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Chronic inflammation, a condition frequently associated with several pathologies, is characterized by angiogenic and fibrogenic responses that may account for the development of granulomatous tissue. We previously demonstrated that the chymase, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties in a model of chronic inflammation sustained by mast cells (MCs), granuloma induced by the subcutaneous carrageenan-soaked sponge implant in rat. In this study, we investigated the effects of palmitoylethanolamide (PEA), an anti-inflammatory and analgesic endogenous compound, on rMCP-5 mRNA expression and Microphtalmia-associated Transcription Factor (MITF) activation in the same model of chronic inflammation. The levels of rMCP-5 mRNA were detected using semi-quantitative RT-PCR; the protein expression of chymase and extracellular signal-regulated kinases (ERK) were analyzed by western blot; MITF/DNA binding activity and MITF phosphorylation were assessed by electrophoretic mobility shift assay (EMSA) and immunoprecipitation, respectively. The administration of PEA (200, 400 and 800 µg/ml) significantly decreased rMCP-5 mRNA and chymase protein expression induced by λ-carrageenan. These effects were associated with a significant decrease of MITF/DNA binding activity and phosphorylated MITF as well as phosphorylated ERK levels. In conclusion, our results, showing the ability of PEA to inhibit MITF activation and chymase expression in granulomatous tissue, may yield new insights into the understanding of the signaling pathways leading to MITF activation controlled by PEA.

In this study we investigated the activation of nuclear factor-kappaB in the carrageenin-induced rat pleurisy. We found that nuclear factor-kappaB DNA binding activity, measured in inflammatory cells which migrated into the pleural cavity, was detectable at 3 and 6 h, markedly increased at 24 h and decreased at 48 h after induction of the inflammation. The increase in nuclear factor-kappaB DNA binding activity paralleled both exudate formation and leukocyte infiltration. Treatment of animals with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappaB activation, inhibited the nuclear factor-kappaB DNA binding activity as well as exudate formation and leukocyte infiltration. These results indicate that nuclear factor-kappaB is activated in the carrageenin-induced pleurisy and suggest that its inhibition may represent a novel strategy for the modulation of inflammatory response.

We studied the role of nuclear factor-kappaB (NF-kappaB) on the tone and on the expression of inducible nitric oxide (NO) synthase, both evaluated in aortas from lipopolysaccharide-treated rats. Thoracic aorta rings from lipopolysaccharide-treated rats (4 mg/kg, i.p.), compared to those from naive animals, showed: (i) reduced contractility to phenylephrine, (ii) progressive loss in tone when contracted with phenylephrine, (iii) increased inducible NO synthase protein expression and NF-kappaB activation. Pyrrolidine dithiocarbamate (10, 30, 100 mg/kg, i.p.), an antioxidant inhibitor of NF-kappaB activation, dose dependently suppressed all these lipopolysaccharide-induced effects. These results demonstrate that in vivo inhibition of NF-kappaB activation prevented the lipopolysaccharide-induced loss of vascular tone, an effect which was correlated to reduced expression of inducible NO synthase protein.

Glucocorticoids inhibit inducible-type NO synthase activity in a variety of cell types. We report here that proteins recovered from the medium of dexamethasone-treated J774 macrophages (1, 10, 100 microg/ml) inhibited lipopolysaccharide-stimulated nitrite generation by 10.0 +/- 3.0\%, 32.3 +/- 5.3\% and 55.0 +/- 6.0\%, respectively, and inducible NO synthase mRNA expression in these cells. Immunoblotting analysis of crude and partially purified glucocorticoid-induced proteins with an anti-lipocortin-1 polyclonal antiserum revealed the presence of lipocortin-1-like immunoreactive species with a molecular mass of 35-37 kDa. Furthermore, inhibition of lipopolysaccharide-induced nitrite production by glucocorticoid-induced proteins in J774 cells was reversed by addition of anti-lipocortin-1 neutralizing polyclonal antibody (1:60 dilution; 4 h before lipopolysaccharide). Comparison of glucocorticoid-induced proteins inhibition of both nitrite production and inducible NO synthase mRNA expression suggests that these effects result mainly from inhibition of lipopolysaccharide-mediated inducible NO synthase gene expression. These results indicate that negative regulation of inducible NO synthase by glucocorticoids is, at least in part, mediated by glucocorticoid-induced proteins that involve also members of the lipocortin-like superfamily.

We have studied the possible involvement of nitric oxide (NO) in granuloma formation induced by subcutaneous implantation in rats of carrageenin-soaked polyether sponges. Modulation of the L -arginine: NO pathway in rats was achieved by treating rats with the NO synthase inhibitor, N G - nitro - L - arginine methyl ester, as well as with L - or D -arginine. Granulomatous tissue formation, cell infiltration and NO2− production were reduced, in a dose-dependent manner, by N G - nitro - L - arginine methyl ester and increased by L -arginine but not by D -arginine. These results suggest that endogenous NO plays a modulating role in granuloma formation.

The effect of cloricromene, a coumarin derivative, was investigated on the lipopolysaccharide-stimulated nitric oxide (NO) synthase induction in intact aortas from endotoxin shocked rats and in the murine macrophage cell line J774. Rings of thoracic aortas from lipopolysaccharide (4 mg/kg, i.v.)-shocked rats, contracted with phenylephrine, showed a progressive decrease in tone, that was of a greater magnitude than that of aortas from naive rats. Moreover, a decreased response to the constrictor effect of phenylephrine was observed in aortas from shocked rats. In vivo treatment with cloricromene (2 mg/kg, i.v.) 30 min before lipopolysaccharide administration partially prevented the loss in tone of aortic rings and improved their reactivity to phenylephrine. Murine J774 macrophages activated with lipopolysaccharide (100 ng/ml) produced significant amounts of nitrites (NO2-; 28.2 +/- 3.5 nmol/10(6) cells per 24 h). Cloricromene (2, 20 or 200 microM) added to the cells concomitantly with lipopolysaccharide inhibited NO2- production in a concentration-dependent manner. Maximum inhibition (84.0 +/- 8.0\%) was observed when cloricromene (200 microM) was added to the cells 6 h before lipopolysaccharide, whereas it was ineffective when given 6 h after endotoxin. These results demonstrate that cloricromene inhibits the expression but not the activity of the inducible NO synthase.

In this study we have shown that dexamethasone induces vasocortin-like proteins in bovine endothelial cells as well as in the bovine aortic endothelial cell line, GM 7373. Vasocortin-like proteins have been characterized by their ability to mimic the glucocorticoid inhibition of rat dextran oedena and histamine release induced by concanavalin-A in rat mast cells. Following partial purification of these proteins by gel filtration, vasocortin activity was found to be associated to proteins with molecular weight between 20–35 kD. This study showed that dexamethasone induces vasocortin-like proteins in endothelial cells and suggests that endothelial cells are target cells of glucocorticoid activity in vascular tissue.

Rat and bovine aorta rings incubated with 10−5 M dexamethasone release proteins which inhibit rat dextran oedema. These proteins seem to be related to vasocortin, derived from the peritoneal fluid of dexamethasone-treated rats, and may contribute to the control that glucocorticoids exert on vascular tonus and permeability.