Generation and Metabolism of Lipoxygenase Products in Normal and Membrane-Damaged Cultured Human Keratinocytes

Floyd A. Green, M.D. Departments of Medicine and Microbiology, State University of New York at Buffalo and Veterans Administration Medical Center, Buffalo, N ew York, U.S.A.

The production and metabolism of lipoxygenase eicosanoids nous lS-HETE by normal keratinocytes was observed. Mea­ were srudied in cultured human keradnocytes. The identiry surable quantities of esterified l S-HETE were found after 1 of these eicosanoid structures was estabLished by a variety of min, but by 18 - 20 h all the esterified IS-HETE was de­ chromatographic and analyti cal techniques. Normal cul­ graded to the extent that 80% o[ the recovered radioacti vity tured kerarinocyrcs did not produce lipoxygenase cicosanoids was found in water-soluble form. In contrast, when JabeJed either spontaneously or when given arachidonic ac id in the or unlabeled 5-HETE was used a much larger fraction was presence of permeabilizing concentrations of ethanol or di ­ esterified intact (30% as opposed to 10%) and at the end of methyl sulfox.ide. Freeze-thawing of human neonatal and 18 - 20 hours a substantial reak of esterified S- HETE re­ adult keratinocytes resulted in a rapid release of linoleic and mained. Intact esterified [3H HETE were recovered only in arachidonic acids over rime. Activation of a latent 15-lipoxy­ the triacy lglycerol fraction. The key findings that W-6Iipox­ genase was demonstrated by the synthesis of l S-hydroxyei­ ygenase products are generated but not esterified by mem­ cosatetraenoic acid (15-HETE) and 13-hydroxyoctadeca­ brane-damaged keratinocytes, whereas these products are es­ dienoic acid. and both these products were greatly increased rerified bur not genera[("d by normal keratinocytes, may be of in amoUJ1[ when the corresponding fatty acid precursor was importance in transcelJular metabolic control.] Intlcst Derma- added. Eicosanoid production by cells of newborn and adult 10/93:486 - 491 , 1989 origin was indistinguishable. Rapid metabolism of exoge-

ince the first description of mammalian Jipoxygenases in undertaken with usc of defined cultures. Membrane damage of the 1974 (I} there has heen continuing interest in the role of ke: ratlnocytes by a freez.e-thaw techni<:lue was enlploycd to activate lipoxygenase . J?roducts as inflammatory mediators in the lipoxygenases. The use of intact cul tured keratinocyres also mammalian 12J as well as in amphibian inflammatory allowed examination in vitro of the metabolic fate of eicosanoids models [3J. Following the initial report of Hammarstrom produced by activated Iipoxygenases where such transcellular me­ eralS that 12-HETE could be recovered from the psoriaric epidermis tabolism could also be expected to occur in vivo. [4]. a variety of different eicosanoids has been reported to be presenr MATERIALS AND METHODS in lesional and non-iesional epidermis 15 - 7}. I nte r pr~tatian of these findings has been complicated by the difficulty in distinguishing Cells, IncubOltions. and HPLC Separations Keratinocy te cul­ between eicosanoid products formed by the epidermis, dermis. in­ tures (from both human adult and newborn donors) Wert' obtained filtrating inflammatory cells, and in addition, by co-processi ng from Dr. Howard Green (Depa,rtmenr of Physiology and Bi ophys­ among the different cell types. As a first step in understanding the ics. Harvard Medical School, Boston, MA) [8- 10]. The cultures rale of lipaxygenasc{s) in human dermatologic diseases. a careful were sh ipped in medium-filled flasks, were confluent at the time. of examination of Iipoxygcnase activity in human keratinocyces was arrival, and continued ro divide until used. On the day of the experi­ ment. the medium waS changed to protein and growth facmr-free Medium 199 containing 40 mM pH 7.6 Hepes buffer. The cells were washed twice .in 10 ml of this media at 37 0 for 30 min. For the Manuscript received December 20. t 988: accepted for publication March freeu-thaw experiments. 1 m! of fresh medium was employed to 20. 1989. just cover the cells, and the plates were air frozen at -26°C for a This nudy was supported by NIH GUilt HL24009 and by the Vetcram; minimum of 1 h. In some experiments, rhe cells were then lyophi­ Administration. lized. The frozen cells were rh en rhawed at 370 and studied with or Reprint requests to: Floyd A. Green, M.D., Department of Medicine. without the add.ition of exogenous arachidonic or linoleic acids Division of Rheumatology, VA Medical Center. 3495 Bailey Avenue. Buf­ (2-20 Jig in 2-100,tt1 of ethanol or dimethylsu lfoxide in 1 ml me­ falo . NY 14215. dium). The ex tent of eicosanoid producti on was fou nd not to be Abbreviations: affected by the presence of 1%-9% ethanol or dimethylsulfoxide, IS-HETE: 15(S)-Hydroxycicosa-SZ,8Z.IIZ.1 3E-tctraenoic acid and in some experiments the exogenous fatty acid was dispersed in a 5-H£TE: 5(S)pH ydroxycicosa-6E.8Z, lIZ.14Z-temenoic acid small volume of 15 mM NH 0 H (prior to media-buffering) with GC/MS: Gas chromatography/ mass spectrometry 4 HPLC, LC: High performance liquid chromatography identical results. T he timed incuba.tions w ere stopped by a.dditions 0 13-HOOD: 13(S)-Hydroxyoctadeca-9Z,11 E-dicnoic acid of 3 volumes of ethanol followed by cooling to -25 and transferral TM$: Trimcthylsil yl to plastic tubes. After the addition of prostaglandin Bz or labeled

0022-202X/89/S03.50 Copyright@ 1989 by The Socic:ty fo r Investigative Dermarology, Inc .

486 VOL. 9J. NO. 4 OCTOBER 1989 KERATINOCYTE IS-LIPOXYGENASE .. 87

H ETE as an imernal standard, tile samples were cenuifuged and the incubation of exogenous labeled or unlabeled eicosanoids with in­ supnnacaOf was rotoevaporared. tnnsferred in methanol, and ulti­ tact keratinocytes was tenninated by rhe addition of 1 volume of mately dissolved in mobile phase for reversed-phase HPlC in a methanol and transferred to glass tubes where the methanol was Hewlett-Packard 1090 lC. This was performed isocratically on gently blown off with argon. The samples were then frozen and Hewlett-Packard ODS-Hypcrsil columns, 10 or 20 em in lyophilized. following lyophilization. the samples were treated by length X 4.6 mm at a flow rate of 0.4 rol / min. The- mobile phase the anhydrous methanolic NaOH method of Kates to guantita­ for the l1lonohyd roxyeicosanoids was 75-80: 25-20: 0.1. metha­ tively transesterify aJI esterified fatry acids and their derivatives to nol/water/ acctic acid; and for leukotrienes 45: 37: 18: 1, water/ their respective methyl esters [141. A single modi.fication was made acetonitrile/ methanol/acetic acid. Slight modifications of these in order to partition any unesterifled fatry acids or their hydroxyl­ conditions arc given in the text. The free acids collected during the ated derivatives into the organic layer: after the addition of water to lC runs were methylated with ethereal di:lZomethane. and rechro­ separate the phases. 100,u1 of 2 M ammonium fonnate buffer (pH matographed on the same HlllC columns (delayed retemion rime 3.2) was added. After washing with methanoljwater (10: 9) saru­ of approximately 15 min). All the lC srudies were run at a wave­ rated with chloroform, the lower phase was reconstituted into the length of236 nm, but recall was available at 270 run and 301 nm as mobile phases indicated above. With this procedure, free hydroxyl­ well as in situ UV spectroscopy. T he ordinate scale is given in ated fatry acids were unchanged and eluted at their nonnaJ times. milliabsorption Ul1it5 throughout, and al l ch romatograms a.re repro­ whereas tht HETE, which had been enzymatically esterified by the duced dirccdy from the LC computer plots. f or each iIlustrative keratinocytes. were el uted as their methyl esters. Retention times chromatogram shown at least three other similar experiments were were confirmed by mixing experiments with authentic labeled free performed which showed virtually identical results. In no case and methylated HETE and by in situ UV spectroscopy and ce. shown was rhe main observation of the experiment not demon­ Estimation of Cell Numbers In some experiments the cells strated in every case. Reproducibiliry of the results of studies with were separated from surface culrures by trypsin-EDTA treatment these cell cultures was a noteworthy feature. Retention rimes (RT) followed by counting in a ZBI Coulter Counter. In other experi­ of :mthentic standards are indicated in the figure legends. Software ments aft er incubation the cell proteins were solubilized in 0.5 N programs for guantitation using published molar extinction coeffi­ sodium hydroxide containing 0. 1% TritOn X t OO followed by esti­ cients were cmplo)'ed to measure concentrations as previously de­ mation of proteins by tbe Folin method [15]. scribed [111 . Unlabeled eicosanoids were obtained from BioMol Re:.earch Labs (Plymouth Meeting. PA) and fatry acids from Nu­ GC/ MS Studies were carried our using a KratOs MS 80 RFA mass C hek Prep. Inc. (Elysian. MN). spectrometer at 50-70 eV. Studies of tbe R elease of Arachidonic and Linoleic Acid Fol­ RESULTS lowing Freezing For these srudies [he incubatjons were inter­ Normal Keratinocytes Normal kerarinocytes did not produce ruptC'd by {he addition of3 volumes of methanol containing 50 pi of any lipoxygenase eicosanoids either spontaneously or .1fter feeding acetic acid . After centrifugation and transfer to glass tubes, ex trac­ arachidonic or linoleic acids in the prese.nce of pt:rmeabilizing con­ tion was performed with chlorofoml/ methanol 2: 1. Water was centrations of erhanol or dimethylsulfoxide or aft er dispt:rsion of added to produce rwo phases, and the lower chloroform layer was the fatty acid in dilute NH .. O H . repeatedly washed with pure upper phase solvents then dried under argon and taken up in ethereal diazomethane with small amounts of Studjes of Frozen and Thawed Keratinocytes When con­ 0 methanol. The rrimethylsilyl ether derivative was prepared by the fluem cul tures of human keratinocytes were frozen at -26 , addition of 20 pi of pyridine and 40 pi of bis(trimethylsilyl)-tri­ thawed, and incubated at 37" arachidonic and linoleic acids were fluoroaceramide (Aldrich Chemical Co., Milwaukee, Wise.). With rapidly rel eased (Fig 1A). By the end of 3 h approximately 25% of rhis procedure rhe released arachidonic and linoleic acids as well as the total esterified fatty acids prescnt were released. 15-HETE was mOllo-HETE were clearly separated by gas chromatography (GC) generated ar approximately the same rate and in amounts of about on an H P 5890 gas chromatograph using a Supelco SP 2330 col­ 10% of the arachidonic acid released (Fig 18). In addition. a hy­ umn. U nder 10 Ibs of column head pressure the retention times droxy fatty acid which was idenri6ed as 13-hydroxyoctadecadie­ were 4.0 min (linoleic acid). 5. 1 min (13-hydroxyoctadecadienoic noic acid (13-HOOD) was also produced in smaUer amounts. No acid). 6.1 min (arachidonic acid), 7.9 min (12-HETE), 8.1 min (15- HETE). and 8.2 min (5-HETE). Known concentrations of lin­ oleic and arachidonic acid methyl esters in hexane were used to 0 0.4 25 ]'4 construct scandard curves from which the amounts of these fatty '0 ~ C ~ .3 acids were calculated. 0 '"'0 ~20 ~3 0.3 ~ Metabolism of Eicosanoids by Normal Keratinocytes The !O• Ii'. '0" eicosanoid.s indicated in the text were incubated with the keratino­ '0 e ~ 15 0.2 "- 0 .g2 cy tes in ethanol or dimethyl sulfoxide (2-40,u1 per 1.0011 of me­ .. w dium) for periods of up to 24 h. P"C] ls-HETE was prepared bio­ • I- '" 10 c W synthetically by rhe addition of carrier free l-['''CJ arac hidonic acid '0 0 0.1 J: '0" , to keratinocytes which had been frozen and thawed (see below). ;j ;/ :E 5 0 '!1 After work-up and HPLC purification the p"e] 15-HETE was ~ 0 added to keratinocyte culture flasks. Synthetic {lH11 5-HETE and ~ ~ [3H] 5-HETE were obtained from Amersham Corporation (Arling­ 0 2 3 ton Heights, IL). After interruption of the incubation, tbe chloro­ 0 60 120 180 TIme (hrs ) Time (mrn) fonn/ methanol extract was washed and applied to one- or two-di­ mensional thin layer chromatographic plates (TLC) for isolation of phospholipids as previously descri bed 11 21 and for di- and triacyl­ Figure I. A: Time courS(" of free farry add production fro m human kerat­ glycerides by the method of Skipski and Barclay [13J. The lipid inoc)'tes following frening and thawing. The ratry acids werc measured by GC as indicated in Methods. Arach idonic acid (opm rirrltos) and linoleic acid spots were visualized with iodine vapor, extracted with chloro­ ((lpcPl squarrs). The figures for rot:lll farry acid contenT were ~pantdy ob­ form/ methanol 2: 1 and transferred to scintillation vials or deriva­ rained by tnn.se.uerincation of thc fatty acids in the total lipids to their tized as indicated below. The radioactivity of the methanol/water methyl cstt:rs: IO.7 I1S for anchidonic acid and 6.6 pg/ S X 10' cells for phase was also examined for water-soluble radioactivity. linoleic acid. B: Release of arachidonic acid (o~ cirdtS) and lS-HETE (ofK" A different technique was used to study incorporation of intact squGm) following freez ing .. nd thawing ofS X 10' ker.ain ocyt~ . Values are mono-HETE fatty acids into phospholipids and glycerides. The mC:lIllS of triplicate determinations. 488 GREEN T HE. JOURNAL OF INVESTIGATIVE. DERMATOLOGY

other lipoxygenase products were observed. T he addition of exoge­ nous arachidonic- or linoleic acids greatly enhanced the production 5 0& f- 8 .120 of the corresponding hydroxyl:ated derivatives, lS-HET E and 13- HODD. These hydroxylared fatty acid derivatives were idenrified ~ by liquid chromatography (LC) utilizing the retention time and UV "c •.. 0 sO&tj c- spectra of authentic standards (Fig 2) and their methylated deriva­ ... ~ "o: tives. by GC on semi-polar columns (see later) and by GCj MS of the • ... a:" trimethylsilyl derivatives. The derivative identified as lS-HETE 3 .. 35. ~ ..ve,led ion fragments at 335 (M-71). 316 (M-90). and 391 222 2 .. u m/z W. .... 1ensth ( n.) (M-IS) in addicion to the characrerinic base peak. m/z 225 and the ~ mass ion .at 406. T he derivative identified as 13-hydroxyoctadec:I­ 2 0& • dienoic acid had ions at m/z 31 1, 225. 292. and 382. with a prepon­ - '-"-;- deranCC" of the fragment ;;a mi l. 311 over that at mil. 225 (ratio = 0 5 10 1. 7). The... e findings are the same as those reported for lS-HET E .. Retention Time (min) \16J .nd for 13-HODD [I7J. When exogenous linoleic acid was added [0 the kerarjnoC)'tcs U\. after freeze-thawing, twO peaks were observed consisting, respec~ "' J" 1\ ,. 20 39 tively, of large amounts of 13-hydroxyoctadecadienoic acid and TI N { ml".) •• lesser amountS of lS-H ETE (data nor shown). From a series of experiments as shown in Fig 2, it was found that the arachidonic FiguTe 3. Products of hum:an k~r:l,[inocytt'$ (5 X 10') following freez.ing, acid product (15~HETE) from totally endogenous farry acids was Iyophiliz:uioll, and the addition of I 011 of water and 10 JIg of arachidonic uniformly eight- to tenfold greater in concentration thar the lino­ acid methyl ester. ;oIIld incub,uion for 3 h. The vc-ry l:a.rg~ peak ..... ith a RT of leic .acid deriv:ltive (t3-HOOD), although the ratio of arachidonic 22 min has a UV spectnlm corresponding to IS-HETE (i...... of 236 nm; .acid to linoleic .acid in the intact cells was only 1.6 by gas chroma­ mst:rt I~r(). Th ~ fractions rr-:presenung this peak were then methylated with tography. The- presence of 10 JIM indomethacin did not affect the dittometh:ane and rechromatographed.. The fractlon1 representlng th~ peak production of hyd roxybted fatry acids (data not shown). :after Inethylanon (4.2 .ug) were collected, and the TMS derivative was ex­ :amined on GC (insm rI:~ lrt) as indu::ated in Methods. T he large GC peOik seen When keratinocytf' freezing was foll owed by lyophilization. and corresponds precisdy with thC' rC'rention time of authentic 15-HETE methyl reconstitution of the aqueous component followed by incubation at c ener. 37 , the twO w-6 hydroxylated fatty acids were also produced from endogenous sources. The concentrations of these products were gready increased (al most 20-fold) in the presence of exogenous fatry acid (free form or as its methyl es ter). In Fig 3, a chromatogram is Because the question had been raised rega rding possible." qualita­ shown of the producrs generated aftt:r addition of to Jig of arachi­ rive dilrerenc('s in posicional specificities of lipo1Cygenases of kerat­ donic acid mcthyl ester to lyophilized and reconstituted kuatino­ inocytes derived from adult as opposed ro Ile-wborn sourcC'\'s /18], a cyte cultures. The very Ia.rge peak (4.2.ug) at 22 min gave the comparative e1Cperimenr was carried our in paralkl. The- hydroxyl­ characteristic UV spectrum with a )-mu of 236 nm, and after meth­ ated fatry acids (I S- HETE and 13-hydroxyoctadecadielloic) were ylation the product gave the chancreristic retention time and UV formed in amounts that differed by Ie-ss than 10% in skin cells from spectra of lS-HET E meth}'1 ester (not shown). The TMS ether adults and newborns (data not shown). Surprisingly, no ('vidence of derivative of the methyl estet gave a single on G C (Fig 3, itUcrl peak a lipox)'genase with orher chan w -6 positional specifi city was ob­ righ/) corresponding to the retention time of authentic lS-HETE served. TMS-methyl ester. After lyophilization, reconstitution, and ultra­ centrifugation. the supernatant contained the w-6lipoxygenase ac­ Metabolism of HETE by N o rmal Keratioocytes When epi­ tivity suggesting that this source can be used as a suitable starting dennal freezing or perhaps other membrane-damaging events occur point for enzyme purification. at a whole organism level in animals or man, aile would anticipate that any lipoxygenase ptoducts genetated as a result of the injury might in turn be subjected to further metabolism by those keratino­ cyees which happen to escape injury. To mimic t~e se conditions. [JH J J 5-HETE was added to normal kerarinocytes and tht:" products ,. formed afreT widely varying periods of incubation were studied. In Fig 4A \'Hj 15-H ETE was .dded ro two sets of healthy cell culrures s. •• and incubated for 1 h. The paired samples werc lyophilized, and one ~ s. • sample was subjected to the complete anhydrous methanolic NaOH 5. ..• •• procedure to quantitatively transesterify all the fatry acids. For the en ' I~2. other sample, NaOH was omitted from the procedure as a conttoL • 2<3 A peak at a RT of 37 min corresponding to 15-H ET E methyl ester 0: •• 228 ", W. .... l .n <" ( nlll ) was observed with the completf' transesterinc:aion procedure, in •• addition to a number of unidentified more polar radioactive peaks . The peak corresponding to 15-HETE methyl esra was totally ab­ 2. sent when the critical NaOH step was omitted (Fig 4A). Asa further conrrol, labeled 15-H ETE was also added to kcutinocytes. which ,. had been incubated at 100· for 5 min (Fig 48). A single radioactive peak corresponding to the labeled lS-H ETE was observed which ,. J5 2. 2. contajned 87% of the recoven~d radioactiviry. This indicated that aJI ,,- ( lft t n. ) [he radioactive products in the previous experiment were enzy~ matically generated. ThC' disappearance t\ of 15-HETE was about Figure 2. HPLC of the products of human ker:..tinocytes (S X 1()&) follow­ 15 min. No evidence of esterified lS-H ETE was observed after ing freezing, thawing. and incubation for 3 h without e.xogenous fatty acids. incubation of lS-HET E with frozen and thawed cells, al­ The pe:l.ks with retention times (RT) of 18 and t 9 min had UV spectra with !lH} )_ of234 nm :and 236 nm filUm) . These values correspond to RT and UV though other unidentified radioactive peaks probably representing sPf"ctr2 of I3-hydroxyoctadecadienoic acid (0.3.ug) :lnd IS-HET E catabolic products were observed (data not shown). No conjugated (0.68 Jig). rC'spectivdy. No other HET E were present. triene or tetraene structures were observed as additional products in VOL. 93. NO. 4 OCTOBER 1989 KERATINOCYTE 15·LlPOXYGENASE 489

200

60 30

50 25 1; ~ 40 20 ::; "'- Q. " C e ~ ?' 15 30 : ~ .E u u 8 0 .., 10 0 20 'D 0 a:0 '" 10 5

5 10 15 5 10 15 20 25 30 35 40 45 20 25 30 35 40 45 50 Time (min) Time ( min)

Figurr S. HPLC of the producrs ofker;ltinocytes (5 X 106) incub2ted with 0.25 pCioqlH] 5-HETE for 1 h. The unchanged [lH} 5-HETEhada RTof 28.5 min and [3H J 5-HETE methyl ester (30% of recovered radjoactivity) f 1 was a[ 42 min. 60 reguhtory role (19]. For these reasons, it was of interest to compare b 50 the metabolism of 5-HETE with that of 15-HETE in normaJ • human cultured keratinocytes. When cultured keratinocytes wcre ::; 40 incubated with !'H ]5-HETE. its methyl ester was recovered (Fig 5), c"'- and the unchanged 5-HETE had a much slower disappearance time (t j = 90 min) than did 15-HETE. A much higher percentage of 30 recovered radioactivity of 5-HETE methyl ester was observed than u'"~ 0 was ehe case for 15-HETE methyl ester. Furthermore, at the end of 0 'D 20 18 - 20 h of incubation rhere was still a substantia] peak of rhe a:0 5-HETE methyl ester, whereas, as indicated above. rh e 15-HETE methyl ester had all been metabolized. difference in merabo­ 10 This lism of the HETE is also demonstrated by the resules of otherexper­ imcllts shown in Fig 6. T wo parallel srudies were carried out in which unlabeled 5- and 15-HET E were serarately incubated with 5 10 15 20 25 30 35 40 45 healthy kcratinocytes for 1 h. At the end 0 this rime much greater nme (min) amounts of 5-HETE and 5-HETE methyl ester were produced/ re­ tained compared with 15-HETE and IS-HETE methyl ester. Simi­ lar results were obtain ed when rhe 5- and 15-HETE were incubated Figure 4. A: HPLC of the products of human keratinocyte5 after a I h together in the same flask. incubation with I pCi ofl)Hj 15-H ETE. The open .squara represent cells that wert' subjected to the fuJI [ranscs[erincation proced ure. The opt1l circl6 repre­ 