Arch Dermatol Res (1991) 283:158-161
Archives of
9 Springer-Verlag1991
The effect of topical retinoids on the leukotriene-B4-induced migration of polymorphonuclear leukocytes into human skin G. Wozel 1'3, A. Chang 1, M. Zultak 2, B. M. Czarnetzki 2'4, R. Happle a, j . Barth 3, and P. C. M. van de Kerkhof 1 1 Department of Dermatology, University Hospital Nijmegen, Javastraat 104, 6524 MJ Nijmegen, The Netherlands 2 Department of Dermatology, Pharma Division, Hoffmann-La Roche & Co Ltd, Basel, Switzerland 3 Department of Dermatology, Medical Academy "Carl Gustav Carus", Dresden, Federal Republic of Germany 4 Department of Dermatology, Free University of Berlin, Rudolf Virchow Hospital, Berlin, Federal Republic of Germany Received September 1, 1990
Summary. Systemic retinoids are effective in a variety of inflammatory dermatoses. Disorders in which polymorphonuclear leukocytes (PMN) are involved, such as psoriasis and acne, respond particularly well to various retinoids. However, side-effects restrict the use of systemic retinoids to severe manifestations. Topical application might provide the possibility of avoiding the systemic sideeffects o f these compounds. In this communication we report on the modulation of transepidermal migration of P M N by topical application of all-trans-retinoic acid, 13cis-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone. Test areas of healthy volunteers were pretreated with these retinoids in a cream base and with corresponding placebo creams, and intraepidermal accumulation of P M N was quantified 24 h after epicutaneous challenge with leukotriene B 4 (LTB4), using elastase as a marker enzyme. Topical treatment with 13-cis-retinoic acid resuited in a marked and statistically significant inhibition of the LTB4-induced migration of PMN. All-trans-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone reduced the accumulation of P M N slightly, but not statistically significantly. Topical treatment with arotinoid methyl sulphone had no effect. Key words: Retinoids B4 - Psoriasis
Neutrophils
-
Leukotriene
only aim at the development of safer analogues, but the topical application of available retinoids should be explored as well [2, 3]. The arachidonic acid cascade may play an important role in inflammation, and one of its main metabolites, leukotriene B 4 (LTB4), is ~ highly potent chemoattractant [25]. In human skin, it has been shown that the epicutaneous application of LTB4 results in an experimentally reproducible accumulation of P M N [6, 34]. Using this approach in an in vivo model, the pharmacological modulation of P M N migration can be studied [20]. Systemic and topical antipsoriatic therapies, including etretinate and its main metabolite acitretin, inhibited the in vivo chemotaxis of P M N [7, 8, 10, 13, 21, 23, 37]. Topical treatment with 13-cis-retinoic acid and alltrans-retinoic acid has been shown to be effective in acne [33]. Recent studies indicate that topical treatment with all-trans-retinoic acid, arotinoid ethyl sulphone and arotinoid methyl sulphone reduce actinic keratoses ([26]; Rufli et al., personal communication). The aim of the present investigation was to study the effect of topical treatment with these retinoids on P M N migration in the skin following epicutaneous application of LTB4. Elastase was used as a marker enzyme for P M N quantification [22].
Materials and methods
Subjects Systemic retinoids are highly effective in a variety of skin disorders [28] and the aromatic retinoids have revolutionized the treatment of disorders of keratinization [4, 16]. Certain retinoids are particularly suited to the management of inflammatory dermatoses in which the migration of polymorphonuclear leukocytes (PMN) is a key feature, such as psoriasis and acne [18, 29]. However, side-effects of retinoids limit their systemic use. For this reason efforts to improve retinoid therapy should not Offprint requests to: P. C. M. van de Kerkhof
Sixteen healthy male volunteers aged 19 to 36 years participated in the study. They had no signs or history of skin diseases. No systemic or topical therapy and no sunbathing were recorded by the selected subjects during a period of 4 weeks before the experiment was started. Application of creams In each volunteer, four test areas (5 x 5 cm) on the outer surface of both the upper arms were marked with a felt pen. The test areas
G. Wozel et al. : Topical retinoids and migration of leukocytes were spaced at least 5 cm apart. Using a double-blind design, a group of eight subjects applied the following creams:
13-cis-retinoicacid 0.1% placebo, containing the vehicle in (1) arotinoid methyl sulphone 0.05% placebo, containing the vehicle in (3). The other eight subjects applied the following creams: (1) all-trans-retinoic acid 0.05% (2) placebo, containing the vehicle in (1) (3) arotinoid ethyl sulphone 0.05% (4) placebo, containing the vehicle in (3).
