Epidermal and Oral Keratinocytes Are Induced to Produce RANTES and IL-8 by Cytokine Stimulation

Jie Li, * Grenham W. Ireland,t Paula M. FarthingJ and Martin H. Thornhill'" · Clinjcal Acadcmic Group of Oral Medicine and Dental Diagnostic Scienccs. University Dental Hospita l of Manchestl!r; tSchool of Biological Sciences, University of Manchester; and :j:Dcpartmcnr of Oral Pathology, The London Hospital Medical College. London, U.K.

RANTES, interleukin-8 (IL-8), and macrophage in­flammatory protein-la (MIP-la) exhibit different and highly selective chemotactic activity for leukocytes. Resting cultured normal oral and skin keratinocytes produced little if any of these chemokines. Stimula­tion with 250-1,000 U/ml of tumor necrosis factor-a (TNF-a) or intel'feron-,), (IFN-')') induced both cell types to produce RANTES. Protein levels peaked after 48 hand n1RNA levels peaked after 8 h of stimulation. Used in combination, TNF-a and IFN-')' synergistically increased mRNA and protein levels. Amounts of 100-1,000 U/ml of TNF-a also induced IL-8 production with peak mRNA levels after 4-24 h of stimulation and maximal protein production after 72 h or more. IL-8 production by oral keratinocytes was significantly greater than that by skin keratino-

C hemokines are a large super f.1m ily of structura lly and functiona lly related molecules with c. hemotactic activity targeted at specific leukocyte populations . They are divided into two subfami lies according to the position of the first two cystei nes w hich al'e

eith er separated by one amino ac id (CXC) or are adjacent (C-C) (Schall, 1991 ; Miller and Krangel, 1992; Baggiolini el 01, 1994). Recently, a third subfamily has beell identified with only one cysteine (C) (Kelner el 01, 1994).

The CXC (a) chemokine subfamily includes inte rl eukin-8 (IL-8) (a lso known as neutrophil activation peptide-l (NAP-l» , plate let facto r 4 (PF-4) , {3-thromboglobulin , GRO, and interferon-ind uc­ible protein 10 (IP-10), many of wh ich have bee n shown to chemoattract and activate neutrophils (Miller and Krangel, 1992; Baggiolini el al; 1994). In contrast, the C-C ({3) subfamily includes RANTES, macrophage inflammatory protein-1 a (MIP-la), MlP-1{3, monocyte chemoattractant protein-l (MCP-l) also known as macrophage chemotactic and activating factor (MCAF) , 1-309 and HC14, which mainly chemoattract and activate mononuclear cells (Schall, 1991; Baggiolini et 01,1994).

IL-8 is a potent chemokine for ne utrophils (Schroder et 01, 1987;

Manuscript received September 29, 1995; revised November 27. 1995; accepted for publication December 11, 1995.

Reprint requests to: Prof. M, I-I. T hornhi ll , Clinical Academic Group of Oral Medicine and Dental Diagnostic Science, University Dental Hospital of Manchester, Higher Cambridge Street. Manchester MIS 6Fl-l, UK.

Abbreviations: MlP-1a, macrophage inllammatory protcin-Ia; RANTES, regulated and normal T ce ll-expressed and secreted: TNF-x. tumor necrosis fac tor-a.

cytes. Although IFN-')' alone did not induce IL-8 production, it enhanced the effect ofTNF-a on bOtll cell types. Stimulation for 24 h with 100-1,000 U/ml ofIL-la also induced IL-8 production by oral but not skin keratinocytes. No MIP-la production was de­tected under the conditions investigated. Keratino­cyte production of RANTES and IL-8, under the influence of cytokines such as TNF-a or IFN-,)" pro­vides a mechanism for the selective accumulation of leukocytes into immunoinflammatory diseases of the skin and oral mucosa. Differences in their production may help to explain differences in the presentation of these diseases on the skin and oral mucosa. Key II101,ds: chetllokiueslMIP-1 altl/lllol' IICCI'osis factol'-alil/teliel'o", a. J Illvest Del'lllato/ 106:661- 666, 1996

