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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.

Here, we have studied the effects of chemically modified tetracyclines (CMTs) on apoptosis both at the level of the cytoplasmic proteolytic caspase cascade, and on Bcl-2 and c-myc mRNA expression in the J774 macrophage cell line. The results indicate that CMTs induce morphological changes consistent with apoptotic events, as clearly demonstrated both by the acridine orange and ethidium bromide staining, and by TUNEL and fragmentation ELISA assays. Furthermore, the analysis of the cell cycle by flow cytometry shows an evident apoptotic sub-G0G1 peak, without important modifications in the cell cycle distribution. CMTs induce programmed cell death (PCD) in a dose-dependent manner and CMT-8 is the strongest among them. CMT-1 and CMT-8 activate mainly caspase-8 as attested by the inhibitory effects of Z-VAD-fmk and Z-IEDT-fmk on CMT-induced apoptosis. Part of CMT-induced PCD is due to the activation of caspase-9, since it is reduced by the specific caspase-9 inhibitor, Z-LEHD-fmk. Besides, CMTs increase Bcl-2 and c-myc mRNA expression. Collectively, these data indicate that CMTs are potentially anti-tumour agents, since they strongly trigger apoptosis both activating the proteolytic system of the caspase family and modulating genes involved in PCD regulation., (Copyright 2002 Elsevier Science B.V.)