Skip to main content
SpringerLink
Log in

Chemical induction of interleukin-8, a proinflammatory chemokine, in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 μmol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 μg), lipopolysaccharide (0.1–100 μg/ml), tumor necrosis factor-α (TNFα) (3.13–50 ng/ml), or interleukin-1α (IL-1α) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNFα induced IL-8 production that coincided with significant cell cycle inhibition. IL-1α had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 μg/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNFα reduced the mitotic index by ∼45%, slowed cell cycle progression by ∼3.5 h, and induced a flat, albeit large, IL-8 response at concentrations ≥12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

B(a)P:

benzo(a)pyrene

BrdU:

5-bromo-2′-deoxyuridine

CHX:

cycloheximide

ICAM:

intercellular adhesion molecules

IL-1α:

interleukin-1α

IL-8:

interleukin-8

KGM:

keratinocyte growth medium

LPS:

lipopolysaccharide

PKC:

protein kinase C

PMA:

phorbol-13-myristate-12-acetate

PMN:

polymorphonuclear neutrophil

ROS:

reactive oxygen species

SCE:

sister chromatid exchange

TNFα:

tumor necrosis factor α

References

  • Adamson GM, Billings RE. Tumor necrosis factor induced oxidative stress in isolated mouse hepatocytes. Arch Biochem Biophys. 1994;294:223–9.

    Google Scholar 

  • Aleen-Hoffman BL, Rheinwald JG. Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture. Proc Natl Acad Sci USA. 1984;81:7802–6.

    Google Scholar 

  • Ansel J, Perry P, Brown J, et al. Cytokine modulation of keratinocyte cytokines. J Invest Dermatol. 1990;94:101S-7S.

    Google Scholar 

  • Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines. In: Dixon FJ, ed. Advances in immunology.Vol.55 New York: Academic Press; 1994:97–179.

    Google Scholar 

  • Barker JNWM, Griffiths CEM, Mitra RS, Dixit VM, Nickoloff BJ. Human epidermal keratinocyte derived interleukin-8: relevance to contact dermatitis. In: Czernielewski JM, ed. Immunological and pharmacological aspects of atopic and contact eczema: pharmacology and the skin. Vol.4. Basel: Karger; 1991a:94–9.

    Google Scholar 

  • Barker JNWN, Mitra RS, Griffiths CEM, Dixit VM, Nickoloff BJ. Keratinocytes as initiators of inflammation. Lancet. 1991a;337:211–4.

    Google Scholar 

  • Barker JNWN, Nickoloff BJ. Leukocyte-endothelium interactions in cutaneous inflammatory processes. Springer Semin Immunopathol. 1992;13:355–67.

    Google Scholar 

  • Boyce D, Ham RG. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free culture. J Invest Dermatol. 1983; 81 (supplement 1):33S-40S.

    Google Scholar 

  • Davidoff AN, Mendelow BV. Unexpected cytokinetic effects induced by puromycin include a G2-arrest, a metaphasemitotic arrest, and apoptosis. Leukemia Res. 1992;16:1077–85.

    Google Scholar 

  • Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol. 1989;44:153–205.

    Google Scholar 

  • Dzarlieva-Petrusevska RT, Fusenig NE. Tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced chromosome aberrations in mouse keratinocyte cell lines: a possible genetic mechanism of tumor promotion. Carcinogenesis. 1985;6:1447–56.

    Google Scholar 

  • Elder PK, Schmidt LJ, Ono T, Getz MJ. Specific stimulation of actin gene transcription by epidermal growth factor and cycloheximide. Proc Natl Acad Sci USA. 1984;81:7476–80.

    Google Scholar 

  • Emerit I, Cerutti P. Clastogenic action of tumor promoter phorbol-12-myristate-13-acetate in mixed human leukocyte cultures. Carcinogenesis. 1983;4:1313–6.

    Google Scholar 

  • Färber B, Petrusevska RT, Fusenig NE, Kinzel V. Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects. Carcinogenesis. 1989;10:2345–50.

    Google Scholar 

  • Grunicke HH, Überall F. Protein kinase C modulation. Semin Cancer Biol. 1992;3:351–60.

    Google Scholar 

  • Guy GR, Chua SP, Wong NS, Ng SB, Tan YH. Interleukin-1 and tumor necrosis factor activate common multiple kinases in human fibroblasts. J Biol Chem. 1991;266:14343–52.

    Google Scholar 

  • Hartley JA, Gibson NW, Zwelling LA, Yuspa SH. The association of DNA strand breaks and terminal differentiation in mouse epidermal cells exposed to tumor promoters. Cancer Res. 1985;45:4864–70.

    Google Scholar 

  • Hawley-Nelson P, Stanley JR, Schmidt J, Gullino M, Yuspa SH. The tumor-promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis. Exp Cell Res. 1982;137:155–67.

    Google Scholar 

  • Ivett JL, Tice RR. Average generation time: a new method for analysis and quantification of cellular proliferation kinetics. Environ Mutagenesis. 1982;4:358.