Walz ct 01, 1987; Yosh.il11ura cl nl, 1987; Leonard et 01, 1991; Swensson et 01, 1991) and may have some chemotactic activ ity for lymphocytes i1l v itro (Larsen el 01, ] 989a; Wilkinson and Newman, 1992; Taub cl 01,1993) . The inttadermaJ injection of IL-8, how­ever, is not accompanied by the accumu lation of lymphocytes (Leonard ct 01, 1991; Swensson cl 01, 1991). In contrast, RANTES is chemotactic for resting CD4 + memory T cells and activated na'ive and m emory T cells (Larsen rt 01, 19893: Schall et 01. 1990; Taub cl 01, 1993), whereas MIP-1 a has chemotactic activity for T and B lymphocytes (Schall et 01, 1993; Tanaka et 01, 1993).

These chemokines arc produced by various cell populations and their pl'oduction can be regulated by certain proinflammatory cytokines . T he precise mix of chemokines released into the tissues may have a powerful effect in determining the composition of iJlflammatory infiltrates and hence the nature of the inflammatory response that results. The identification of the cellular sources of the chemokines and the way in which their production may be modulated by cytokines is far f1-OI11 complete. The precise ro le of chemokines in specific inflammatory processes is poorly under­stood. We therefore investigated the production and release of lL-8, RANTES, and Mn>-l a by oral and skin keratinocytes and studied the effect of cytokines in regulating their production . T his may provide important insights into the way in w hich keratinocytes regulate immune and inflammatol")' processes. Differences in che­mokine production by ora l and skin keratinocytes may help to explain the different clinical co urse and nature of immunoinflam­matory diseases, such as lichen planus, psoriasis, and graft-versus­host disease, that affect the skin and oral mucosa (Scully and El-Kolll, 1985; Eversole, 1994) .

0022-202X/96/S10.50 • Copyright © 1996 by The Socicry for Investigative Dermatology, Inc.

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Cytokincs R ecombinant human (rll) interfe ron-ga l11ma (IFN-y) was obtained from J3iogen SA, Geneva , Switzerland. Rh tumor necros is 61ctor-n (TNF-a ) was a gift fi'om D r. B.A . J3cut1 er, University of T ex as Health Sciences Center, Dal);15, T X. Intcrl eukin- 'I a (I L-lcr) and IL-4 were pur­chased from n .. &D SY,5 tem , Cowley. Oxfo rd. UK. R.h IL-S was the kind g ift of Dr . .I.M .A. Wil ton . University of Illinois. UrbaJl a, It. . Ith tr311 ,fo rl11ing growth facto r-{3 (TGF-{3) was obta in ed fro m Austral Diologicals , San Ramoll , C A. COllccntrario.lls used were 0-1 .000 U / 1111 of IFN-y, T NF-a , and IL-l a; 0 -1 00 ng/ml ofl L-4; 50 ng/m l of lL-8 and 10 ng/ 1111 orTG F- f3.

Kera tinocyte Culture Kcratinocy tes were prepared (i'om fresh biopsies of normal human ski!) o r oral mucosa using a m odifi cation (Li el nl. 1995b) of the method described by R.hein wald and Green (1975) . Skin keratino­cytes were obtained fi'onl [hree W0111ell and one m;m (age r,mge: 32- 56) using excess skjJ] rCJll o v cd during breas t reducti o n o r apr0l1 cct0 I11 Y proce­dures . O ral ker<1tin ocyrc" were prep :lred from normal oral mucOsa removed durjng fi-cn cctOJll Y, g ing ival surge ry. Or fro lll Hap ,narg ins '"rOlll one l11an and three women (age range: 28 - 72) . All e"perin1ents w ere performed on third to fifth p:lssage cultures. For experiments the ccUs were re-plated into serum-frce medium based on MC D131 53 (Imperial Lnboratorics. Hants, UK) plus 0.2% bo vine serum albumin, 10 ng/ml ep ider",al cell grow th factor, 0 .4 mg/ml hydrocorti ~one, 5 m g/ml insulin ,S mg/1111 transferrine . ·\. S X 1 O-~ M "denine , SO U/1l11 penicillin. 50 m g /1111 strepto m yc in (all M'om Sigma. Poole . Dorset, U K) and 0.2 5 I11g /ml fungi zo ne (G lBCO , Paisley, Scotland , UK) in the absence of feeder b yers. The ceUs were culnlrcd until subconflucnt and were then tre<1ted for various t imes wi th cytokiJl es in fTe" h serum-M'ce medium no t containing hydrocorti sone. The supernaWllts were coll ec ted and stored at - 70°C un til required for chcm o­kine quan tifi cation . T he kcratinocytc monolayers were lysed fo r R.N A a naJ )'s is.

Quantification of Chemol<ines IL-8 . I1..ANTES and MlI)-h~ concen­tr;lti o ns in kerat inocytc cu lture supc rn ntants w e re n1 casllrcd using cy tokinc­specifi c sandwich enzyme-linked immllnosorbent ass;Jy (ELI SA) kits , Quan­tikine (D8000 , DRN{)O , and OMAOO), from R &D Syste111 s, Abingdon , Oxon, UK. A standard Cllrve was produced fo r each "ssay using kn own con centrati ons o f the recombinant hum"n ch et110kine provided in each kit. T h e concentration o f lL-S. I:t.ANTES , 01' MIP-l n in th e supernat:1nts was determined from the standard curve . T he detectio n limit of the assays was 3.13- 2.000 pg /ml fo r I'tANTES and lL-8 and 3 .13-1 ,OOll pg/ ml fo r MIP-1 a . Where neccss"ry, samples were diluted to bring th em into the detecti on range . (The supernatants were diluted 50 limes with serulll- fr ee medium for the IL- 8 assay and n"at supernatants were used in the ['lA NT ES and MI.I'-1 CI 'ISsays).

Northern Blot Analysis T ota l ce llular RNA was ex tracted fi'om sub­conflu e n t 1110 ll o1aycrs of untreated and cytokinc treated k crarinocytcs lI sing U hr"sepc l'lNA isolation system (Biotecx, AMS Bio technology UK Ltd. , Itlll ey, O x fo rd). N orthern blo t analysis was performed using a nonradioac­ti ve digoxigcnin- Iabcling sys tcm essentially as described by Doehringcr Mannheim user' s guide. Briefl y, the l'lNA was fractionated over a 1.2'Vr, agarose/ fof1naldeh yde gel and transferred to a positi vely ch ;1rged nylon m embrane (Boehringer Mannheim , East Sussex . UK). The m embrane was pre-hybridized for 4 h at 42°C with blocking reagent and po ly(A} (Boehr­inger Manl1heim) to block nonspec if, c binding. Oigo xigcnin-Iabeled 30-base oligonucleotide probes fo r IL-8 (13PR 100) "nd RANTES (BPR 24 6) (R&D System). containing an equimolcr mixture of 5' , mid-, "nd 3' regions. were used to detect IL-8 and RANTES Il1 I~As, rcspcc tively. After overnight hybridizatio n at 42°C, the membrane was w ashed under increasin gly strin gent condi t io ns and trea ted w ith blocking reage n t to prevent nonspecifi c attraction of antibody. The 111 embrane was then incubated with anti-digoxigenin Fab fragments and conjugated to alkaline phospha tase (Boehringer Mannheim) fo r 30 min at 1'00111 tempe1';ltllTe. After tw o 1 5-min wash es, the m C111br;\1)e Was reacted with the substrate disodium 3-( 4-methoxyspiro p .2-dio,;etane- 3 ,2' -(5 ' -chloroltricyd o(3.3.1 .1 J.7·)decan}_ 4-y\)phcny\phosph"te (CSPO) (Boehringer Mallnhe.im) fo r 5 min and t.hen incubated at 37°C for '15 min to promote the reaction . T he chenlil1111linnescellt sigllals w e re recorded by exposing the me mbrane to x-ray filill at 1'0 0111 tcmpcratme to r 15-30 min. IL-8 and fl..ANTES ml~As were quantified using a. laser densitometer (Uill Vltrascaner). The intensity of each band on th e x-ray fil.ll1 ,vns expressed as the area 1Ilider the curve obtained fro lll I;ls~r densitom­etry. Ethidiu111 bromide staining of 18 S ribosom<11 Il...NA was used to indicate equal loading of RN A onto cach lalle.

Statistics Differences between the resul ts of experimental trea tm ents were c\l ~lu"tcd h y n1cans of the. 2- t.<1. ilcd Stude nt 's 1 tcst.

TH E JO UR.NA L O F INVEST IGATIVE D ERMATOLOGY

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Figure 1. TNF-a and IFN-y induce RANl'ES production by oral (OK) altd skill (SK) keratinocytes . OK and SK were treated with m edium onl y (BL), T NF-tv 250 U / ml . IFN-y 250 U / 1111. lL-l a lOO U/ml, IL-4 '10 ng/1l11 or T GF-J3 10 ng /1111 , or TNF-n + IFN- y together. respecti vely, fo r 24 h. RANTES production in the supernatant.s was guantifl ed by sandwich E U SA. For cach point the standard dev iation did no t exceeel 2. 5% of the value . This experiment is reprcsel1t:t tivc of (our replicate cxp cri,nlCllts with sirnilar results.

R E SULTS

TNF-a and IFN- l' Induce RANTES Production by Kcrati­nocytes N e ither oral nor skin kcratinoc ytes produ ced KANTES without stimulation, but wh ell scilTIula ted with TNF-a or IFN-y for 24 h, signifi C<lIIt level s o f RANTE S w e rc d e tecte d in c ulture supe rnatants of both cell types (Fig 1). Peak produ c cion b y both cell t yp e s o c c urre d afte r stimulation w ith 250-1,000 U / ml of TNF-a (data n o t shown) for 4 8 h or 250-1 ,000 U / m l of IFN-y (dat,1 not shown) for 4 8 -72 h (Fig 2). No e ffec t 011 r~NTES produc tion was found afte r stimulation with IL-l a , IL-4 , or TGF-{3 (Fig 1) .

When u sed in combination , T NF-a and IFN-y had a h1rge synergisti c e ffec t (I' < 0 .01) on RANTE S production by both oral and skin k e ratinoc ytes, whic h was about 4 .5-7 .5 times g reater than the e/fect of TNF-a alone. (Fig 1). The r e was no significant dilfe rence betwee n oral and skin keratinocytes Ul this respe ct. Other

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Figure 2 . Kinetics of TNF-a or IFN- ')'- illduced RANTES produc­tion by oral (OK) and skill kcratinocytes (SK). OK and SK wen' incubated with 250 U/ml TNF-a ("Ji) or 250 U / ml IFN-y (light) for di(/c rcnt pe riods o f t ime between 0 'lIld 72 h. RANTES production in rhe supernatants was measured by sandwich ELISA. For each poill t rhe standard deviation did IIOt exceed 2.5% of rhe value. The experiments shown "t<'

re prese ntati ve of fo ur re plica te sets of CXPCriJ11CJ1tS w ith sinJilnf r esu.lts.

VOL. 106. NO.4 APRJLI996

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combinations including TNF-a + IL-1a, TNF-a + IL-4, IFN-l' + IL-la, and IFN-l' + IL-4 had no significant synergistic effect (data not shown).

Consistent with the results for RANTES protein production, unstimulated oral and skin keratinocytes contained little if any mRNA for l~NTES . TNF-a or IFN-l' induced RANTES mRNA synthesis (Fig 3). which peaked after 8 h of stimulation (by TNF-a) for both cell types (Fig 4) . When used in combination, TNF- a and IFN-l' acted synergistically to increase RANTES mRNA transcrip­tion in oral and skin keratinocytes (Fig 3).

TNF-a and IL-1a Induce IL-8 Production by Keratinocytes IL-8 production by unstimulated oral and skin keratinocytes was very low and often undetectable. Stimulation with 250 U/ml TNF-a for 24 h induced IL-8 produ ction by both cell types, although the level of 1L-8 produced by oral keratinocytes was significantly greater than that produced by skin keratinocytes (p <

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Figure 4 . Kinetics ofTNF-a-induced RANTES (left) and IL- 8 (';gl.t) mRNA synthesis by oral (OK) and skin (SK) keratinocytes. OK and SK were stimulated with TNF-a 250 U /m l for differcnt pcriods of time between 0 and 72 h . Total cellular R.NA was cxtracted and mR.NA for IL-8 or RANTES were hybridized with digoxigenin-Iabelcd specific oligonu­ceotide probes and quantified by densitomctry of Northern blots. The results show the relative density of the signals from each lan e of the Northern blots as a pcrcentage of the maximal signal. This expcriment is repre sentative of t\vo replicate cXpCril11cnts w'ith silllilar results.

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KERATINOCYTE RANTES AND IL-8 PRODUCTION 663

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Figure 3. TNF- a and IFN- I' induce RANTES mRNA synthesis in oral (OK) and skin (SK) keratinocytes. Northern blot analysis was performcd for RANTES mRNA (top track) extractcd from OK and SK stimu­lated for 20 h with medium only (BL). TNF-a 250 U/ml. IFN-y 250 U/ml. IL-l a 100 U/ml. lL-4 10 ng/ml. or TNF-a + lFN-y. Equal loading of the lanes is shown by ethidium bromide staining of 18 S ribosomal RNA (bottolll tl"nck). The relative density of the signals from each lane of the upper track was deter­mined by scanning laser densitometry and plot­ted as a perccntage of the maximum signal (qrap"). TIllS expcrimcnt is representativc of two repHcate experiments with similar results.

0.01) (Fig 5). Dose-response and kinetic studies showed that IL-8 production was maximal for both cell types when stimulated with 100-1,000 U / ml ofTNF-a for 72 h or more (Fig 6). Although, under the conditions used, 1L-1 a had no significant effect on 1L-8 production by skin keratinocytes, it induced a significant increase in 1L-8 production by oral keratinocytes (p < 0.05). A dose-response study showed that maximal 1L-8 secretion occurred when oral keratinocytes were stimulated with 1,000 U/m.l of 1L-1 a (Fig 7) .

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F ig ure 6. Dose r esponse and kinetics o f TNF-O'-induce d IL-8 pro­duction by oral (OK) and skin (SK) ker atillocytes. O K and SK were ,incubated with di ffe rent concentrations ofTNF-O' for 48 h (lift) or with 250 U/ml TNF- cx fo r differen t periods of time between 0 and 72 h (rig"t). The concentration of 1L-8 in the culture supernatan t was determined by sandwich ELISA. For each poin t the standard deviation did not exceed 3% of the value. The experiments shown are representative of two se ts of replicate experiments with similar results.

IFN-)" IL- 4, and TGF-f3 h ad no significant eff'ect o n IL- 8 produc­tion by either cell type (F ig 5), altho ugh there were sm all non-signi fica nt in creases in som e skin keratin ocyte cul tures treated with l FN-)'.

When used in combination , T N F- a + IFN- )' induced a sm all extra increase in IL- 8 production by bo th cell types. T his was significant for skin kera tinocytes (p < 0.05) but not for oral keratinocytes (Fig 5). Combin ati ons ofTN F- a + lL-1 a, T N F- a + IL-4, IFN -)' + IL-l a, IFN-)' + IL-4 had no synergistic eff'ect o n IL- 8 production by either cell typ e (data no t sho wn) .

Unstimulated keratinocytes contained very low le vels of IL-8 rnRNA. T N F- O' stimulation produced a rapid ri se in IL - 8 mRNA synthesis, which was optimal be tween 4 and 24 h o f stimulatio n for bo th cell types (F ig 4) . T he increase in lL-8 mRN A level w as abo ut 4-fold higher for o ral than skin keratin ocytes (F igs 4, 8). When llSed in combin atio n , T N F- a and IFN-)' had a syne rgistic eff'ec t on IL- 8 mR N A productio n in both cell types (F ig 8) . Under the condi tions used , IL-1a also induced a sm all increase in IL- 8 mRNA transcriptio n in o ral bu t n ot in skin ke ratinocytes (F ig 8) .

Failure o f MIP-la P roduction by Keratinocyt es Similar experim ents were pe t-formed to look fo r the productio n of MIP-1 a

Figure 7. IL-la induces IL-8 production by oral kera tinocytes (OK). OK and SK were incubated with increasing doses oflL-1 IX from 0 to 1000 U/ml fo r 48 h and the level of IL-8 in the supernatant was measured by sandwich ELISA. For c;,ch poin t the standard devia tion did not exceed 3% of the va lue. Tllis experimcnt is reprcsenta ti ve of two rep lica te ex­pcrilllcnts with sinl il ar resul ts .

T H E JOURNAL OF INVESTIGATIVE DER.M ATO LOGY

by o ral and skin ke ratin ocytes. N o MIP- l a production was de­tected in c ultures of un sti mul ated keratinocytes o r cultu res stimu­la ted w ith IL-1a, IL- 8, TGF- {3, TNF- a , or IFN- y (data not shown) .

DISCUSSrO N

Inflammato ry mu cocu taneous diseases are characte rized , according to the nature of the disease, by the accumulation of diff'e rent popul atio ns of leukocytes in the tissues . T hese leave the circul ation selectively to accumul ate in the connective tissue and m ay invade the epidermi s or epithelium. Endo thelial ce ll adh es ion molecules including [CAM-I , E- selectin, and V CA M-l are im portant in initia ting this p rocess by fac ili tating the t rans-endo thelial migration of leukocytes and , according to the patte rn of adhesio n m olecules expressed , exert a degree of selecti vity over dus process (Berg et ai, 1989; Luscin skas et ai, 1989; Tonnesen , 1989, Po ber and Cotran, 1990). T he in duction o f ICAM-l express io n on kera tin ocytes may be impo rtan t in permi tting le ukocy te invasion of the e pidermis or epi thelium (D ustin et ai , 1988; Ni cko lo ff et ai, 1990; Li ct ai, 1995b). T he consequences of this are tho ught to be central to th e patho­physiology of skin disease (Nicko loff' e£ ai, 1990) . T his mechanism is unl ikely to exert any selectivity over the nature of the leukocytes interacting with the kera tinocytes .

C hem okines released from keratinocytes have the potential to targe t thi s traffic on the epidermis or epi thelium and, according to

the types of chemokines released , exert a further degree of selectivity over tb e types of le ukocytes that accumulate and hence o ver th e n ature of the disease .

To date epiderm al kera tinocytes have been fOllnd capable of producing the CXC chem okin es IL- 8 (La rsen et ai, 1989b; Barker et ai , 1991), GR Oa (R.ichm ond and T ho m as, 1988) and [PI0 (Kaplan et ai, 1987), all of w hi ch are pr im arily chenlOtactic for ne utrophils. Indeed , chro nic overprodu ction of IL- 8 by epidenllal kera tinocytes has been fo und in pso ri as is and is th ought to account fo r the accumulatio n of neutrophil s, and the development of the neutrophilic micro-abscesses that characterize the disease (N ick­olo ff' et ai, 1990; Barker el ai, 1991; Schroder, 1992).

In tins study we dem o nstra ted that oral keratinocytes could also be ind uced to produ ce IL-8. Altho ugh the o ral mucosa is only rarely affected by psorias is, a reversibl e accumula tion of neutrophils w ith invasion of the epithelium is seen il1 o th er mu cosal condi tions such as oral candidias is and IL- 8 co uld play a role in this.

lL-8 exhibi ts chem o tac tic activity for lymphocytes ill lI i l l'o (La.rsen el ai , 1989a; Bacon and Camp, 1990; Wilkin son and Newman, 1992) and has bee n linked with th e accumula tio n oflymphoc)'tes in psoriasis (Gri.ffi th s ct ai , 1991; Nickoloff' et ai, 1991) although intraderm al injecti on studies do no t suppo rt a ro le for IL-8 in hum an lymphocyte chemotaxis ill IJ i IJO (Leonard et ai, 199 1; Swens­son e( ai, 199 1) . It has been suggested that the co-p roduction of C - C chem oki.11 es in psorias is could account fo r the lymphocytic component of the inAamm ato l'Y infiltrate (Baggio lini el ai, 1994). A previo us study has shown that skin keratinocytes are able to produce M C P-1 after IFN-)' stimulation (B arker cl ai, 1990) . T h.is p articular C -C chem okin e appears to have li ttle if any chemotactic activi ty for lymphocytes (Simo n , 1988; Bischo ff' el ai, 1993; Taub er ai, 1993 ).

In con t rast , l~NTES is markedly chem o tactic fo r lymphocytes w ith activity for res ting C D 4 + m em ory T ceJls and ac ti va ted naive an d m em ory T cells of CD 4 and C D 8 phenotype (Larsen el ai, 1989a; SchaU el ai, 1990; T aub el ai, 1993). It may therefore be importan t in the recruitment o f T cells in psorias is and other dermatoses where lymphocytes are an important feature of the inAammatory in ftltrate. Sin ce RAN TES w as also produ ced by oral ke ratin ocytes, it could playa ro le i.n the recruitment ofl ym phocytes into the o ral les io ns o f conditions such as lichen planus, graft­versus-host disease, or lupus erythem atosus. Further studies are required to confirm the relationship of RAN TES production to specifi c mucocu taneous condi tio ns. Altho ugh MTP-l a is also a C-C chem okine wi th activi ty for lym phocytes, no MIP-l a p roduction w as detected fo rm oral o r skin ke ra tinocytes.

Previo us studies have dem on strated tha t TN F- a and IL- l stim-

VOL. 106, NO. 4 APR1L 1Y%

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I-+

u.. Z I-

ulation of epidermal keratinocytes induces IL- 8 mRNA produ ction (Larsen et (/1, 1989b; B~ rke r et ai, 1991 ; Kristenscn et ai, 1991 ) and IFN-')', a ltho ugh not active by itself, syn ergizes with TN I~-O' for IL- 8 production (Barker el ai, 1990). Simi lar results w ere fo und in this study, a lthough IL-8 synthes is and message levels were mini­mal, if present, after IL-IO' stimul ation . In contrast , o ral keratino­cytes produced IL-8 in response to both IL-IO' and TNF- O' stimu­lation and the levels of IL-S produced by o ral keratinocytes we re signi ficantly higher than skin keratinocytes fo llowing TNF-O' stim­ulation.

In contrast, skin and o ra l keratinocytes both produced .RANTES after stim ulation w ith IFN-')' as well as TNF-O' and ill combin ation the two cytokin es had a marked syne rg istic effect. T h e level of RANTES produced b y the two cell types was simila ... O ral and skin keratinocytes, however, m ay not be the on ly ce LIs of epithelial origin able to pro du ce RANTES since bronchial epithe lia l celJ s and cell lines are also able to produce this cytokin e (Davies ei ai, 1995; Kwon el ai, 1995; Stellato ('/ ai, 1995).

Interesti ngly, in keratinocytes, although mRNA levels peaked after S h , lu\NTES protein levels did not peak un ti l afte r 4S h of sti.m.u lation w ith TNF-O'. T lli s delay suggests thatMNTES may undergo post-transcriptional modification in these celJs. T he syn­ergy between T N F- O' and IFN-')' for Iu\NTES production would appear to be a pre-transcriptional event, since messagc levels appear ro equate to levels of chemokine release. Indeed, other evidcnce sugges ts tha t the effect may occur at th c Icvcl of tran scription regulation . In a gastric cell lin c T N F-O' and IFN-,), have been fo und to act synergistica lly by prom oting the binding of the tran scriptio n regulato ry elem ents activator protcin- 'I (AP- 'I) and nuclear factor K13 (NFKB) to the IL-S gen e (Yasumoto ci (/1, 1992) . Since th c RANTES gcnc also con tains binding sites for AP-l and NFKB, it is possible th at the same m echanism operatcs for RANTES (Mukaida el ai, 1992; Marfaing-Koka et ai, 1995).

