(4) on endotoxin-induced uveitis (EIU) in rats. The inflammatory cell number and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), and protein, as well as expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), in the anterior chamber of the eye were determined 24 h after lipopolysaccharide (LPS; 200 mug/paw) intradermal injection. The immunohistochemical reactivities of nuclear factor-kappaB (NF-kappaB) and c-Jun were also examined. Topical LXA(4) (1-10 ng/eye) pretreatment decreased the number of inflammatory cells and the protein leakage into the aqueous humor (AqH). In addition, topical LXA(4) (10 ng/eye) inhibited the LPS-induced production of IL-1beta, TNF-alpha, and PGE(2), and expression of COX-2 and VEGF. A decreased activation of NF-kappaB and c-Jun was also found in LXA(4)-treated eyes. It is very interesting that an anti-inflammatory effect was achieved even when LXA(4) (10 ng/eye) was applied topically after LPS challenge, as indicated by the reduction in the cellular and protein extravasations into the AqH. Moreover, topical treatment of corticosteroid prednisolone (200 mug/eye) beginning before or after LPS injection reduced all of the molecular and biochemical alterations promoted on EIU rats in an efficacy similar to that of LXA(4). Together, the present results provide clear evidence that pharmacological activation of LXA(4) signaling pathway potently reduces the EIU in rats. Therefore, LXA(4) stable analogs could represent promising agents for the management of ocular inflammatory diseases.     

Role of p38 MAP kinase in LPS-induced airway inflammation in the rat

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED(50)=15.8 mg kg(-1)) plasma levels of TNF-alpha in rats challenged with LPS (1.5 mg kg(-1), i.p.). Aerosolized LPS induced a peak in TNF-alpha levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL-1beta levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100 mg kg(-1)), had no effect on peak TNF-alpha levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg(-1)) significantly reduced both TNF-alpha levels and neutrophilic inflammation. However, the BAL fluid from rats pre-treated with either compound significantly inhibited TNF-alpha release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100 mg kg(-1)) produced dose-related inhibition of BAL IL-1beta levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4 h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL-1beta and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.