BACKGROUND AND PURPOSEAzithromycin 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 APPROACHHuman blood monocytes were primed with IFN-g for 24 h and activated with LPS for 24 h. Azithromycin, anti-inflammatory and lysosome-affecting agents were added 2 h before IFN-g. 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 RESULTSAzithromycin (1.5-50 mM) dose-dependently inhibited gene expression and/or release of M1 macrophage markers (CCR7, CXCL 11 and IL-12p70), but enhanced CCL2, without altering TNF-a 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-kB 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 IMPLICATIONSAzithromycin 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.

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Tyrosine phosphorylation represents a unique signaling process that controls metabolic pathways, cell activation, growth and differentiation, membrane transport, apoptosis, neural, and other functions. We present here the three-dimensional structure of Fyn tyrosine kinase, a Src-family enzyme involved in T-cell receptor signal transduction. The structure of Fyn was modeled for homology using the Sybyl-Composer suite of programs for modeling. Procheck and Prosa II programs showed the high quality of the obtained three-dimensional model. Rosmarinic acid, a secondary metabolite of herbal plants, was discovered as a new Fyn kinase inhibitor using immunochemical and in silico methods. Two possible binding modes of rosmarinic acid were evaluated here, i.e., near to or in the ATP-binding site of kinase domain of Fyn. Enzyme kinetic experiments revealed that Fyn is inhibited by a linear-mixed noncompetitive mechanism of inhibition by rosmarinic acid. This indicates that rosmarinic acid binds to the second "non-ATP" binding site of the Fyn tyrosine kinase.

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