roxithromycin and Inflammation

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

In addition to their antibiotic effects, tetracyclines have anti-inflammatory action that is often beneficial in the control of inflammatory skin disorders. In this study, we examined the effects of tetracycline (TET) and two of its derivatives, doxycycline (DOX) and minocycline (MIN), on the production of interleukin-8 (IL-8) elicited by the activation of protease-activated receptor 2 (PAR2) in normal human epidermal keratinocytes (NHEK). In NHEK, the production of IL-8 stimulated by an agonist peptide of PAR2, SLIGKIV-NH(2), at 100 microM was significantly reduced by TET, DOX, or MIN at 5 and 10 microM, concentrations that are noncytotoxic. The tumor necrosis factor alpha (TNF-alpha)-induced production of IL-8 was synergistically augmented by SLIGKIV-NH(2), and that synergistic increase in the production of IL-8 was suppressed by 100 nM PAR2-specific small interfering RNA. It was also suppressed by TET, DOX, or MIN but not by the 14-membered-ring macrolide antibiotics erythromycin, roxithromycin, and clarithromycin, which also have anti-inflammatory activities, at 10 microM. These results suggest that tetracyclines attenuate the PAR2-IL-8 axis in keratinocytes and thereby effectively modulate proinflammatory responses in the skin.

Topics: Anti-Bacterial Agents; Cells, Cultured; Epidermal Cells; Epidermis; Humans; Inflammation; Interleukin-8; Keratinocytes; Receptor, PAR-2; Tetracyclines