title: Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes.
abstract: Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.
authors.affiliation: Department of Dermatology, Kuopio University Hospital, Kuopio, Finland. ilkka.harvima@kuh.fi
authors.forename: Ilkka T
authors.initials: IT
authors.lastname: Harvima
authors.forename: Katriina
authors.initials: K
authors.lastname: Lappalainen
authors.forename: Maija-Riitta
authors.initials: MR
authors.lastname: Hirvonen
authors.forename: Mikko
authors.initials: M
authors.lastname: Mättö
authors.forename: Petri K
authors.initials: PK
authors.lastname: Kivinen
authors.forename: Mika
authors.initials: M
authors.lastname: Hyttinen
authors.forename: Jukka
authors.initials: J
authors.lastname: Pelkonen
authors.forename: Anita
authors.initials: A
authors.lastname: Naukkarinen
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004848:Epithelium / Q000187:drug effects / Q000254:growth & development; D016193:G1 Phase / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; C032253:involucrin; D006493:Heparin