It has bccn suggested that thc naturc of imllluno-inflamlllatory dermatoses is dctcrmin ed by the re lease of cytokines in to the tissues. By inducin g chcm okin c production , cytokin es ma y deter­mine the nature of the inflamm atory in fil trate that develops (Schro­der, 1995). TNF-a and IL- l may bc produced by keratinocytes, both from skin (Bell el ai, 19S7; Kock et ai, 1990) and oral mucosa (Yamamoto el ai, 1994; Li ei ai, 1995a), Poten tia ll y these co uld stimulate ke rati nocytes to secrcte IL- S and R A N TES and initiate

-t ...J

KERATINOCYTE I,-ANTES AND IL-S PRODUCT ION 665

I L-H 111 RN 1\

IHSrRN/\

Figurc S. Cytokinc induction ofIL-S mRNA synth csis in oral (OK) and skin (SK) kcrati­Ilocytcs. Northern blot analysis was performed fo r Il-H mlt.NA (IIPpc/" t/"nek) extracted from OK and SK stimulated for 20 h with medium only (ill). TNF-O' 250 U/ ml. IFN-y 250 U/ ml, Il-10' '100 U/ ml, 1l-4 10 ng/ ml or TNF-O' + IFN-y . Equal load ing of the lanes is shown by ethidiul11 bromide staining of 18 S ribosomal RNA (I",,,,,,. l/"lIck). TIll: n:laLive density of the signals fj'OIll each lane of the upper o'ack was derennincd by scanning laser densitometry and plottccf as a pcrcent.'gc of the maximum signal (~rnJlIt). T his experiment is repre­sentative of two replicate cxpcrinlcncs ,vith sil11ilnr results.

the accum ulation of inA amm atory cell s. In tUnl these wou ld re lease mot'e cyto kincs.

T h e presence of T cell s wou ld be required for the re leasc of IFN-')' w hich m ay have a m o re specific cffect in indu cing keratino­cytes to re leasc MNTES as well as syne rgi zin g with TNF- O' for the furthe r re lease ofrL-S and lu\NTES . I FN-')' in particul ar appcars to bc important in preparing th e ep ithclium! epiderm is to take part in immun o-inflammatory even ts, in ducing keratinocytes to express ICAM-1 and MHC class 11 molecul es , and the re leasc of chemo­kincs and cytoki ncs that wi ll enhance Ic ukocytc-keratinocyte intcr­actions. It is poss ible that the prescn ce of sti mul atory cytokincs is not csscntia l to initiate the inflammatot-y process, sincc activators of protein kinase C can directly inducc IL-8 production and ICAM-1 exprcssio n by kcratin ocytes (Barker ct ai, 1990; Griffiths I?t ai, 1990). T lus activatio n , w hi ch can be indu ced by a hos t of intrinsic as well as cxtcrn al stim uli, could ini tiatc the influ x and reten tion of inflammatory ceUs via ch e m otactic f<lctors and adhesion m olecu les (Griffiths an d Nickoloff, 1989; Barker et ai, 1990).

In conclus.io n , the observation that ora l an d skin keratinocytes can p roducc R.ANTES as well as lL-S . after appropriate cytokinc stimu lation , provides cv idence that kcratinocytes have thc potential to rcgulate th c nature of inflammato ry in fi ltrates th at develop in the skin and ora ll1lucosa. It is possi ble that difFerenccs in the production of chemokincs by skin and ora l kcratinocytes cou ld h elp to cxplain differen ccs in the prescntation of mu cocutaneous inflammatory di scases in thc mouth and on th e skin.

The "",I",,.s ncl."."",lcdge the sIIpp"rt pmJlidrd iJy Tht' Fnn tll), BCQllC5t Fllll tls ~r the Uttitl{'l"sity '!l Mallchesler Fant/I)I 41\1[edidll ll nlld till' tl'rllllirni ntl"irt' I}/"O"ided "), D,.. N. T lttlkk",..

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