    Google Scholar 

  • Kinzel V, Richards J, Stöhr M. Tumor promoter TPA mimics irradiation effects on the cell cycle of HeLa cells. Science (Wash). 1980;210:429–31.

    Google Scholar 

  • Köck A, Schwarz T, Kirnbauer R et al. Human keratinocytes are a source for tumor necrosis factor α: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. J Exp Med. 1990;172:1609–14.

    Google Scholar 

  • Kupper TS. Role of epidermal cytokines. In: Oppenheim JJ, Schevach EM, eds. Immunophysiology: the role of cells and cytokines in immunity and inflammation. New York: Oxford University Press; 1990:285–305.

    Google Scholar 

  • Kuroki T, Nemoto N, Kitano Y. Metabolism of benzo(a)pyrene in human epidermal keratinocytes in culture. Carcinogenesis. 1980;1:559–65.

    Google Scholar 

  • Lankas GR Jr, Baxter CS, Christian RT. Effect of tumor promoting agents on mutation frequencies in cultured V79 Chinese hamster cells. Mutat Res. 1977;45:153–6.

    Google Scholar 

  • Lawrence JN, Benford DJ. Chemically-induced unscheduled DNA synthesis in cultures of adult human epidermal keratinocytes. Mutat Res. 1993;291:105–15.

    Google Scholar 

  • Lazutka JR, Rudaitiené S. Effect of tumor necrosis factor on cell proliferation kinetics and sister chromatid exchange frequency in human lymphocytes. Carcinogenesis. 1991;12: 1355–7.

    Google Scholar 

  • Little JB, Nagasawa H, Weichselbaum RR, et al. Differing patterns of cytotoxicity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in various human cell strains. Carcinogenesis. 1985;6:1703–8.

    Google Scholar 

  • Loveday KS, Latt SA. Effect of a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister chromatid exchange formation in cultured Chinese hamster cells. Mutat Res. 1979;67:343–8.

    Google Scholar 

  • Matsushima K, Oppenheim JJ. Interleukin-8 and MCAF: novel inflammatory cytokines inducible by IL-1 and TNF. Cytokine. 1989;1:2–13.

    Google Scholar 

  • McKenzie RC, Sauder DN. The role of keratinocyte cytokines in inflammation and immunity. J Invest Dermatol. 1990;95: 105S-7S.

    Google Scholar 

  • Nagasawa H, Fornace D, Little JB. Induction of sister-chromatid exchanges by DNA-damaging agents and 12-O-tetradecanoylphorbol-13-acetate (TPA) in synchronous Chinese hamster ovary (CHO) cells. Mutat Res. 1983;107:315–27.

    Google Scholar 

  • Pardee AB. G1 events and regulation of cell proliferation. Science. 1989;246:603–8.

    Google Scholar 

  • Parkinson EK, Emmerson A. The effects of tumor promoters on the multiplication and morphology of cultured human epidermal keratinocytes. Carcinogenesis. 1982;3:525–31.

    Google Scholar 

  • Parkinson EK, Newbold RF. Benzo(a)pyrene metabolism and DNA adduct formation in serially cultivated strains of human epidermal keratinocytes. Int J Cancer. 1980;26:289–99.

    Google Scholar 

  • Perry P, Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature (Lond). 1974;251:156–8.

    Google Scholar 

  • Petrusevska RT, Fürstenburger G, Marks F, Fusenig NE. Cytogenetic effects caused by phorbol ester tumor promoters in primary mouse keratinocyte cultures: correlation with the convertogenic activity of TPA in multistage skin carcinogenesis. Carcinogenesis. 1988;9:1207–15.

    Google Scholar 

  • Pillai S, Bickle DD, Eessalu TE, Aggarwal BB, Elias PM. Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes. J Clin Invest. 1989;83:816–21.

    Google Scholar 

  • Pittelkow MR, Scott RE. New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc. 1986;61:771–7.

    Google Scholar 

  • Raetz CRH, Ulevitch RJ, Wright SD, Sibley CH, Ding A, Nathan CF. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 1991;5:2652–60.

    Google Scholar 

  • Ringold GM, Dieckmann B, Vannice JL, Trahey M, McCormick F. Inhibition of protein synthesis stimulates the transcription of human β-interferon genes in Chinese hamster ovary cells. Proc Natl Acad Sci USA. 1984;81:3964–8.

    Google Scholar 

  • Sehgal PB, Tamm I, Vilcek J. On the mechanism of enhancement of human interferon production by actinomycin D and cycloheximide. Virology. 1976;70:256–9.

    Google Scholar 

  • Schneider EL, Lewis J. Aging and sister chromatid exchange. VIII. Effect of environment on sister chromatid exchange induction and cell cycle kinetics on Ehrlich ascites tumor cells. Mech Aging Dev. 1981;17:327–30.

