RESUMO
BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Fatores Imunológicos/farmacologia , Complexo Antígeno L1 Leucocitário/efeitos dos fármacos , Mucosa Bucal/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/efeitos dos fármacos , Northern Blotting , Calgranulina A/análise , Calgranulina A/efeitos dos fármacos , Calgranulina B/análise , Calgranulina B/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Medicamentos de Ervas Chinesas/administração & dosagem , Células Epiteliais/efeitos dos fármacos , Humanos , Fatores Imunológicos/administração & dosagem , Interleucina-1alfa/antagonistas & inibidores , Interleucina-1alfa/farmacologia , Complexo Antígeno L1 Leucocitário/análise , Mucosa Bucal/citologia , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
Squamous mucosal epithelial cells constitutively express calprotectin in the cytoplasm. To study how this antimicrobial protein complex confers epithelial resistance to invading bacteria, an epithelial cell line was stably transfected to express the calprotectin complex. Cells expressing calprotectin resist invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium. Calprotectin expression was accompanied by altered actin organization, increased alpha3 integrin expression, and spreading cell morphology. In this study, we assessed whether calprotectin expression affects bacterial binding and uptake. Threefold-fewer Listeria organisms bound to the surfaces of calprotectin-expressing cells, and 10-fold fewer were localized intracellularly by immunofluorescence. Similarly, fewer Salmonella organisms bound to cells expressing calprotectin. Calprotectin-expressing and sham-transfected cells showed similar levels of expression of surface E-cadherin and intracellular adhesion molecule 1 (ICAM-1) by flow cytometry. Calprotectin-expressing transfectants expressed calprotectin on the cell surface as well as in the cytosol. In conclusion, two bacterial pathogens showed reduced binding to calprotectin-expressing epithelial cells. Calprotectin-expressing cells appeared to have internalized disproportionately fewer Listeria organisms, suggesting that reduced binding and translocation supplemented direct antimicrobial effects in calprotectin-expressing cells.