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.
Department of Physiological Sciences, University of Newcastle, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Marina.Smirnova@ncl.ac.uk
M G
MG
Smirnova
S L
SL
Kiselev
J P
JP
Birchall
J P
JP
Pearson
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