Luca Parente

Annexin 1: more than an anti-phospholipase protein.

Annexin 1 (ANXA1) is the first characterized member of the annexin family of proteins able to bind (i.e. to annex) to cellular membranes in a calcium-dependent manner. ANXA1 may be induced by glucocorticoids in inflammatory cells and shares with these drugs many anti-inflammatory effects. Originally described as a phospholipase A2 (PLA2)-inhibitory protein, ANXA1 can affect many components of the inflammatory reaction besides the metabolism of arachidonic acid. Recent data have shown that ANXA1 may specifically target cytosolic PLA2 by both direct enzyme inhibition and suppression of cytokine-induced activation of the enzyme. ANXA1 inhibits the expression and/or activity of other inflammatory enzymes like inducible nitric oxide synthase (iNOS) in macrophages and inducible cyclooxygenase (COX-2) in activated microglia. The inhibition of iNOS expression may be caused by the stimulation of IL-10 release induced by ANXA1 in macrophages. Like glucocorticoids, ANXA1 exerts profound inhibitory effects on both neutrophil and monocyte migration in inflammation. Several mechanisms may contribute to the protein effect on cell migration, namely the activation of receptors like the formyl peptide receptor (FPR) and the lipoxin A4 receptor (ALXR), the shedding of L-selectin, the binding to α4β1 integrin and carboxylated N-glycans. Furthermore, again mimicking the action of glucocorticoids, ANXA1 promotes inflammatory cell apoptosis associated with transient rise in intracellular calcium and caspase-3 activation. Finally, ANXA1 has been recently identified as one of the ‘eat-me’ signals on apoptotic cells to be recognised and ingested by phagocytes. Thus, ANXA1 may contribute to the anti-inflammatory signalling that allows safe post-apoptotic clearance of dead cells.

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U‐937 cells

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U‐937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U‐937 cells. This constitutive level was increased time‐ and dose‐ dependently by incubation with phorbol myristate acetate (PMA). In U‐937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 μM for 16 h) caused an increased synthesis of the mRNA level of LCT‐1 and 2, but not of LCT‐5, over the level induced by PMA. DEX had no effect in undifferentiated cells, Moreover, DEX stimulated the extracellular release of LCT‐1 and 5, but not of LCT‐2, and inhibited the release of PGE2 and TXB2 only in the differentiated U‐937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U‐937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.

Down‐regulation of microglial cyclo‐oxygenase‐2 and inducible nitric oxide synthase expression by lipocortin 1

Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). The anti‐inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo‐oxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS), respectively. Dexamethasone (DEX, 1–100 nM) and LC1‐derived N‐terminus peptide (peptide Ac2‐26, 1–100 μg ml−1) dose‐dependently inhibited the production of both PGE2 and NO from LPS‐stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N‐terminus of human LC1. The peptide Ac2‐26 did not affect arachidonic acid release from control and LPS‐stimulated microglial cultures. Western blot experiments showed that the LPS‐induced expression of COX‐2 and iNOS was effectively down‐regulated by DEX (100 nM) and peptide Ac2‐26 (100 μg ml−1). In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.

α-melanocyte-stimulating hormone reduces interleukin-lβ effects on rat stomach preparations possibly through interference with a type I receptor

Abstract Contractions elicited by CaClz on isolated rat stomach strip preparations have been reported to be potentiated by interleukin-1β (IL-1β). We have investigated whether this effect can be reduced by the putative IL-1β antagonist, a-melanocyte-stimulating hormone (aMSH). Additionally, the effects of αMSH on the specific binding of IL-1β to B- and T-cells have been investigated to further clarify its inhibitory activities. Both αMSH and its carboxyl terminal tripeptide concentration dependently reduced the potentiation of CaCl 2 -induced contractions caused by IL-1β but not those caused by leukotriene D 4 , the parent molecule being approximately 250 times more active. Additionally, both peptides potently and selectively reduced 125 I-IL-1β binding to the T-cell sub-clone EL4-6.1 but not to the B-cell sub-clone 1H7. The results indicate that IL-1β effects on rat stomach may be mediated through a type-I (80 kDa) IL-1β receptor.

Relationship between the anti-phospholipase and anti-inflammatory effects of glucocorticoid-induced proteins

Glucocorticoid-induced anti-phospholipase proteins were partially purified by using ion-exchange and molecular sieve chromatography. These proteins, as well as dexamethasone itself, inhibited the hind-paw rat oedema induced by carrageenin. This inhibition was reversed by arachidonic acid, Anti-phospholipase proteins as well as hydrocortisone, also reduced the formation of prostaglandin E2 and leukotriene B4 by phagocytosing leucocytes. A specific monoclonal antibody was able to reverse the inhibition of eicosanoid formation. The mechanism of the anti-inflammatory effect of glucocorticoids and anti-phospholipase proteins is discussed in the light of these results.

The anaphylactic release of platelet-activating factor from perfused guinea-pig lungs

1 The release of platelet‐activating factor (Paf‐acether) and of its inactive precursor/metabolite lysoplatelet activating factor (lyso‐Paf) from control and sensitized guinea‐pig isolated lungs challenged with antigen was investigated. 2 Control guinea‐pig lungs perfused either through the pulmonary circulation or through the airways and challenged with antigen did not release Paf‐acether. 3 Sensitized guinea‐pig isolated lungs perfused through the pulmonary circulation and challenged with antigen released lyso‐Paf but not Paf‐acether. 4 Sensitized guinea‐pig isolated lungs perfused through the airways and challenged with antigen released three times more lyso‐Paf and also Paf‐acether. 5 These results support a possible role for Paf‐acether in respiratory anaphylaxis in the guinea‐pig.

Dexamethasone-induced translocation of lipocortin (annexin) 1 to the cell membrane of U-937 cells.

Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U‐937 cells were incubated with or without 10 nm phorbol 12‐myristate 13‐acetate (PMA) to induce cell differentiation. Then 1 μm dexamethasone was added and incubation carried out for increasing times (1–24 h). Dexamethasone caused a time‐dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U‐937 cells the steroid‐induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U‐937 cells. Since extracellular lipocortin 1 has anti‐inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.

Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.

Annexin-1 (ANX-1) is an anti-inflammatory protein induced by glucocorticoids. Like glucocorticoids, ANX-1 and derived peptides inhibit eicosanoid synthesis, block leukocyte migration and induce apoptosis of inflammatory cells. Cytokines may possess either pro-inflammatory, i.e. interleukin(IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12 or anti-inflammatory properties, i.e. IL-4, IL-10. The experiments described in the present study have been performed to answer the question whether the anti-inflammatory action of ANX-1 may be mediated, at least in part, by the release of IL-10. In macrophage (J774) cell line cultures primed with lipolysaccharide (LPS), recombinant ANX-1 stimulated IL-10 release in a dose- and time-dependent manner. In the same cells, the protein and its derived N-terminal peptide (amino acids 2-26) dose-dependently inhibited the release of nitric oxide (NO). Furthermore, both the whole protein and the peptide down-regulated the mRNA expression of the inducible nitric oxide sythase (iNOS). The peptide was also able to inhibit the expression of IL-12 mRNA. These results suggest that some of the anti-inflammatory effects of ANX-1 may be mediated by the release of IL-10, which, in turn, inhibits iNOS mRNA expression and, hence, NO release. In addition, ANX-1-stimulated IL-10 release may also be responsible for the inhibition of IL-12 mRNA expression and, consequently, IL-12 synthesis.

Evidence that endogenous interleukin-1 is involved in leukocyte migration in acute experimental inflammation in rats and mice

As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect ofin vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1β antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1β serum inhibited selectively the number of neutrophil that migrated to the inflamed site (∼40%) and the IL-1 activity recovered in (∼70%) the exudate.In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1α or anti-murine IL-1β sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the α species, whereas IL-1β was the only species apparently present in the mouse peritoneal exudate.

Cl-IB-MECA inhibits human thyroid cancer cell proliferation independently of A3 adenosine receptor activation

A3 adenosine receptor (A3AR) agonists have been reported to modulate cellular proliferation. This work was aimed to investigate the expression and the possible action of A3 receptor in the human thyroid carcinomas. Normal thyroid tissue samples did not express A3 adenosine receptor, while primary thyroid cancer tissues expressed high level of A3AR, as determined by immunohistochemistry analysis. In human papillary thyroid carcinoma cell line, NPA, at concentrations ≥10µM, the A3AR-selective agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5’-N-methylcarboxamide (Cl-IB-MECA) produced inhibition of cell growth, by blocking the G1 cell cycle phase in a concentration- and time-dependent manner. This effect was well correlated with a reduction of protein expression of cyclins D1 and E2 after 24 hours of Cl-IB-MECA treatment. Moreover Cl-IB-MECA induced dephosphorylation of ERK1/2 in a time- and concentration-dependent manner, which in turn inhibits cell proliferation. The effect of Cl-IB-MECA was not prevented by A3AR antagonists, MRS1191 or MRS1523 or FA385. Furthermore, neither nucleoside transporter inhibitors, Dypiridamole and NBTI, nor the A1, A2A and A2B receptors antagonists were able to block the response to Cl-IB-MECA. Although Cl-IB-MECA has been shown to influence cell death and survival in other systems through an A3AR-mediated mechanism, in NPA cells the growth inhibition induced by micromolar concentrations of Cl-IB-MECA appeared not mediated through activation of this receptor and hence that its effects on human papillary carcinoma cell line seem to be independent of the presence of this receptor subtype.