Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatorycytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in t he pathogenesis of otitis media with effusion (OME), A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the acc umulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effus ions, To determine the effect of IL-6 and TNF-a on MUC5AC and MUC5B secreti on we have used HT29-MTX goblet cells, which secrete both types of mucins, MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosor bent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and po lyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time de pendent manner, both in preconfluent and post-confluent cells, IL-6 (15 ng/ ml and 20 ng/ml) produced a;low and prolonged stimulation of mucin secretio n that persisted for 72 hours, with peak response at 24 hours after inducti on. The IL-6-mediated mucin secretion at 24 hours was concentration-depende nt, with a maximal effect at 15 ng/ml, TNF-alpha (20 ng/ml) induced rapid s timulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction, IL-6 and TNF-alpha exposure significantly incr eased MUC5AC secretion, but not MUC5B secretion, Maximal levels of cytokine -induced mucin secretion were detected in pre-confluent cells that showed o ne and a half- and two-fold increases in MUC5AC secretion after IL-6 and TN F-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a diffe rential up-regulation of mucin secretion and thus contribute to the express ion of mucin genes in inflammatory responses.