0022 -202X/79/7302-0180$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 73:1 80-183, 1979 Copyright © 1979 by Th e Williams & Wilkins Co. Vol. 73, No . Printed in U.S'''\ Arachidonic Acid Transformations in Normal and Psoriatic Skin SVEN HAMMARSTROM, M.D., JAN AKE LINDGREN, B .Sc ., CYNTHIA MARCELO , PH . D., ELIZABETH A. DUELL, PH.D ., THOMAS F. ANDERSON, M.D., AND JOHN J. VOORHEES, M.D. Departmen.t of Chemistry, Karolinslw In:;titutet, Stochholm, Sweden (S H and JL) and the Department s of Derm.atology and Bio chemistry, The University of Mi chigan, Medical School, Ann Arbor, Mi chigan, U.S.A. (CM, ED, TA , and JV) The lev e ls of free arachidonic acid and 12L-hydroxy- 5,8,10,14-eicosatetraenoic acid (12-HETE) are elevated in lesional epidermis of psoriasis (Hammarstrom et al : Proc Nat! Acad Sci USA 72:5130 - 5134, 1975). In the present study, transformations of arachidonic acid by normal and psoriatic involved and uninvolved epidermis were qualitatively compared with similar ' transformations in adult mouse epidermis and dermis and neonatal mouse keratinocyte suspensions. Metabolites were identified by mass s pectrome try or by comigration with reference compounds on thin-layer chromatography . Human epi- dermis and mous e dermis converted arachidonic acid mainly to 12- HETE and prostaglandin E z • Mouse epider- mis, formed HETE, PGD 2 and PGE 2 • The keratinocyte suspensions formed the same products as mouse epider- mis but also PGF2" . Cultures of these cells exhibited stimulated synthesis of PGE z in response to tetradeca- noyl phorbol acetate. This stimulation was prevented by triamcinolone, indomethacin and 5,8,11,14-eicosatetray- noic acid (ETA) but not by 5,8,1l-eicosatriynoic acid . (ETI). The latter compound stimulated basal PGEz for- mation. In conclusion: (1) the tissues investigated all conv e rted arachidonic acid to HETE and PGEz (2) throm- Bz and 6-keto-PGF I .. wer e not formed in appre- ciable amounts under the conditions used; which might s ugge st (3) that HETE, PGE z , PGF 2 .. and their precursors are mor e likely to be regulators of epidermal function than thromboxane Az or PGlz and finally (4) cultured k era tinocytes which produce HETE, PGE2 and PGFz" may be a suitable model for detailed studies of growth regulation by arachidonic acid metabolites. Arachidonic ac id is the precurs or of a numb er of biologi c ally act ive compounds s uch as prostag landin (PG) PGl z, thromboxane Az (Fig 1), a nd 12L-hydroxy-5,8, 10, 14-ei cos - atetrae noic acid (1 2 -HETE) [1]. Thromboxan e A z and PGIz are unstable in a qu eo us medi a a nd yield thromboxane Bz a nd 6- k eto-PGF I ", respectively, upon r eact ion with wat er (Fig 1). Prev iou sly, we reported in cr ease d levels of arachidonic ac id in involved p soriatic epidermis [2]. In a ddition, marke dl y ele- Manu script received December 11 , 1978; acce pt ed for publication Febru ary 2 1, 1979 Th is work was s upporte d by the Swedish Medical Researc h Co un c il (03X-217) and NIH program proj ect grant 2 POI AM 15740-06. Reprint requests to: John J. Voorhees, M.D., Departments of Der - mato logy and Biochemistry, Th e University of Mich igan, Medica l Sc hool, Ann Arbor, Michigan 48109. Abbr ev iat ions: ETA: 5,8, 11,14-e i cosatet r ay noic ac id ETI : 5,8,l1-eicosatriynoic ac id GC-MS: gas-liquid chr omatogra ph y- mass spectro met ry HETE: h ydroxyeicosatetraenoic ac id 12- H ETE: 12L-hydr oxy-5,8,10,14-eicosatet r ae noic acid 1M: in domethacin PC: prostaglandin 'fA: triamc in olone TLC: t hin laye r