5~------~~=------, se nt cells which were incubated and worked-ur in rhe same way bur without S-HETE NaOH. The large radioactiv(, peak at J:lT 0 34 min represents [lHj 15- HETE methyl ('ster (10% of recovered radioactivity). The peak representi.ng unmctabolized [lHJ IS-HETE h:l.s:I. RT of21 min. B: HPLC of the products 4 ofkcrarinoc),tcs (5 X 106) after addition of 1 ,uCi of 15-HETE. The cdls h.ad previously been incubated at 100 · for 5 min. 87% of the recovered activity W2S loc2ted in lh e pe2k corresponding to ullchange.d [lHj 15-HETE (RT . S-HETE HE ESTER 21.4 min). 3B any of the/resenr studies with fresh or froz.en cells even after the ZB addition 0 lS(S) -HPETE. The course of production and disappearance of esterified HETE lS - HETE HE ESTER was eX:l.m ined. Evidence of esterified lS-HETE production from IB the normal kerarinocyres could be observed after 1 min and in­ creased up to 30 min. T hereafter the esterified 15-HETE concen­ tration decreased and by the end of 18-20 h there was very little radioactiviry left in this chromatogram: approximately 80% of the ZB 4B radioactiv iry was recovered in water-soluble form. The interaction of neutrophil s with kerarinocytes is thought to Figure 6. Superimposed HPLC of paired experiments in which 2.511g of playa fundamental role in the pathogenesis of psoriasis [6]. Trans­ IS-HETE and S.HETE. respectively. are added to 5 X to' fresh kentino­ cellular metabolism of lipoxygenase products may be involved in cyte~ and incubared for I h. The recoveries were 1S-HETE (24 ng), S-HETE this interaction. For example. the 5-lipoxygenase of the neutrophil (320 ng), IS-HETE methyl ester (S7 ng). and 5-HETE methyl ester may g('nerate, among other produces, 5-HETE. which may have a (2. 3 ng). 490 G..... N THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

In ex periments with the [I·e] and [3H] 5- and 15-HETEs, exten­ previously fo und that freezing activates the 5-lipoxygenase of sive radioactivity was recovered from phospholipids which had human ncutrophils to produce leukotriene B~ /39] and evidence has been separated by class. Phosphatidylerhanolamine concained rhe been found that cell dam age may also activate 15-lipoxygenase of greatest amount of radioactivity. How('ver, when separation of tbe human neutrophils 140l phospholipids was followed by transesrerincarion it became appar­ Suggested functional effects of J 5-HETE can be divided into em that very little radioactivity was acrually in intact but esterified effects of the free hydroxylated fatty acid and effects resulting &om 15-HETE. The rriacylglyceroJ fraction, however, did reveal sub­ its esterified form. Although nothing is yet known of the latrcr, it is stantial radioactivity corresponding co esterified HETE. This is known thal 15-HETE may modulate muCus secretion l4 1J and can demonstrated for PH] 5-HETE methyl csrer in Fig 7. also affect the activity of the other lipox-ygenases 142]. There have been few reported examples that firmly documeor the DISCUSSION cellular esterification of HETE. Stenson and Parker (441 fi rst pro­ Among the reported studies on kerarinocyres, either with cultured vided evidence of esterification of 5-HETE into phospholipids and cells or cel ls removed from human skin, the only srudies rhat appear gl ycerides of acti vated neutrophils. Bonser et al 145J found the 5- to reflect rhe same Jipoxygenase as that observed in the present srudy HETE was estcrified i.n the phospholipids and acyl glycerides of rhe arc those of BurraH et al i 18,20}. These investigators sonicated diffe rentiared human myelocytic cell-line HL60. These studies 011 human neonataJ keratinocytes and found lS-lipoxygenase