Glucosamine sulfate inhibits leukocyte adhesion in response to cytokine stimulation of retinal pigment epithelial cells in vitro

Abstract

Glucosamine is an amine-containing sugar that exhibits immunosuppressive effects in vitro and in vivo, although its mechanism of action is unknown. We investigated whether glucosamine sulfate (GS) modulates the proinflammatory cytokine interleukin (IL)-1β-induced expression and production of intercellular adhesion molecule (ICAM)-1, the mechanism responsible for this effect, and whether GS inhibits leukocyte adhesion to the monolayer of retinal pigment epithelial (RPE) cells stimulated with various cytokines. We used flow cytometry and an ARPE-19 cell model to determine the effect of GS on the production of ICAM-1 in response to IL-1β, IL-6, tumor necrosis factor (TNF)-α plus IL-1β, TNF-α plus IL-6, and TNF-α plus interferon (IFN)-γ. We also used semiquantitative RT–PCR to determine the effect of GS on IL-1β-induced expression of the ICAM-1 gene, and immunocytochemistry and western blotting to measure the effect of GS on the activation and nuclear translocation of the nuclear factor NF-κB and the degradation of cytoplasmic IκB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell–neutrophil adherence assay. IL-1β increased the expression of ICAM-1 at the mRNA and protein levels in ARPE-19 cells. GS downregulated the production of ICAM-1 induced by IL-1β, IL-6, TNF-α, and IFN-γ at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-κB subunit p65 and partially prevented the degradation of cytoplasmic IκB in IL-1β-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1β, IL-6, TNF-α, and IFN-γ. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1β by blocking NF-κB subunit p65 translocation and by partially preventing IκB degradation. GS also decreases leukocyte adhesion to the monolayer of ARPE-19 cells stimulated with various cytokines by decreasing ICAM-1 production. Our study demonstrates a potentially important property of GS in reducing ICAM-1-mediated inflammatory mechanisms in the eye.