(1) (2) (3) (4)
These preparations were applied thinly and evently twice a day for 14 days at 9 a.m. and 9 p.m. Only the last application on day 14 was carried out at 12 noon, During the treatment phase following the first visit, all subjects were seen on two occasions by the investigators in order to achieve optimal compliance; the clinical appearance of the test areas was assessed, and the subjects applied the creams under the investigator's supervision.
Application of LTB4 Eight hours after the last application of the creams, 10 ng aliquots of LTB~ (Passel GmbH, Frankfurt, FRG) dissolved in ethanol (10 gl) were applied through glass cylinders (5.5 ram) on untreated and treated test areas [26]. Ethanol was evaporated by a stream of helium. The LTB4 application sites were carefully marked with eosin and covered with semipermeable dressings (Silverpatch, Van der Bend, Brielle, The Netherlands). After the application of LTB4, subjects refrained from bathing until the biopsy had been taken 24 h later. Biopsies (3 mm diameter, weight 2 - 3 mg) were taken freehandedly using a razor in conjunction with a metal guard and chlorethyl spray as local anaesthetic.
159 PMN/IO pg skin 400
300
200
t00
0
13-RA
Control
]
drug
AMS
AES
[]
alI-RA
placebo
Fig. 1. LTB4-induced accumulation of PMN (mean _+SEM) in skin areas pretreated with different retinoids in comparison to the corresponding placebo creams. 13RA, 13-cis-retinoic acid; AMS, arotinoid methyl sulphone; AES, arotinoid ethyl sulfone; allRA, all-trans-retinoic acid. * Statistically significant at the levelp < 0.02
where (1), (2), (3) and (4) represent the fluorescence obtained from the respective incubation mixtures. The density of infiltrating PMN per 10 gg skin was calculated using the value of 0.66 pmol 7-amino4-methylcoumarin released per h per PMN.
Statistical evaluation Statistical evaluation was carried out using a Wilcoxon ranking test for paired data.
Analytical procedures
Results
Biopsies were processed immediately for elastase measurements as described previously [9, 22]. The skin specimens were rinsed in calcium-free phosphate-buffered saline (PBS), weighed and dried between filter papers. The biopsies were then homogenized in buffer containing cetrimide using an all-glass Potter grinder. After centrifugation (3000 rpm, 10 rain) the supernatants were stored at -20~ Aliquots of the supernatants (20 gl) were incubated with 20 gl cetrimide buffer and 20 gl of the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-N-methytcoumarin. Elastase activity of the supernatant was determined by measurement of the fluorescence of 7-amino-4-methylcoumarin released from the substrate. Inhibition of the skin samples was corrected by the inclusion of an internal standard of elastase (equivalent to 500 PMN). The following incubation mixtures were set up in duplicate:
The application o f placebo creams 13-cis-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone did n o t induce any clinical change. However, in six subjects, the c r e a m containing all-trans-retinoic acid elicited a slight-to-moderate erythema, scaling and occasionally some itching. The LTB4-induced a c c u m u l a t i o n o f P M N in the pretreated areas is summarized in Fig. 1. The 13-cis-retinoic acid caused a substantial inhibition o f the a c c u m u l a t i o n o f P M N c o m p a r e d with the c o r r e s p o n d i n g placebo (p < 0.02). All-trans-retinoic acid and arotinoid ethyl sulphone reduced the a c c u m u l a t i o n o f P M N slightly. The reduction, however, was n o t statistically significant (p > 0.05). A r o t i n o i d methyl sulphone did n o t elicit any effect. The e n d o g e n o u s elastase-inhibiting activities ( E I A ) o f the retinoid- and placebo-treated skin are presented in Table 1. N o significant change o f E I A was observed in any test area.
