The link between met-enkephalin-induced down-regulation of APN activity and the release of superoxide anion.

We have previously shown that methionine-enkephalin (MENK) differentially alters the production of superoxide anion (O(2)(-)) from neutrophils of different donors. This effect could be due to variable activity of proteolytic enzymes involved in the degradation of this neuropeptide. In this study, we investigated the possible association between the effect of MENK on O(2)(-)release and the two neutrophil associated hydrolytic enzymes that participate in enkephalin degradation; aminopeptidase N (APN) and neutral endopeptidase (NEP). We have demonstrated that APN but not NEP activity was down-regulated by MENK. This might be due to internalization, since APN down-regulation was observed only with intact neutrophils and not with the respective membranes. Preincubation of neutrophils with inhibitory anti CD13 MoAb (WM15) abbrogated the suppressive effect of MENK (10(-12), 10(-10)and 10(-8)M). These facts, show that in the periphery (as well as the brain) the dominant role in MENK hydrolysis can be attributed to APN. Also, they further support the idea of the link between the membrane associated CD13 and binding of the ligand to the opioid receptor.

Azithromycin distinctively modulates classical activation of human monocytes in vitro.

BACKGROUND AND PURPOSE: Azithromycin has been reported to modify activation of macrophages towards the M2 phenotype. Here, we have sought to identify the mechanisms underlying this modulatory effect of azithromycin on human monocytes, classically activated in vitro. EXPERIMENTAL APPROACH: Human blood monocytes were primed with IFN-γ for 24 h and activated with LPS for 24 h. Azithromycin, anti-inflammatory and lysosome-affecting agents were added 2 h before IFN-γ. Cytokine and chemokine expression was determined by quantitative PCR and protein release by ELISA. Signalling molecules were determined by Western blotting and transcription factor activation quantified with a DNA-binding ELISA kit. KEY RESULTS: Azithromycin (1.5-50 µM) dose-dependently inhibited gene expression and/or release of M1 macrophage markers (CCR7, CXCL 11 and IL-12p70), but enhanced CCL2, without altering TNF-α or IL-6. Azithromycin also enhanced the gene expression and/or release of M2 macrophage markers (IL-10 and CCL18), and the pan-monocyte marker CD163, but inhibited that of CCL22. The Toll-like receptor (TLR) 4 signalling pathway was modulated, down-regulating NF-κB and STAT1 transcription factors. The inhibitory profile of azithromycin differed from that of dexamethasone, the phosphodiesterase-4 inhibitor roflumilast and the p38 kinase inhibitor SB203580 but was similar to that of the lysosomotropic drug chloroquine. Effects of concanamycin and NH4Cl, which also act on lysosomes, differed significantly. CONCLUSIONS AND IMPLICATIONS: Azithromycin modulated classical activation of human monocytes by inhibition of TLR4-mediated signalling and possible effects on lysosomal function, and generated a mediator expression profile that differs from that of monocyte/macrophage phenotypes so far described.