    Google Scholar 

  • Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1. EMBO J. 1991;10:2247–58.

    Google Scholar 

  • Schulze-Osthoff K, Bakker C, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial function: evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992;267:5317–23.

    Google Scholar 

  • Schütze S, Machleidt T, Krönke M. Mechanisms of tumor necrosis factor action. Semin Oncol. 1992;19(supplement 4):16–24.

    Google Scholar 

  • Schwartz JL, Banda MJ, Wolff S. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induces sister-chromatid exchanges and delays in cell cycle progression in Chinese hamster and human cell lines. Mutat Res. 1982;92:393–409.

    Google Scholar 

  • Schwarz T, Luger TA. Pharmacology of cytokines in the skin. In: Mukhtar H, ed. Pharmacology of the skin. Boca Raton: CRC Press; 1992:283–313.

    Google Scholar 

  • Shacter E, Beecham EJ, Covey JM, Kohn KW, Potter M. Activated neutrophils induce prolonged DNA damage in neighboring cells. Carcinogenesis. 1988;9:2297–304.

    Google Scholar 

  • Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. San Francisco: WH Freeman; 1969.

    Google Scholar 

  • Stadler J, Bentz BG, Harbrecht BG, et al. Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration. Ann Surg. 1991;216:539–46.

    Google Scholar 

  • Stryer L. Biochemistry, 3rd edn. San Francisco: WH Freeman, 1988.

    Google Scholar 

  • Sugarman BJ, Aggarwal BB, Hass PW, Figari IS, Palladino MA Jr, Shepard HM. Recombinant human tumor necrosis factor-α effects on proliferation of normal and transformed cells in vitro. Science (Wash). 1985;230:943–5.

    Google Scholar 

  • Symington FW. Lymphotoxin, tumor necrosis factor, and gamma interferon are cytostatic for normal human keratinocytes. J Invest Dermatol. 1989;92:798–805.

    Google Scholar 

  • Thompson LH, Baker RM, Carrano AV, Brookman KW. Failure of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to enhance sister chromatid exchange, mitotic segregation, or expression of mutations in Chinese hamster cells. Cancer Res. 1980;40:3245–51.

    Google Scholar 

  • Trefzer U, Brockhaus M, Loetscher H, et al. 55-kd tumor necrosis factor receptor is expressed by human keratinocytes and plays a pivotal role in regulation of human keratinocyte ICAM-1 expression. J Invest Dermatol. 1991;97:911–6.

    Google Scholar 

  • Tsutsui T, Kawamoto Y, Suzuki N, Gladen BC, Barrett JC. Cytotoxicity and chromosome aberrations in normal human oral keratinocytes induced by chemical carcinogen: comparison of inter-individual variations. Toxicol in Vitro. 1991;5:353–61.

    Google Scholar 

  • Vaca CE, Wilhelm J, Harms-Ringdahl M. Interaction of lipid-peroxidation products with DNA: a review. Mutat Res. 1988;195:137–49.

    Google Scholar 

  • Van Pelt FNAM, Haring RM, Overkamp MJI, Wetterings PJJM. Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide. Mutat Res. 1991;252:45–50.

    Google Scholar 

  • Vessey DA, Lee K-H, Blacker KL. Characterization of the oxidative stress initiated in cultured human keratinocytes by treatment with peroxides. J Invest Dermatol. 1992;99:859–63.

    Google Scholar 

  • Villa LV, Vieira KBL, Pei X-F, Schlegel R. Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18. Mol Carcinogen. 1992;6:5–9.

    Google Scholar 

  • Weitzman SA, Gordon LI. Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis. Blood. 1990;76:655–63.

    Google Scholar 

  • Wille JJ Jr, Pittelkow MR, Scott RE. Normal and transformed human prokeratinocytes express divergent effects of a tumor promoter on cell cycle-mediated control of proliferation and differentiation. Carcinogenesis. 1985;6:1181–7.

    Google Scholar 

  • Wilmer JL, Bloom SE. A cytogenetic approach for detecting the selective toxicity of drugs in avian embryonic B and T lymphocytes in vivo. Mutat Res. 1991;253:161–72.

    Google Scholar 

  • Wilmer JL, Burleson FG, Kayama F, Kanno J, Luster MI. Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin. J Invest Dermatol. 1994;102:1–8.

    Google Scholar 

  • Wong GHW, Elwell JH, Oberley LW, Goeddel DV. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell. 1989;58:923–31.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Environmental Immunology and Neurobiology Section, National Institute of Environmental Health Sciences, PO Box 12233, 27709, Research Triangle Park, North Carolina, USA

    J. L. Wilmer & M. I. Luster

Authors
  1. J. L. Wilmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. I. Luster
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wilmer, J.L., Luster, M.I. Chemical induction of interleukin-8, a proinflammatory chemokine, in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity. Cell Biol Toxicol 11, 37–50 (1995). https://doi.org/10.1007/BF00769991

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00769991

Key words

Search

Navigation