(1) (2) (3) (4)
20 gl sample, 20 ~tl cetrimide buffer, 20 gl substrate 20 gl elastase standard, 20 gl cetrimide buffer, 20 lal substrate 20 gl sample, 20 Ixl elastase standard, 20 ~tl substrate 40 I-tlcetrimide buffer, 20 gl substrate.
After incubation for i h, the reaction was stopped by the addition of 1 ml carbonate buffer (0.1 M, pH 10.5). Fluorescence was then measured and inhibition calculated as follows: (2) -Elastase activity (corrected) = (1)- (4) x ~ . (4)
Percentage endogenous inhibition = [(2) - (4)] - [(3) - (1)] x 100,
(2)-(4)
Discussion The present study demonstrates that topical retinoids inhibit the LTB4-induced a c c u m u l a t i o n o f P M N in hu-
G. Wozel et al. : Topical retinoids and migration of leukocytes
160 Table 1. Endogenous elastase inhibiting activity of pretreated skin Pretreatment
Inhibition (mean _+SEM) (%)
13-Cis-retinoic acid Placebo Arotinoid methyl sulphone Placebo Arotinoid ethyl sulphone Placebo All-trans-retinoic acid Placebo Without pretreatment
3.4 + 4.3 8.8 • 3.4 7.8 -t- 4.2 1.7 ___3.0 11.7 • 4.3 16.1 • 4.8 13.5 • 4.8 9.2 • 3.9 14.4 • 3.9
man skin: 13-cis-retinoic acid showed a marked inhibition, whereas all-trans-retinoic acid and arotinoid ethyl sulphone yielded only a weak inhibition (not statistically significant). Our results are consistent with previous investigations on the effect of topical and systemic retinoids on P M N chemotaxis. Dubertret et al. demonstrated a highly significant inhibition of in vivo migration of P M N by topical etretinate and acitretin, using the skin chamber approach with autologous serum as chemoattractant [13]. In line with the effects of the topical route of administration, systemic treatment with 13-cis-retinoic acid, etretinate and its main metabolite, acitretin, has been shown to inhibit the in vivo migration of P M N [13, 27, 31, 32, 37]. In actinic keratoses, topical applications of creams containing arotinoid methyl sulphone 0.05%, arotinoid ethyl sulphone 0.05%, and aIl-trans-retinoic acid 0.05% have been reported to be equally effective ([32]; T. Rufli, personal communication). In the mouse papilloma model, topical application of 13-cis-retinoic acid cream proved to be half as active and less irritant compared to all-trans-retinoic acid (W. Bollag, personal communication). Therefore, a 0.10% concentration was selected for 13-cis-retinoic acid. Although topical application of the four retinoids proved to have an ant• effect, the present study demonstrates that only the 13cis-retinoic acid cream significantly inhibits the in vivo migration of PMN. This implies that the ant• potential of a retinoid is not indicative of its anti-inflammatory potential. Elastase is a marker enzyme for PMN. However, monocytes contain about 5% of the activity found in P M N [12]. Histological studies in LTB4-induced inflammation of the skin indicate that P M N are the dominating cell type during the first 24 h after LTB 4 application. Subsequently mononuclear ceils invade the skin. In an ultrastructural study it has been shown that the intraepidermal accumulations of P M N still possess their granules following epicutaneous application of LTB4, whereas degranulation of some P M N is sporadically restricted to the phase of extravasation [30]. Therefore, elastase can be regarded as a reliable marker for P M N in biopsies taken 24 h after epicutaneous application of LTB4.
Migration of P M N was inhibited by 13-cis-retinoic acid in vivo but apparently not in vitro [5]. An explanation for the discrepancy between the in vivo and in vitro situation might be a direct interference by retinoids with the tissue, for example as a modification of the stratum corneum. The retinoic acid treated skin might have different pharmacodynamic properties, resulting in a modified bioavailability of LTB4. An alternative explanation might be an interaction of retinoids with metabolic events in the P M N themselves which are required for the invasion of these cells into the tissues. Evidence to support this would be the observation that amongst a series of retinoids, 13-cis-retinoic acid proved to be the most efficient inhibitor of lysosomal enzyme release from the specific and azurophyllic granules of the P M N following activation in vitro [5]. Furthermore, it has been shown that elastase and cathepsin G are of importance for the passage of P M N into the tissue, and the inhibitory effect of 13-cis-retinoic acid could be explained by a decreased release of this enzyme [1]. Additionally it has been suggested that 13-cis-retinoic acid inhibits the migration of P M N through endothelial cell coated filters [36]. The exact mechanisms involved in this inhibition are still unclear. Our knowledge of the mechanism of action of topical retinoids in psoriasis and acne is still fragmentary. The ant• effect of all-trans-retinoic acid has been suggested by several uncontrolled studies [14, 15, 24]. In psoriasis it has been proposed as an adjunct therapy to topical corticosteroids [35]. However, no reduction in epidermal turnover has been demonstrated in psoriasis treated with topical retinoids [19]. Similarly in ache no change in sebum excretion rate has been reported [17]. The mechanism of action might be non-specific keratolysis [11]. The exploration of LTB4-induced P M N chemotaxis might elucidate alternative mechanisms of action of topical retinoids. Topical application of retinoids provides the possibility of bypassing the systemic side-effects of these drugs. Based on the present study it seems worthwhile to investigate the effect of topical 13-cis-retinoic acid in psoriasis.
References
1. Briggaman RA, Schechter NM, Fr/iki J, Lazarus GS (1984) Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes. J Exp Med 160:1027-1042 2. Bollag W (1983) The development of retinoids in experimental and clinical oncology and dermatology. J Am Acad Dermatol 9: 797- 805 3. BollagW(1987) Experimentaldata on new retinoids. In: Farber EM, Nall L, Morhenn V, Jacobs PH (eds) Psoriasis. Proceedings of the 4th International Symposium, Stanford University, July 6-11, 1986. Elsevier, New York, pp 197- 203 4. Bruge SM, Wilkinson JD (1984) The use of Tigason (Etretinate) in Darier's disease. In: Cunliffe WJ, Miller AJ (eds) Retinoid therapy. A review of clinical and laboratory research. MTP Press, Lancaster, pp 61 --62 5. Camisa C, Eisenstat B, Ragat A, Weissmann G (1982) The effects of retinoids on neutrophil functions in vitro. J Am Acad Dermatol 6 : 620 -- 629
G. Wozel et al.: Topical retinoids and migration of leukocytes 6. Camp R, Russel Jones R, Brain S, WooUard P, Graeves M (1984) Production of intradermat microabscesses by topical application of leukotriene B4. J Invest Dermatol 82:202-204 7. Chang A, KerkhofPCM van de (1988) PUVA and UVB inhibit the intraepidermal accumulation of polymorphonuclear leukocytes. Br J Dermatol 119:281 - 287 8. Chang A, Kerkhof PCM van de (1988) Topical application of clobetasol-17-propionate inhibits the intra-epidermal accumulation of polymorphonuclear leukocytes. Acta Derm Venereol (Stockh) 68 : 57 - 60 9. Chang A, Jongh GJ de, Mier PD, Kerkhof PCM van de (1988) Enzymatic quantification of polymorphonuclear leucocytes in normal and psoriatic skin following standardized injury. Clin Exp Dermatol 13 : 6 2 - 66 10. Chang A, Alkemade H, Kerkhof PCM van de (1989) Dithranol modulates the leukotriene B4-induced intra-epidermal accumulation of potymorphonuclear leukocytes. J Invest Dermatol 92: 8 0 6 - 808 11. Cunliffe W J, McDonald Hull S (1988) Lack of effect of topical retinoic acid on sebum excretion rate in acne. Lancet II : 503 12. Davies M, Hughes KT, Andrew PW, Smith GP, Peters TJ (1984) The activities and subcellular localization of neutral proteinases in human monocytes and polymorphonuclear leukocytes. Cell Mol Biol 30:337--343 13. Dubertret L, Lebreton C, Touraine R (1982) Inhibition of neutrophil migration by etretinate and its main metabolite. Br J Dermatol 107:681 - 685 14. Frost P, Weinstein GD (1969) Topical administration of vitamin A acid for ichthyosiform dermatoses and psoriasis. J Am Acad Dermatol 207:1863 - 1868 15. G/inther S (1973) The therapeutic value of retinoic acid in chronic discoid, acute guttate, and erythrodermic psoriasis: clinical observations on twenty-five patients. Br J Dermatol 89:515-517 16. Happle R, Kerkhof PCM van de, Traupe H (1987) Retinoids in disorders ofkeratinization: their use in adults. Dermatologica [Suppl 1] 175 : 107 - 124 17. Harms M, Phillipe I, Ceyrac D, Saurat JH (1985) Isotretinoin ineffective topically. Lancet I: 8 18. Hennes R, Mack A, Schell H, Vogt HJ (1984) 13-cis-retinoic acid in conglobate acne. A follow up study of 14 trial centers. Arch Dermatol Res 276: 2 0 9 - 215 19. Hodgson C, Hell E (1976) The action of retinoic acid in psoriasis. Clin Exp Dermatol 1:215-220 20. Lammers AM, Kerkhof PCM van de (1987) Response of polymorphonuclear leukocytes to topical leukotriene B4 in healthy and psoriatic skin. Br J Dermatol 116:521 - 5 2 4 21. Lammers AM, Kerkhof PCM van de (1987) Etretinate modulates the leukotriene B4 induced intra-epidermal accumulation of polymorphonuclear leukocytes. Br J Dermatol 117: 2 9 7 300
161 22. Lammers AM, Kerkhof PCM van de, Schalkwijk J, Mier PD (1986) Elastase, a marker for polymorphonuclear leukocytes in skin infiltrates. Br J Dermatol 115:181 - 186 23. Lammers AM, Kerkhof PCM van de, Mier PD (1987) Reduction of leukotriene B4-induced intradermal accumulation of polymorphonuclear leukocytes by methotrexate in psoriasis. Br J Dermatol 116: 6 6 7 - 671 24. Macdonald A, Fry L (1972) Retinoic acid in the treatment of psoriasis. Br J Dermatol 86:524-527 25. Malmsten CL, Palmblad J, Ud6n AM, Ramark O, Engstedt L, Samuelsson B (1980) Leukotriene B4: a highly potent and stereospecific factor stimulating migration of polymorphonuclear leukocytes. Acta Physiol Scand 110:449-451 26. Misicwisz IG, Sendagorta E, Gulebiowska E, Loaienc B, Czarnetzki BM, Jablonska S (1990) Topical treatment of multiple actinic keratoses on the face with arotinoid methyl sulfone (Ro 14-9606) cream versus tretinoin cream. A double-blind comparative study. J Am Acad Dermatol (in press) 27. Norris DA, Osborn R, Robinson W, Tonnesen G (1987) Isotretinoin produces significant inhibition of monocyte and neutrophil chemotaxis in vivo in patients with cystic acne. J Invest Dermatol 89:38-43 28. Orfanos CE (1987) An introductory comment: retinoids - 10 years later. Dermatologica 175 [Suppl 1]: 2 - 3 29. Orfanos CE (1989) Retinoide: der neue Stand. Hautarzt 40:123-129 30. Paulissen M, Copius Peereboom-Stegeman JHJ, Kerkhof PCM van de (1990) An ultrastructural study of transcutaneous migration of polymorphonuclear leukocytes following application of leukotriene B4. Skin Pharmacol 3:236-247 31. Pigatto PD (1988) Effect of isotretinoin on the neutrophil chemotaxis in cystic acne. J Invest Dermatol 90:885-886 32. Pigatto PD, Fioroni A, Riva F, Brugo MA, Morandotti A, Altomare GF, Finti A F (1983) Effects of isotretinoin on the neutrophil chemotaxis in cystic ache. Dermatologica 167:1618 33. Plewig G, Ruhfus A, K16vekorn W (1983) Sebum suppression after topical application of retinoids. J Invest Dermatol 80:357 34. Soter NA, Lewis RA, Corey EJ, Amsten K F (1983) Local effects of synthetic leukotriencs (LTC4, LTD4 and LTB4) in human skin. J Invest Dermatol 80:115-119 35. Thomas JR, Doyle JA (1981) The therapeutic uses of topical vitamin A acid. J Am Acad Dermatol 4: 505- 513 36. Tonnesen MG, Osborn RL, Norris DA (1986) Inhibition of neutrophil chemotaxis by 13-cis-retinoicacid may be endothelial cell dependent. Clin Res 34:165 37. Kerkhof PCM van de, Chang A, Dooren-Greebe R van, Geiger JM, Happle R (1988) Intra-epidermal accumulation of polymorphonuclear leukocytes in persistent palmoplantar pustulosis during treatment with acitretin. Acta Derm Venereol (Stockh) 68: 4 4 9 - 503
Archives of
9 Springer-Verlag1991
The effect of topical retinoids on the leukotriene-B4-induced migration of polymorphonuclear leukocytes into human skin G. Wozel 1'3, A. Chang 1, M. Zultak 2, B. M. Czarnetzki 2'4, R. Happle a, j . Barth 3, and P. C. M. van de Kerkhof 1 1 Department of Dermatology, University Hospital Nijmegen, Javastraat 104, 6524 MJ Nijmegen, The Netherlands 2 Department of Dermatology, Pharma Division, Hoffmann-La Roche & Co Ltd, Basel, Switzerland 3 Department of Dermatology, Medical Academy "Carl Gustav Carus", Dresden, Federal Republic of Germany 4 Department of Dermatology, Free University of Berlin, Rudolf Virchow Hospital, Berlin, Federal Republic of Germany Received September 1, 1990
Summary. Systemic retinoids are effective in a variety of inflammatory dermatoses. Disorders in which polymorphonuclear leukocytes (PMN) are involved, such as psoriasis and acne, respond particularly well to various retinoids. However, side-effects restrict the use of systemic retinoids to severe manifestations. Topical application might provide the possibility of avoiding the systemic sideeffects o f these compounds. In this communication we report on the modulation of transepidermal migration of P M N by topical application of all-trans-retinoic acid, 13cis-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone. Test areas of healthy volunteers were pretreated with these retinoids in a cream base and with corresponding placebo creams, and intraepidermal accumulation of P M N was quantified 24 h after epicutaneous challenge with leukotriene B 4 (LTB4), using elastase as a marker enzyme. Topical treatment with 13-cis-retinoic acid resuited in a marked and statistically significant inhibition of the LTB4-induced migration of PMN. All-trans-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone reduced the accumulation of P M N slightly, but not statistically significantly. Topical treatment with arotinoid methyl sulphone had no effect. Key words: Retinoids B4 - Psoriasis
Neutrophils
-
Leukotriene
only aim at the development of safer analogues, but the topical application of available retinoids should be explored as well [2, 3]. The arachidonic acid cascade may play an important role in inflammation, and one of its main metabolites, leukotriene B 4 (LTB4), is ~ highly potent chemoattractant [25]. In human skin, it has been shown that the epicutaneous application of LTB4 results in an experimentally reproducible accumulation of P M N [6, 34]. Using this approach in an in vivo model, the pharmacological modulation of P M N migration can be studied [20]. Systemic and topical antipsoriatic therapies, including etretinate and its main metabolite acitretin, inhibited the in vivo chemotaxis of P M N [7, 8, 10, 13, 21, 23, 37]. Topical treatment with 13-cis-retinoic acid and alltrans-retinoic acid has been shown to be effective in acne [33]. Recent studies indicate that topical treatment with all-trans-retinoic acid, arotinoid ethyl sulphone and arotinoid methyl sulphone reduce actinic keratoses ([26]; Rufli et al., personal communication). The aim of the present investigation was to study the effect of topical treatment with these retinoids on P M N migration in the skin following epicutaneous application of LTB4. Elastase was used as a marker enzyme for P M N quantification [22].
Materials and methods
Subjects Systemic retinoids are highly effective in a variety of skin disorders [28] and the aromatic retinoids have revolutionized the treatment of disorders of keratinization [4, 16]. Certain retinoids are particularly suited to the management of inflammatory dermatoses in which the migration of polymorphonuclear leukocytes (PMN) is a key feature, such as psoriasis and acne [18, 29]. However, side-effects of retinoids limit their systemic use. For this reason efforts to improve retinoid therapy should not Offprint requests to: P. C. M. van de Kerkhof
Sixteen healthy male volunteers aged 19 to 36 years participated in the study. They had no signs or history of skin diseases. No systemic or topical therapy and no sunbathing were recorded by the selected subjects during a period of 4 weeks before the experiment was started. Application of creams In each volunteer, four test areas (5 x 5 cm) on the outer surface of both the upper arms were marked with a felt pen. The test areas
G. Wozel et al. : Topical retinoids and migration of leukocytes were spaced at least 5 cm apart. Using a double-blind design, a group of eight subjects applied the following creams:
13-cis-retinoicacid 0.1% placebo, containing the vehicle in (1) arotinoid methyl sulphone 0.05% placebo, containing the vehicle in (3). The other eight subjects applied the following creams: (1) all-trans-retinoic acid 0.05% (2) placebo, containing the vehicle in (1) (3) arotinoid ethyl sulphone 0.05% (4) placebo, containing the vehicle in (3).
(1) (2) (3) (4)
These preparations were applied thinly and evently twice a day for 14 days at 9 a.m. and 9 p.m. Only the last application on day 14 was carried out at 12 noon, During the treatment phase following the first visit, all subjects were seen on two occasions by the investigators in order to achieve optimal compliance; the clinical appearance of the test areas was assessed, and the subjects applied the creams under the investigator's supervision.
Application of LTB4 Eight hours after the last application of the creams, 10 ng aliquots of LTB~ (Passel GmbH, Frankfurt, FRG) dissolved in ethanol (10 gl) were applied through glass cylinders (5.5 ram) on untreated and treated test areas [26]. Ethanol was evaporated by a stream of helium. The LTB4 application sites were carefully marked with eosin and covered with semipermeable dressings (Silverpatch, Van der Bend, Brielle, The Netherlands). After the application of LTB4, subjects refrained from bathing until the biopsy had been taken 24 h later. Biopsies (3 mm diameter, weight 2 - 3 mg) were taken freehandedly using a razor in conjunction with a metal guard and chlorethyl spray as local anaesthetic.
159 PMN/IO pg skin 400
300
200
t00
0
13-RA
Control
]
drug
AMS
AES
[]
alI-RA
placebo
Fig. 1. LTB4-induced accumulation of PMN (mean _+SEM) in skin areas pretreated with different retinoids in comparison to the corresponding placebo creams. 13RA, 13-cis-retinoic acid; AMS, arotinoid methyl sulphone; AES, arotinoid ethyl sulfone; allRA, all-trans-retinoic acid. * Statistically significant at the levelp < 0.02
where (1), (2), (3) and (4) represent the fluorescence obtained from the respective incubation mixtures. The density of infiltrating PMN per 10 gg skin was calculated using the value of 0.66 pmol 7-amino4-methylcoumarin released per h per PMN.
Statistical evaluation Statistical evaluation was carried out using a Wilcoxon ranking test for paired data.