chromatography TMS: tri met hyls il yl TPA: tetradeca noyl ph orbol acetate 180 vated 12- HETE lev els were found wh er eas the levels of PGE., a nd PGFt" were only minim a ll y elevated. This led us to quali = tat iv ely inv estigate the patte rn of ar ac hidonic ac id transfor. mation s in epidermis. The res ul ts s how that HETE and PGE. are ma jor produ cts form ed by normal and psoriatic human e pid e rmi s and by adult mou se dermi s. Mous e epidermis con. ve rt ed arac hidoni c acid mainly to HETE , PGD 2 and PGE., whereas n eo natal mouse k erat ino cy te suspens ions in form ed PGF 2". METHODS Chemi cal8 [l - "' C]Arac hidonic acid (55 Ci/ mol) was purchased from Th e Radi _ ochemical Centr e, Amersham. [l - "'C]pCH, was prepared as previously described [3]. Th e co mpounds were pure as judged by t hin -layer chr omatogra ph y. Unl abe led prostaglandins and 6- keto-PG F, ,, wer e kindly provided by Dr. J . Pike of the Upjohn Co mpany, Kalamazoo Michigan. ' Shin biopsies Epidermis biopsies from uninvolved and involved psori at ic s kin (sta ble psoriasis vulgaris; plaques prese nt for > 2 mo a nd not tr eated for > 2 weeks) and from normal human skin were removed by a Cast roviejo ker ato me as previous ly descr ibed [4]. Adult BALB/c mi ce were anest hetized by intraperiton ea l injec ti on of pentobarbital (20 m g). The fur was mechani ca lly clipped and the skin was shaved. Twen ty_ fo ur hours lat er, ep idermis was removed by a keratom e (depth = 0.1 mm). Der mis was subsequ ently removed by ker atome (depth = 0.2 mm ). The ti ssues were immedi ate ly snap frozen in liquid nitrogen and s tored at -70°C. Cell cultures Ker at inocyte cult ures were prepared as previously reported [5]. Briefly, 50-60 neo natal ' BALB/c mice were washed in pHi sohex, 70% et hanol and anest het ize d on ice. Afte r removal of full thickness skin th e epidermis was separate d fr om the dermi s by digestion with 0. 25 ' trypsin at 37° for 1 hr . The ke ratino cytes were sepa rated from de bri and any de rmal fibrobl asts by use of a disc ontinuous FicoU gradient. Fres hly dissocia te d kerat inocytes were suspe nd ed in medium 199/ 13% f eta l calf se rum/ penicillin C/streptomycin (Flow Laboratories, Rock- ville, Maryland , USA). Aliqu ots were frozen for dete rmin at ions of a ra chidonic ac id transformations or plated in 1'-25 fl asks and grown at 33°C with 5% CO, in air gassing. The medium was changed every second da y. Incubation:; S kin bi opsies were powdered at - 78 ° and suspend ed in 6 volumes of 0.1 M KpO, ,, pH 8.0/0 .03 M EDTA. [l - "' C]Arachidonic acid was added in et hanol to a concentr at ion of 4 It g/ ml and the mixtures were incu- bat ed at 37° for 10 min with st irring under air. Inc ubat ions were sto pped by addition of 5 volumes of et hano l. Alt ern atively, the mixtur es were preincubat ed for 30 sec at 24°, [I-I "C] pCH, in aceto ne was added to a concentr at ion of 25 Itg/ml and th e mi xt ures were in cubated for 1 min at 24 °. Ethanol containing 5 mg/ ml of SnCll (5 volumes) was add ed to stop these rea ct ions. Keratinocyte suspensions (1. 5 x 10' cells) were thawn and centrifuged at 100,000 xg for 60 min . Aft er rinsing with buffer the se diment was resuspend ed in 0.5 ml of 0.1 M KPO., pH 8.0/ 0.03 M EDTA a nd preincubated for I min at 37°. [l- 14 C]Arac hidonic acid was a dded, the incub at ion was continued and stop ped as described above. The mix t ur es co ntaining et hanol were filtered, diluted with water, ac idified to pH 3 a nd extracte d with diethyl et her.