title: 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)-1beta-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-1beta, IL-6, tumor necrosis factor (TNF)-alpha plus IL-1beta, TNF-alpha plus IL-6, and TNF-alpha plus interferon (IFN)-gamma. We also used semiquantitative RT-PCR to determine the effect of GS on IL-1beta-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-kappaB and the degradation of cytoplasmic IkappaB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell-neutrophil adherence assay. IL-1beta 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-1beta, IL-6, TNF-alpha, and IFN-gamma at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-kappaB subunit p65 and partially prevented the degradation of cytoplasmic IkappaB in IL-1beta-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1beta, IL-6, TNF-alpha, and IFN-gamma. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1beta by blocking NF-kappaB subunit p65 translocation and by partially preventing IkappaB 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.
authors.affiliation: Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, #325 Section 2 Cheng-Kung Road, Taipei 114, Taiwan. jt66chen@ms32.hinet.net
authors.forename: Jiann-Torng
authors.initials: JT
authors.lastname: Chen
authors.forename: Po-Liang
authors.initials: PL
authors.lastname: Chen
authors.forename: Yun-Hsiang
authors.initials: YH
authors.lastname: Chang
authors.forename: Ming-Wei
authors.initials: MW
authors.lastname: Chien
authors.forename: Yi-Hao
authors.initials: YH
authors.lastname: Chen
authors.forename: Da-Wen
authors.initials: DW
authors.lastname: Lu
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology*; D004357:Drug Synergism; D005136:Eye Proteins / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005944:Glucosamine / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D053583:Interleukin-1beta / Q000032:analysis; D007962:Leukocytes / Q000187:drug effects* / Q000276:immunology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA / Q000235:genetics; D014178:Translocation, Genetic
chemical_list: D016207:Cytokines; D005136:Eye Proteins; D020672:I-kappa B Proteins; D007166:Immunosuppressive Agents; D053583:Interleukin-1beta; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D005944:Glucosamine