activity cdls that are very shorr-lived afrer activation did nor suggest specific in the presence of exogenous substrate. Other cell types have also functional consequences of esterification to these investigators. revealed 15-1ipoxygenase activity [2 1 - 32}. The failure to find evi­ Howevcr. the present studie5. in which much longer-lived cdls dence of other lipoxygcnase activity [37] in the present studies could were used. also showed that such cells can esterify hydroxylated indicate that these cultured cells do not possess such enzymes. or farry acids. These ('vents may have functional implications ill disease that they were not activated by the membrane damage which is stares. Convincing evidence is presented of esterification of HETE central to the process of freezing. when 15- or 5-HETE. either in labeled or unlabeled form, was The mechanism of tissue damage that results from freezing is added to intact kerarinocyte cultures. Other labdcd products were unknown 133J. Intracell ular hyperroniciry and/ or intracellular ice seen. especially with 15-HETE, virtually all of which had been crystal format\on hJ.ve been incriminated :1;5 cntical events preced­ degraded to warer soluble radioactiviry by the end of 18 h. This ing the accual damage [34J. At a ptacticallevel. freezing of the skin contrasted to the metabolism of exogenous 5-HETE by healthy and subcutaneous tissues of man (frostbite) continues to be an im­ keratinocyres in thar de-gradation was much slower and the esteri­ portant cause of morbidiry / mortaliry [35}. Although the freezing of fied S-HETEconstituted a rnuch higher percent of the total radioac­ isolated keratinocytes in no way simulates frostbite necrosis, the tivity recovered. In some situations. protracted incubation of ke­ 15-lipoxygenase ofkeratinocytes could reasonably expect to be acti­ ratinocyre with exogenous 5-HETE stimulated the endogenous vated. In the present studies, freezing was simply employed as one production of 15-HETE in esterified form. Small amounts of this m erhod of cell membrane perturbation. The documentation of re­ product were also found in orher long-term experiments. which lease of free arachidonic acid and linoleic acids is indicative of acti­ giv{"s rise to the possibility that endogenous 15-lipoxygC'llasc activa­ vation of phospholipase(s). Human keratinocytes appear to have a tion may be a common reaction to keracinocytc stimulation/ d:un­ number of functional phospholipases {431 . The- sOu.rce of the re­ age and may result in the prod.uction of 15-HETE, which is then leased (my acids, diacyl- versus l-alkyl, 2-acyl-phospholipids 1361 esterified and ultimately degraded by healthy keratinocytes. On the was not identified. The fo rmation of the respective products of w-6 other hand. cdlular incorporation of 5-HETE, perhaps released by lipoxygenation. lS-HETE, and 13-hydroxyocradecadicnoic acid, infiltrating activated neutrophils or m:tcrophages. may result in could be grc:I;tly enhanced by rhe addition of exogenous unsarurated metabolically stable esterified 5-HETE. The mccabolic conse­ fatty acids indicating that the enzyme could acr on both arachidonic quences and possible effects 011 cell division and function of rhis and linoleic acid. It is a point of central imerest that frozen and stable esterified hydroxylated farty acid remain to bt" determined. thawed cdls resulted in l S-lipoxygenase activation to produce the HETE. buronJy normal cells could esterify these products. W e have TJl r CCIMS analyses Wf'rr kinJly perJormtd in ,Ir, Ma )'s Spmrom!!lr)' Laboratory (Drpa,,,,ml! oj Diaphysiu) by Dr. Jam!!s McRryllo/Js. 35

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