Chemokine IP-10 expression in cultured human keratinocytes.
IP-10, a member of the CXC family of chemokines, is considered to play an important role in inflammation via its T-cell chemotactic and adhesion-promoting properties. Elevated IP-10 levels in the epidermis of psoriasis, delayed-type hypersensitivity reactions, cutaneous T-cell lymphoma and fixed drug eruptions prompted us to study its expression in keratinocytes. IP-10 mRNA could be detected using the sensitive RT-PCR method, but not by Northern blotting in RNA preparations from unstimulated normal cultured keratinocytes, indicating a low steady-state level of IP-10 mRNA. Upon stimulation with IFN-gamma, IP-10 mRNA was found to accumulate in high amounts in a time- and dose-dependent manner. Superexpression was found with the combination of IFN-gamma and TNF-alpha or IL-1, although these latter cytokines by themselves did not induce accumulation of IP-10 mRNA. Nuclear run-on experiments performed to investigate the regulation of IP-10 mRNA expression, showed a very high constitutive transcriptional activity of the IP-10 gene in unstimulated keratinocytes, which was not affected by stimulation with IFN-gamma, TNF-alpha, or a combination of IFN-gamma and TNF-alpha. Protein kinase C (PKC) was shown to be involved in IP-10 mRNA expression since the PKC inhibitor H7 decreased IP-10 mRNA accumulation. A protein was isolated from culture supernatants of stimulated keratinocytes using HPLC techniques and, by sequence analysis, was found to be identical to IP-10. The dynamics of secretion of IP-10 protein as monitored by ELISA was shown to parallel the mRNA expression.
Department of Dermatology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands. dm.boorsma@azvu.nl
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DM
Boorsma
J
J
Flier
S
S
Sampat
C
C
Ottevanger
P
P
de Haan
L
L
Hooft
R
R
Willemze
C P
CP
Tensen
T J
TJ
Stoof
D000970:Antineoplastic Agents / Q000494:pharmacology; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000970:Antineoplastic Agents; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate