Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6.
The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal 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 dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.
Smirnova
M G
MG
Department of Physiological Sciences, University of Newcastle, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Marina.Smirnova@ncl.ac.uk
Kiselev
S L
SL
Birchall
J P
JP
Pearson
J P
JP
D002455:Cell Division; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D019073:HT29 Cells; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D055271:Mucin 5AC; D055257:Mucin-5B; D009077:Mucins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
D015850:Interleukin-6; C524678:MUC5AC protein, human; C524578:MUC5B protein, human; D055271:Mucin 5AC; D055257:Mucin-5B; D009077:Mucins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha