doi:10.1006/cyto.2000.0713, available online at http://www.idealibrary.com on

SHORT COMMUNICATION

GROWTH-REGULATED PEPTIDE-� (GRO-�)PRODUCTION BY ORAL KERATINOCYTES: ACOMPARISON WITH SKIN KERATINOCYTES

Jie Li and Martin H. Thornhill

Growth regulated peptide (GRO-�) is chemotactic for neutrophils. It also stimulates keratino-cyte proliferation and migration, and angiogenesis in cutaneous wound healing. We comparedGRO-� production by normal human skin and oral keratinocytes, and the effects of cytokinestimulation. Resting keratinocytes produced little, if any, GRO-�. TNF-� induced a largeincrease in GRO-� mRNA and protein production in both cell types (P<0.001). However,the response of oral keratinocytes was significantly higher (P<0.01). Oral, but not skin,keratinocytes also produced significant amounts of GRO-� in response to IL-1� (P<0.005) andIL-4 (P<0.01) stimulation. Indeed, there was an additive effect on GRO-� production when oralkeratinocytes were stimulated with combinations of TNF-� and IL-1� or TNF-� and IL-4.Neither cell type responded to interferon �. Keratinocyte GRO-� production may helpselectively recruit neutrophils in mucocutaneous inflammatory diseases, and differences inproduction by skin and oral keratinocyte could explain the different presentation of thesediseases at the two sites. The increased GRO-� responsiveness of oral keratinocytes may alsohelp explain the excellent wound healing properties of oral mucosa.

� 2000 Academic Press

GRO is a member of the CXC subfamily ofchemokines and is chemotactic for neutrophils. Threedistinct GRO genes, designated GRO-�, �, and �, havebeen identified and these are clustered together withother CXC chemokine genes on human chromosome4q21.1

All three GRO peptides possess potent neutro-phil and basophil chemotactic activity in the orderGRO-�>GRO-�>GRO-�,2 and intradermal injectioninduces an acute inflammatory reaction with accumu-lation of neutrophils at the site of injection.3 GRO-�has also been found to stimulate keratinocyteproliferation and angiogenesis in cutaneous woundhealing.4–6

CYTOKINE, Vol. 12, No. 9 (September), 2000: pp 1409–1413

Skin keratinocytes have been reported to produceGRO-� when stimulated with TNF, IL-1, phorbolesters or UV light.7–9 High levels of keratinocyteGRO-� production have been found in psoriasis7

and healing skin lesions.4–6 In psoriasis, GRO-� overproduction is thought to be associated with bothneutrophil recruitment and the epidermal hyperpro-liferation characteristic of the condition. However, ourknowledge of the regulation and production of GRO-�by epidermal keratinocytes is far from complete and weknow even less about the production of GRO-� byoral mucosal keratinocytes. The aim of this studywas to compare GRO-� production by oral and skinkeratinocytes.

RESULTS

From the Oral Disease Research Centre, St Bartholomew’s and The

Royal London School of Medicine and Dentistry, Queen Maryand Westfield College, London, UK

Correspondence to: Prof. Martin H. Thornhill, Oral DiseaseResearch Centre, Clinical Sciences Building, St Bartholomew’sand The Royal London School of Medicine and Dentistry,2 Newark Street, London E1 2AD, UK. E-mail:M.H.Thornhill@mds.qmw.ac.uk

Received 12 November 1999; received in revised form 6 March 2000;accepted for publication 5 April 2000

� 2000 Academic Press1043–4666/00/091409+05 $35.00/0

KEY WORDS: IL-1/IL-4/Interferon-�/TNF

TNF-� induces GRO-� production by oral andskin keratinocytes

Neither oral nor skin keratinocytes produced sig-nificant levels of GRO-� constitutively. Stimulationwith TNF-� (250 U/ml) for 20 h resulted in a bigincrease in GRO-� production by both cell types(P<0.001), although the level produced by oral

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1410 / Li and Thornhill CYTOKINE, Vol. 12, No. 9 (September, 2000: 1409–1413)

keratinocytes was significantly greater than that byskin keratinocytes (P<0.01; Fig. 1). GRO-� mRNAparalleled protein production (Fig. 2). Dose andkinetic studies showed that for both cell types optimalproduction of GRO-� occurred after stimulationwith 250–1000 U/ml of TNF-� (data not shown)for 48–72 h, while mRNA levels peaked after 8 h(Fig. 3). Stimulation with interferon � (IFN-�) had nosignificant effect on either cell type.

IL-1� and IL-4 induce GRO-� production by oralbut not skin keratinocytes

Stimulation with IL-1� (100 U/ml) and IL-4(10 ng/ml) induced a significant increase in GRO-�production (IL-1�, P<0.005; IL-4, P<0.05) by oralkeratinocytes but had no significant effect on skinkeratinocytes (Figs 1 and 2). GRO-� production wassignificantly increased after 8 h and continued to riseuntil 72 h (Fig. 4). Dose-response studies showed thatoptimal stimulation occurred with 250 U/ml IL-1� and10–100 ng/ml IL-4 (data not shown).

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Figure 1. The effect of different cytokines on GRO-� production by oral ( ) and skin ( ) keratinocytes.

Oral and skin keratinocytes were incubated for 20 h with plain medium (BL), TNF-� 250 U/ml, IFN-� 250 U/ml, IL-1� 100 U/ml, IL-4 10 ng/mlor combinations of these cytokines. The GRO-� concentration in the culture supernatants was then determined by ELISA. The results show themean�SD and are representative of four similar experiments.

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Figure 2. The effect of different cytokines on keratinocyte GRO-�mRNA synthesis.

Total cellular RNA was extracted from oral (OK, ) and skin (SK,) keratinocytes incubated for 20 h with plain medium (BL), TNF-�

250 U/ml, IFN-� 250 U/ml, IL-1� 100 U/ml, IL-4 10 ng/ml orcombinations of these cytokines. GRO-� mRNA was detected byNorthern blot analysis using a 32P-labelled Oligonucleotide probe(5�-CTGGCATGTTGCAGGCTCCTCAGAAATAT-3�).1 The rela-tive density of the signals from each lane was quantified by scanninglaser densitometry and plotted as a percentage of the maximumsignal. Densitometry of ethidium bromide stained 18S rRNA wasused to correct for inequalities of RNA loading onto each lane. Theresults shown are representative of two similar experiments.

Additive effect of cytokine combinations onGRO-� production by oral but not skinkeratinocytes

When used in combination with TNF-�, IL-1�and IL-4 had an additive effect on GRO-� productionby oral keratinocytes. This was also reflected bychanges in GRO-� mRNA levels. No such effect wasobserved with skin keratinocytes. Although IFN-�

alone had no effect on GRO-� production, it tended toinhibit TNF-�-induced GRO-� production in oral andskin keratinocytes (Figs 1 and 2).

Keratinocyte GRO-� production / 1411

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Figure 3. Kinetics of TNF-�-induced GRO-� production by keratinocytes.

Oral (OK, — —) and skin keratinocytes (SK, – – – – ) were incubated with 250 U/ml of TNF-� for different period of times between 0–72 h.GRO-� production in culture supernatants was measured by ELISA (left graph). For each point plotted the standard deviation did not exceed5% of the mean. Total cellular RNA was extracted from OK and SK at each time point. GRO-� mRNA was detected by Northern blot analysisusing a GRO-� specific 32P-labelled oligonucleotide probe. The relative density of the signals from each lane was quantified by scanning laserdensitometry and plotted as a percentage of the maximum signal (right graph). The results shown are representative of three similar sets ofexperiments.

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Figure 4. The kinetics of IL-1� and IL-4 induced GRO-� production.

Oral (OK, — —) and skin keratinocytes (SK, – – – – ) were stimulated with 100 U/ml of IL-1� (left graph) or 10 ng/ml IL-4 (right graph) fordifferent periods of times between 0–72 h. GRO-� production in the culture supernatants was measured by ELISA. For each point on the graphthe standard deviation did not exceed 5% of the mean value. The results shown are representative of three similar sets of experiments.

DISCUSSION

In this study we have shown that oral keratino-cytes are able to produce GRO-�, a CXC chemokine,which has potent chemotactic activity for neutrophils.We have also revealed differences in GRO-� produc-tion between oral and skin keratinocytes in response to

cytokine stimulation. These differences are similar tothose we reported for IL-8, and contrast with those forthe CC chemokines RANTES10 and MCP-1.11

Although oral and skin keratinocytes produce GRO-�and IL-8 following TNF, and to a lesser extent IL-1�,stimulation, they exhibit no responsiveness to IFN-�and no synergy between TNF and IFN-�. In contrast,

1412 / Li and Thornhill CYTOKINE, Vol. 12, No. 9 (September, 2000: 1409–1413)

TNF and IFN-�, but not IL-1�, induce both cell typesto produce RANTES and MCP-1 and there is markedsynergy between TNF and IFN-�. Also, although oraland skin keratinocytes exhibit similar responsivenessfor RANTES and MCP-1 production, oral keratino-cytes are far more responsive than skin keratinocytesfor GRO-� and IL-8 production.

Inflammatory mucocutaneous diseases are charac-terised by the accumulation of specific populations ofleukocytes in the tissues. Circulating leukocytes firsttether to the endothelial lining of blood vessels viaselectin adhesion molecules. They then cross theendothelial barrier by interacting with ICAM-1. Thisrequires activation of the cells and chemokines havebeen shown to play an important role in activatingthese tethered leukocytes. GRO-� does this and maythereby selectively enhance the accumulation of neu-trophils in the tissues.12 Once in the tissues, neutrophilswill be subject to a concentration gradient of GRO-�,and other chemokines released by neighbouringkeratinocytes. This will direct leukocyte traffic towardsthe epidermis/epithelium and exert a further degree ofselectivity over the type of leukocytes that accumulateand hence the nature of the disease.

Clinically, high levels of keratinocyte GRO-� pro-duction have been found in psoriasis, UV irradiation,burns and other wounds where neutrophils are promi-nent.4,6,7,13 In the mouth neutrophil infiltrates are seenin Candida infections, apthous and oral ulceration,geographic tongue, gingivitis and other non-specific oracute inflammatory lesions. Oral keratinocytes mayproduce GRO-� and IL-8 in these situations butfurther studies will be needed to confirm this.

Finally, as well as being chemotactic for neu-trophils, GRO-� can act as a growth factor andchemokine for keratinocytes4,6 and endothelial cells.5

Indeed, keratinocyte-derived CXC chemokines appearto be important mediators of wound healing. Early on,they recruit the neutrophil dominated infiltrate that isimportant in the catabolic phase of wound healingand later, in the repair phase, they stimulatere-epithelialization and angiogenesis.4,6 The increasedGRO-� responsiveness of oral keratinocytes tocytokine stimulation could help explain the excellentwound healing properties of oral mucosa compared toskin. However, further studies of the role of GRO-�and pro-inflammatory cytokines in regulating woundhealing in the skin and oral mucosa will be needed toconfirm this.

MATERIALS AND METHODS

Recombinant human IFN-� was obtained from BiogenSA, Switzerland. TNF-� was a gift from Dr B. A. Beutler(University of Texas Health Sciences Centre). IL-1� and IL-4

were purchased from R&D Systems. Concentrations usedwere 0–1000 U/ml of IFN-�, TNF-� and IL-1�; 0–100 ng/mlof IL-4.

Keratinocytes were prepared from fresh biopsies ofnormal human skin or oral mucosa as described previously.14

Before experiments, keratinocytes were re-plated into serumfree medium in the absence of feeder layers and treated withcytokines. GRO-� concentration in the culture supernatantswas quantified using a sandwich ELISA assay (QuantikineDGR00; R&D Systems). Total cellular RNA was extractedfrom keratinocyte monolayers and Northern blot analysiswas performed as described previously.10

Differences between the results of experimental treat-ments were evaluated by means of the two-tailed Student’st-test.

Acknowledgement

We would like to acknowledge the supportprovided by a Royal Society Research Grant.

REFERENCES

1. Haskill S, Peace M, Morris J, Sporn SA, Anisowicz A, LeeSW, Smith T, Martin C, Ralph P, Sagor R. (1990) Identification ofthree related human GRO genes encoding cytokine functions. ProcNatl Acad Sci USA 87:7732–7736.

2. Geiser T, Dewald B, Ehrengruber MU, Clark-Lewis I,Baggiolini M (1993) The interleukin-8 related chemotactic cytokinesGRO�, GRO� and GRO� activate human neutrophil and basophilleukocytes. J Biol Chem 268:15419–15424.

3. Zwahlen R, Walz A, Rot A (1993) In vitro and in vivoactivity and pathophysiology of human interleukin-8 and relatedpeptides. Int Rev Exp Pathol 34:27–42.

4. Nanney LB, Mueller SG, Bueno R, Peiper SC, Richmond A(1995) Distributions of melanoma growth stimulatory activity ofgrowth-regulated gene and the interleukin-8 receptor B in humanwound repair. Am J Pathol 147:1248–1260.

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6. Rennekampff HO, Hansbrough JF, Woods V, Jr., Dore C,Kieesig V, Schroder JM (1997) Role of melanoma growth stimu-latory activity (MGSA/gro) on keratinocyte function in woundhealing. Arch Dermatol Res 289:204–212.

7. Kojima T, Cromie MA, Fisher GJ, Voorhees JJ, Elder JT(1993) GRO-alpha mRNA is selectively overexpressed in psoriaticepidermis and is reduced by cyclosporin A in vivo, but not incultured keratinocytes. J Invest Dermatol 101:767–772.

8. Boorsma DM, de Haan P, Willemze R, Stoof TJ (1994)Human growth factor (huGRO), interleukin-8 (IL-8) and interferon-gamma-inducible protein (gamma-IP-10) gene expression in culturednormal human keratinocytes. Arch Dermatol Res 286:471–475.

9. Venner TJ, Sauder DN, Feliciani C, McKenzie RC (1995)Interleukin-8 and melanoma growth-stimulating activity (GRO) areinduced by ultraviolet B radiation in human keratinocyte cell lines.Exp Dermatol 4:138–145.

10. Li J, Ireland GW, Farthing PM, Thornhill MH (1996)Epidermal and oral keratinocytes are induced to produce RANTESand IL-8 by cytokine stimulation. J Invest Dermatol 106:661–666.

11. Li J, Farthing P, Thornhill M (1999) Oral and skin kerati-nocytes are stimulated to secrete monocyte chemattractant protein-1by tumour necrosis factor-� and interferon-�. J Oral Pathol Med(in press).

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12. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M,Ding HA, Gimbrone MA, Jr., Luster AD, Luscinskas FW,Rosenzweig A (1999) MCP-1 and IL-8 trigger firm adhesion ofmonocytes to vascular endothelium under flow conditions. Nature398:718–723.

13. Engelhardt E, Toksoy A, Goebeler M, Debus S, BrockerEB, Gillitzer R (1998) Chemokines IL-8, GROalpha, MCP-1, IP-10,

and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing.Am J Pathol 153:1849–1860.

14. Li J, Mahiouz KL, Farthing PM, Haskard DO, ThornhillMH (1996) Heterogeneity of ICAM-1 expression, and cytokineregulation of ICAM-1 expression, in skin and oral keratinocytes.J Oral Pathol Med 25:112–118.