Eicosanoids in skin of patients with atopic dermatitis: Prostaglandin E2 and leukotriene B4 are present in biologically active concentrations

sanoids in skin of patients with atop rmatitis: Prostaglandin E, and ~e~kotrie re present in biologically ive concentrations rsten Fogh, MD, Troels Herlin, Aarhus.

MD, PhD, and Knud Kragballe,

MD,

Denmark

The biochemical events leading to atopic dermatitis (AD) are unknown. Certain eicosanoids derived from arachidonic acid are potent mediators of skin inf?ammation and modulators of certain T-lymphocyte activities. The purpose of the present study was to determine whether eicosanoids are present in biologically active concentrations in the skin of adult patients with AD. The levels of the cyclooxygenase product, prostaglandin E, (PGE,) and the lipoxygenase products, leukotriene B, (LTB,), 12- and 15.hydroxyeicosatetraenoic acid were determined in biopsy specimens obtained by keratome from lesional, perilesional, and clinically unaffected skin of patients with AD. Methods for identification of eicosanoids included reversed-phase high-performance liquid chromatography combined with radioimmunoassays. Eicosanoid levels were at the same level in normal skin and in uninvolved skin of AD. Compared with uninvolved skin, both lesional and perilesional skin contained markedly elevated concentrations of PGE, and LTB,: PGE,, 97.2 t 15.6 nglgm of lesional skin and 128.3 i- 27.2 nglgm of perilesional skin; LTB,, 5.2 -t 1.6 nglgm of lesional skin and 3.2 +- 0.6 nglgm of perilesional skin. Compared with uninvolved skin, the levels of 12- and 15-hydroayeicosatetraenoic acid were elevated sevenfold and elevenfold, respectively, in lesional skin, but did not reach biologically active concentrations. The results demonstrate that the in~ammato~y mediators PGEz and LTB, are present in lesional skin of atopic subjects in biologically active concentrations. Because these mediators are able to induce cutaneous inJammation and to modulate cellular immunity, they may be involved in the biochemical processes leading to AD. (J ALLERGY CLIN IMMUNOL

1989;83:450-5.)

AD is a common chronic and pruritic inflammatory skin disease.’ The histolopathologic changes are nonspecific but consist of acanthosis, spongiosis, and a dermal infiltrate mainly comprised of mononuclear leukocytes (lymphocytes and monocytes) and, to a lesser degree, of eosinophils. Furthermore, increased number of mast cells are present.’ In some patients the disease is associated with allergy, and it has been demonstrated that T cell-mediated reactions in the skin as well as in the peripheral blood are depressed in

From the Department of Dermatology, University of Aarhus, Marselisborg Bospital, Aarhus, Denmark. Supported by Danish Medical ResearchCouncil Grants 12-6262, 12-7066, and 12-7437, and by the Institute for Experimental Clinical Research,University of Aarhus, Aarhus, Denmark. Received for publication May 2, 1988. Accepted for publication July 6, 1988. Reprint requests:Karsten Fogh, MD, Department of Dermatology, Marselisborg Hospital, DK-8000 Aarhus C, Denmark.

I

Abbreviations used AA: Arachidonic acid AD: Atopic dermatitis co: Cyclooxygenase HETE: Hydroxyeicosatetraenoic acid LO: Lipoxygenase LT: Leukotriene PG: Prostaglandin RIA: Radioinnnunoassay RP-HPLC: Reversed-phase high-performance liquid chromatography Ultraviolet

AD.’ The biochemical changes underlying these inflammatory and immunologic abberations in AD are unknown. Several inflammatory mediators have the capacity to induce cutaneous inflammation. As mentioned above, increased numbers of mast ceils have

VOLUME NUMBER

Skin eicosanoids

83 2, PART 1

been detected in AD, and early studies have demonstrated elevated histamine levels in AD.2,3 However, recently Ruzicka et a1.4found no elevation of the skin histamine level. Although histamine can induce wheal and flare after intradermal injection into human skin, its role in AD is questionable because the therapeutic effectiveness of antihistamines is minimal. Therefore, other mediators may be involved in the inflammatory processes of the disease. Certain eicosanoids derived from AA have been suggested to play an important role in inflammatory skin diseases.4-7The 5-LO product, LTB,, can induce chemotaxis of leukocytes-” and increase vascular permeability,” which can lead to cutaneous inflammation after topical application’* or intradermal injection.13 The cyclooxygenase product, PGE, can inhibit certain T-lymphocyte functionsI and induce wheal and flare by intradermal injection into human skin. l5 Furthermore, PGE, can amplify the wheal-and-flare reaction induced by intradermal injection of LTB, into human skin”; 12-HETE formed by the 12-LO has properties similar to LTB4, although it is far less potent than LTB4,16, I7 and 15-HETE formed by the 15-LO has no proinflammatory capacity but has the potential to inhibit both the formation of and the chemotactic effect of LTB,,18, l9 thereby serving as a potential regulator of inflammation elicited by LTB,. In the present study we have determined whether these eicosanoids are present in biologically active concentrations in the skin of adult patients with AD.

Eight adult patients (mean age, 29 years; range, 18 to 48 years) with AD took part in the investigation. Diagnosis was made according to the method of Hanifin and Rajka.” Informed consent was obtained from all patients, and the study was approved by the local Ethical Committee. The patients had received no topical treatment and no systemic treatment 4 weeks before the study. Skin biopsy specimens from lesional skin, from perilesional skin (unaffected skin adjacent to affected skin), and from clinically unaffected skin at least IO cm from lesional skin were obtained with a keratome, as previously described.6 Briefly, the skin sites were infiltrated with local anaesthetic (lidocaine 1%). Skin biopsy specimens were then obtained by tangential shaving with a motor-driven keratome. The keratome was set to obtain biopsy specimens measuring 0.2 mm in thickness that were immediately placed in liquid nitrogen and stored at -70” C until analysis. Analysis

of eicosanoids

Skin biopsy specimens (100 to 200 mg per sample) were homogenized, and lipids were extracted on octadecylsilyl silica columns and eluted, as described previously.6 The

d~F~~~i~~~

methanol fraction containing AA metabolites was taken to dryness under a stream of N, and resuspended in l@Op1 of 70% methanol.

RP-HPLC Extracted lipids were separated by RP-HPLC with a system from Waters equipped with a Hypersil C,, column (5 pm, 100 by 4.6 mm internal diameter) (Waters Associates, Millipore Corp., Milford, Mass.).6 The column was eluted isocratically with methanol/water/acetic acid, 70: 30:0.01 (by volume) as the mobile phase. The eluent was collected by a fraction collector programmed to collect 1-minute fractions.

RIA for PGEz, LTB,,, and 15-HET Eluate fractions cochromatographing with authentic ‘HPGE, were evaporated under a stream of N, and resuspended in assay buffer (0.9% of NaCl, 0.01 mol/L of ethylenediaminetetraacetic acid, 0.3% of bovine gamma globulin, 0.005% of Triton X-100, 0.05% of sodium azide, 0.0255 mol/L of NaH,PG,,H,Q, and 0.0245 mol/L of Na,,HP0,,7H,O, pH 6.8). The antibody was rabbit antiPGE, with high specificity for PGE, exhibiting crossreactivity of 3.7% with PGE,,
Neutrophil ATlENTS AND METHODS iopsy specimens

in atopic

chemokinesis

Chemokinetic activity was measured by use of the neutrophil microdroplet chemokinesis assay.” Briefly, eluate fractions coeluting with authentic IZHETE were collected, pooled, evaporated, and resuspended in assay buffer; 100 p.1 of this mixture was placed over a 2 ~1 microdroplet containing neutrophils in a solidified agarose gel. After incubation for 2 hours at 37” C in a humidified atmosphere, the radius of the microdroplet was measured with a concentric ruler in an inverted microscope. Neutrophil movement was expressed as the chemokinetic index (CI) as follows: CI = radius in test well/radius in control well.

Statistical

analysis

Results are expressed as mean 2 standard error of the mean. Statistical significance was assessed by Wilcoxon’s rank-sum test. A p value CO.05 was considered significant.

RESULTS A representative RP-HPLC chromatogram of lipids extracted from 200 mg of keratomed lesional AD skin is illustrated in Pig. 1. Recorded as UV absorption, RP-HPLC produced five peaks. Peak I and III coeluted with authentic 13-hydroxyoctadecenoic acid

45

Fogh et aj.

-

270 NM __lf_____

235 NM----i

20

2’5

I

30

ELUTION TIME (MINI PIG. 1. RP-HPLC of lipids extracted from 200 mg of homogenized lesional skin obtained by keratome from a patient with AD. Column: Hypersil, C,,, 5 pm (100 x 4.6 mm, internal diameter). Flow rate: 0.7 mlimin (0 to 10 minutes) and 1.2 mlimin (10 to 30 minutes). The UV detector was set at 270 from 0 to 15 minutes and at 235 nm from 15 to 30 minutes. Retention times of authentic eicosanoids are indicated by arrows.

and 11-HETE, respectively, and both had maximum UV absorption at 235 nm. No bioassay or RIA is available to confirm the identity of these compounds. Peak II and IV had retention times similar to authentic 15-HETE and 12-HETE, respectively, and maximum UV absorption at 235 nm. One minor peak eluted at 8 minutes and had maximum UV absorption at 270 nm but did not coelute with any known eicosanoid standards. The identity of this compound is unknown. No peaks coeluted with authentic 20-hydroxy LTB,, 20-carboxy LTB4, or 5-HETE. Because the UV detector was set at 270 nm from 0 to 1.5minutes, PGE? exhibiting maximum absorption at 190 nm was not detected by UV absorption. No peaks coeluted with authentic LTB,. In order to identify and quantify PGE, and LTB,, the eluate fractions coeluting with authentic PGEl and LTB, were collected and analyzed by RIA (see below). The eluate fraction coeluting with 15-HETE was collected, and the identity of 15HETE

was confirmed by RIA. The eluate fraction coeluting with authentic 12-HETE was collected, pooled, and assayed for chemokinetic activity by use of the neutrophil microdroplet chemokinesis assay. This fraction contained chemokinetically active material (data not presented). Subsequently, 12-HETE and 15HETE were quantified by integrated optical density. The levels of eicosanoids in normal nonatopic skin and uninvolved AD skin is presented in Table I. PGE, and LTB4 were quantified by RIA after RP-WPLC: 1% HETE, and 1%HETE were quantified by integrated optical density. For all eicosanoids there was no statistical difference between the levels in uninvolved AD skin and in normal nonatopic skin. The levels of eicosanoids in biopsy specimens obtained by keratome from lesional, perilesional, and uninvolved skin of patients with AD are presented in Table II. Compared with uninvolved AD skin, the levels of all eicosanoids in both lesional and perilesional skin were signifi-

VOLUME NUMBER

“T/a

Skin eicosanoids in atopic der~at~~~s 453

83 2. PART 1

I. Comparison

between

eicosanoid

levels in normal

PGEP Skin site

Normal nonatopic skin (N = 5) Uninvolved AD skin (N = 4)

nonatopic

skin and uninvolved

LTB,

12-HETE

nglgm of wet tissue

nglgm of wet tissue

nglgm of wet tissue

27.1 + 9.8

co.05

120.0 i

17.8 c 1.5”

-co.05

101.0 r+ 38.01

14.2

AD skin

wet tissue

133.0 it 44.2 62.0 + 16.0”

Results are expressedas mean f SEM. *Normal nonatopic skin versus uninvolved AD skin, not significant. PGE,, and LTB, were quantified by RIA after RP-HPLC; 12-HETE and 1%HETE were quantified by integrated optical density.

II. Eicosanoid levels in biopsy specimens and uninvolved skin of patients with AD

Skin site

Lesional skin (N = 8) Perilesional skin (N = 5) Uninvolved skin (N = 4)

obtained

by keratome

from lesional,

PGE,

LTB,

12-HETE

nglgm of wet tissue

ng/gm of wet tissue

nglgm of wet tissue

97.2 i:

periiesionai,

15.6*

5.2 !I 1.6*

717.0

+ 186.0t

687.0

128.3 k 27.2f

3.2 + 0.64

413.0

t

177.0 t

17.8 * 1.5§

<0.05§

106.0#

101.0 c 38.09

-i- 197.0-v

117.0$

62.0 rt IS.O?j

Results are expressedas mean ? SEM. * Lesional versus perilesional, not significant. tLesiona1 versus perilesional, p < 0.05. #Ferilesional versus uninvolved, p < 0.05. 8Lesional versusuninvolved, p < 0.05. PGE, and LTB, were quantified by RIA after RP-HPLC, 12-HETE, and 15-HETE were quantified by integrated optical density.

cantly elevated. The level of PGE, in lesional and perilesional skin was 5.5-fold and 7.2-fold elevated, compared to uninvolved AD skin. The level of LTB, in uninvolved AD skin was below the detection limit (0.05 ng per sample), and in both lesional and perilesional AD skin, the level was at least 100 times elevated, reaching nanogram amounts per gram of wet tissue. In both lesional and perilesional skin, PGE, and LTB, were present in biologically active concentrations.15 For both PGE, and LTB, there was no statistical difference between the levels in lesional skin and the levels in perilesional skin. The level of 12HETE in lesional and perilesional skin was sevenfold and fourfold increased, compared to uninvolved skin, respectively, and the level of 15-HETE in lesional and perilesional skin was elevenfold and threefold increased, respectively, compared to uninvolved skin. The levels of 12-HETE and 15-HETE were significantly higher in lesional skin, compared to perilesional skin (Table 1I). However, neither 12-HETE nor 15 HETE were present in biologically active concentra-

tions as compared to in vitro concentrations to cause chemokinesis ( 12-HETE)24 and to inhibit fo~atjo~ of LTB, (15HETE).18

DISCUSSION In the present study we have found elevated levels of PGE2, LTB4, lZHETE, and 15-HETE in lesional and perilesional skin of patients with AD. However, only PGE, and LTB4 reached biologically active concentrations. Since both PGE, and LTB4 have the potential to elicit cutaneous inflammation after intraderma1 injection or topical application, their presence in biologically active concentrations may indicate that they may be of importance in the inflammato~ processes in AD. In a previous study, Ruzicka et al4 found PGE, and LTB,, present in suction-blister fluid obtained from lesional skin of patients with AD. PGE, was found to be lower in lesional than in clinically unaffected skin, which is in contrast to our results dem~~s~ati~g a 5.5 fold increase in the lesional content of PGE2. They

4

Fogh et al

also found an elevation of LTB, compared to uninvolved AD skin. However, LTB, levels were approximately 10 times lower than in the present study. The difference in eicosanoid levels between their and our results may be related to differences in sampling techniques, because the analytic procedures applied were identical. Eicosanoid levels found in blister fluid may not be directly comparable to the intralesional levels and may particularly be the case, if the eicosanoids are present in the scale, as in psoriasis.” PGE, was 5.5-fold and 7.2-fold increased in lesional and perilesional skin, respectively, reaching concentrations capable of causing biologic effects.15 PGE, causes wheal and flare when it is injected intradermally into human skinI Furthermore, PGE, has been demonstrated to inhibit a number of Tlymphocyte functions. I4 Therefore, PGE, may be involved in the erythematous appearance of AD and may, at least in part, account for the depressed cellular immunity in AD skin. Also, the lesional and perilesional levels of LTB, were markedly increased and present at concentrations able to cause biologic effect. ‘*, I33I5 LTB, is chemotactic and chemokinetic for monocytes’ and eosinophils,’ and it has been demonstrated that LTB, is a specific chemoattractant for lymphocytes. ‘OThe lesional and perilesional concentrations of LTB, correspond to approximately 1.5 x 10e8 mol/L and 9.5 X 10e9 mol/L. Because of the bell-shaped dose-response curve for LTB,-induced chemotaxis,8 these concentrations of LTB, are optimal for the induction of leukocyte chemotaxis.‘-lo Furthermore, LTB, can increase vascular permeability.” When LTB, is injected into human skin, it results in wheal and flare. Simultaneous injection of LTB, and PGE, results in a marked amplified reaction.15 Therefore, PGE, may potentiate the inflammatory reaction induced by LTB,. For both PGE, and LTB4, the levels were similar in Iesional skin and in perilesional skin (Table II). These findings may reflect that the lesions of AD are poorly defined. Also subclinical dermatitis may be present in perilesional skin. Therefore, it would have been of interest to know whether there was any microscopic signs of inflammation in biopsy specimens of perilesional skin. Another fact that further supports the role of PGE, and LTB, is the improvement of AD after glucocorticosteroid treatment. Glucocorticosteroids are believed to act, at least in part, by inducing the synthesis of a phospholipase A, inhibitor, lipocortin, thereby leading to a reduction in the release of AA.22 The reduced availability of AA may in turn lead to decreased synthesis of PGE, and/or LTB,. Elevated levels of 12-HETE-like material was de-

J. ALLERGY

CLIN. IMMUNOL. FEBRUAW 1989

tected in lesional and perilesional AD skin. The peak on RP-HPLC coeluting with authentic 12-HETE contained chemokinetically active materia!. In psoriatic skin lesions the 12-HETE peak on RP-HPLC is supposed to consist of two isomers, namely 12(R)-HETE and 12(S)-HETE23; 12(R)-HETE is chemokinetically more potent than 12(S)-HETE.*” Therefore, an attempt to quantify 12-HETE by chemokinesis may lead to an underestimation of 12(S)-HETE. In contrast, an attempt to quantify 12-HETE by RIA may lead to an underestimation of 12(R)-HETE because the antibody for 12-HETE is specific for 12(S)-HETE. Whether both isomers are present in AD skin is unknown. Therefore, we have quantified 12-HETE-like material by integrated optical density. Both 12(R)-HETE and 12(S)-HETE have properties similar to LTB4161I7 but appear to be 100 to 1000 times less potent as chemoattractant.% Therefore, the iesional concentration (2.2 X 10s6 mol/L) and the perilesionai concentration (1.3 X 10T6 mol/L) of 12-HETE in AD appear to be too low to contribute to the inflammatory process. These findings indicate that IZI-IETE is not involved in the cutaneous inflammation in AD. Although the level of 15-HETE in lesional skin is elevated, the mean level of 15HETE (2.1 X lo-” mol/L) would be unable to cause any inhibition of LTB, formation and/or inhibition of LTB,-induced chemotaxis. 18% l9 Therefore, 15-HETE in skin of atopic subjects does not appear to be of any importance in the regulation of LTB, formation. In conclusion, the results of the present study reveal that PGE, and LTB4, both potent mediators of cutaneous inflammation, are present in lesional and perilesional skin of AD in biologically active concentrations. Therefore, they may be involved in the biochemical processes occurring in AD. To elucidate further the role of 5-LO products in AD, studies are in progress to determine the level of peptidoleukotrienes (LTC, and LTD,) in skin of patients with A We thank Mrs. W. Heidemann for expert technical assistance. REFERENCES 1. Hanifin JM. Pathophysiology of atopic dermatitis. In: Soter NA, Baden HP, eds. Pathophysiology of dermatological diseases. New York: McGraw-Hill, 1984:135-44. 2. Johnson HH, de Oreo G, Lascheid WP, Mitcheli F. Skin histamine levels in chronic atopic dermatitis. J Invest Dermatol 1960;34:237-8. 3. Juhlin L. Localisation and content of histamine ir? normal and diseased skin. Acta Derm Venereol 1967;47:383-91. 4. Ruzicka T, Simmet T, Peskar BA, Ring J. Skin levels of arachidonic acid-derived inflammatory mediators and histamine in atopic dermatitis and psoriasis. J Invest Dennatol 19%; 86:105-8.

VOLUME NUMBER

Skin eicosanoids

83 2, PART 1

5. Brain S, Camp R, Dowd P, Black AK, Greaves M. The release of leukotriene B&e material in biologically active amounts from the lesional skin of patients with psoriasis. .I Invest Dermatol 1984;83:70-3. 6. Fogh K, Kiil .I, Herlin T, Temowitz T, Kragballe K. Heterogeneous distribution of lipoxygenase products in psoriatic skin lesions. Arch Dermatol Res 1987;279:504-11. 7. Barr RM, Brain S, Camp RDR, Cilliers J, Greaves MW. Levels of arachidonic acid and its metabolites in the skin in human allergic and irritant contact dermatitis. Br 3 Dermatol 1984; II 1:23-S. 8. Temowitz T, Berlin T, Fogh K. Human monocyte and polymorphonuclear leukocyte chemotaxis and chemokinetic responses to leukotriene B,. Acta Path01 Microbial Immunol 1987;95:47-54. 9. Czametzki BM, Rosenbach T. Chemotaxis of human neutrophils and eosinophils towards leukotriene B, and its 20-omegaoxidation products in vitro. Prostaglandins 1986;31:85 l-8. 10. Payan DG, Goetzl EJ. The dependence of human Tlymphocyte migration on the 5-lipoxygenation of endogeneous arachidonic acid. .I Clin Immunol 1981 ;1:266-70. 11. Bjork .I, Hedqvist P, Arfors KE. Increase in vascular permeability induced by leukotriene B, and the role of polymorphonuclear leukocytes. Inflammation 1982;6:189-200. 12. Camp RDR, Jones RR, Brain S, Woollard P, Greaves M. Production of intraepidermal microabscesses by topical application of leukotriene B,. .I Invest Dermatol 1984;82:202-4. 13. Soter NA, Lewis RA, Corey EJ, Austen KF. Local effects of synthetic leukotrienes (LTC,, LTD,, LTE,, and LTB,) in human skin. 5 Invest Dermatol 1983;80:115-9. 14. Parker CW. Leukotrienes and prostaglandins in the immune system. In: Zor U, et al., eds. Advances in prostaglandin, thromboxane, and leukotriene research, vol 16. New York Raven Press, 1986:113-34. 15. Archer CB, Page CP, Juhiin L, Morley J, MacDonald DM. Delayed synergism between leukotriene B, and prostaglandin E, in human skin. Prostaglandins 1987;33:799-805.

in atopic

dermatitis

16. Dowd PM, Kobza Black A, Woollard PM, Greaves NW. Cutaneous responses to 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 5,12-dihydroxyeicosatetraenoic acid (leukotriene B4) in psoriasis and normal human skin. Arch Dermatol Res 1987;279:427-34. 17. Ruzicka T, Burg G. Effects of chronic intracutaneous administration of arachidonic acid and its metabolites: induction of leukocytoclastic vasculitis by leukohiene B, and 12hydroxyeicosatetraenoic acid and its prevention by prostaglandin E,. J Invest Dermatol 1987;88:120-3. 18. Vanderhoek JY, Bryant RW, Bailey JM. Inhibition of leukotriene biosynthesis by the leukocyte product 15hydroxy-5,8,11,13-eicosatetraenoic acid. J Biol Chem 1.980; 255:10064-6. 19. Temowitz T, Fogh K, Kragbaile K. 15hydroxyeicosatetraenoic acid (15-HETE) specifically inhibits LTB,induced chemotaxis of human neutrophils. Skin Pharmacol 1988;1:93-9. 20. Hanifin JM, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol 1980;92:44-8. 211 Fogh K, Herlin T, Kragballe K. Eicosanoids in early psoriatic lesions: leukotriene B,, but not 12-hydroxyeicosatetraenoic acid (12-HETE) is present in biologically active amounts in acute guttate lesions [Abstract]. J Invest Dermatol 1988;91: 401. 22. Blackwell GJ, Camuccio R, DiRosa M, Flower RJ, Parente L, Persico P. Macrocortin: a polypeptide causing antiphospholipase effect of glucocorticoids. Nature 1980;287: 147-9. 23. Woollard PM. Stereochemical difference between 12-hydroxy5,8,10,14eicosatetraenoic acid in ptatelets and psoriatic lesions. Biochem Biophys Res Commun 1986;136:169-76. 24. Cunningham FM, Woollard PM. 12(R)-hydroxy-5,8.10,14eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leukocytes in vitro. Prostaglandins 1987;34: 71-8.

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