Analytical procedures
Results
Biopsies were processed immediately for elastase measurements as described previously [9, 22]. The skin specimens were rinsed in calcium-free phosphate-buffered saline (PBS), weighed and dried between filter papers. The biopsies were then homogenized in buffer containing cetrimide using an all-glass Potter grinder. After centrifugation (3000 rpm, 10 rain) the supernatants were stored at -20~ Aliquots of the supernatants (20 gl) were incubated with 20 gl cetrimide buffer and 20 gl of the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-N-methytcoumarin. Elastase activity of the supernatant was determined by measurement of the fluorescence of 7-amino-4-methylcoumarin released from the substrate. Inhibition of the skin samples was corrected by the inclusion of an internal standard of elastase (equivalent to 500 PMN). The following incubation mixtures were set up in duplicate:
The application o f placebo creams 13-cis-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone did n o t induce any clinical change. However, in six subjects, the c r e a m containing all-trans-retinoic acid elicited a slight-to-moderate erythema, scaling and occasionally some itching. The LTB4-induced a c c u m u l a t i o n o f P M N in the pretreated areas is summarized in Fig. 1. The 13-cis-retinoic acid caused a substantial inhibition o f the a c c u m u l a t i o n o f P M N c o m p a r e d with the c o r r e s p o n d i n g placebo (p < 0.02). All-trans-retinoic acid and arotinoid ethyl sulphone reduced the a c c u m u l a t i o n o f P M N slightly. The reduction, however, was n o t statistically significant (p > 0.05). A r o t i n o i d methyl sulphone did n o t elicit any effect. The e n d o g e n o u s elastase-inhibiting activities ( E I A ) o f the retinoid- and placebo-treated skin are presented in Table 1. N o significant change o f E I A was observed in any test area.
(1) (2) (3) (4)
20 gl sample, 20 ~tl cetrimide buffer, 20 gl substrate 20 gl elastase standard, 20 gl cetrimide buffer, 20 lal substrate 20 gl sample, 20 Ixl elastase standard, 20 ~tl substrate 40 I-tlcetrimide buffer, 20 gl substrate.
After incubation for i h, the reaction was stopped by the addition of 1 ml carbonate buffer (0.1 M, pH 10.5). Fluorescence was then measured and inhibition calculated as follows: (2) -Elastase activity (corrected) = (1)- (4) x ~ . (4)
Percentage endogenous inhibition = [(2) - (4)] - [(3) - (1)] x 100,
(2)-(4)
Discussion The present study demonstrates that topical retinoids inhibit the LTB4-induced a c c u m u l a t i o n o f P M N in hu-
G. Wozel et al. : Topical retinoids and migration of leukocytes
160 Table 1. Endogenous elastase inhibiting activity of pretreated skin Pretreatment
Inhibition (mean _+SEM) (%)
13-Cis-retinoic acid Placebo Arotinoid methyl sulphone Placebo Arotinoid ethyl sulphone Placebo All-trans-retinoic acid Placebo Without pretreatment
3.4 + 4.3 8.8 • 3.4 7.8 -t- 4.2 1.7 ___3.0 11.7 • 4.3 16.1 • 4.8 13.5 • 4.8 9.2 • 3.9 14.4 • 3.9
man skin: 13-cis-retinoic acid showed a marked inhibition, whereas all-trans-retinoic acid and arotinoid ethyl sulphone yielded only a weak inhibition (not statistically significant). Our results are consistent with previous investigations on the effect of topical and systemic retinoids on P M N chemotaxis. Dubertret et al. demonstrated a highly significant inhibition of in vivo migration of P M N by topical etretinate and acitretin, using the skin chamber approach with autologous serum as chemoattractant [13]. In line with the effects of the topical route of administration, systemic treatment with 13-cis-retinoic acid, etretinate and its main metabolite, acitretin, has been shown to inhibit the in vivo migration of P M N [13, 27, 31, 32, 37]. In actinic keratoses, topical applications of creams containing arotinoid methyl sulphone 0.05%, arotinoid ethyl sulphone 0.05%, and aIl-trans-retinoic acid 0.05% have been reported to be equally effective ([32]; T. Rufli, personal communication). In the mouse papilloma model, topical application of 13-cis-retinoic acid cream proved to be half as active and less irritant compared to all-trans-retinoic acid (W. Bollag, personal communication). Therefore, a 0.10% concentration was selected for 13-cis-retinoic acid. Although topical application of the four retinoids proved to have an ant• effect, the present study demonstrates that only the 13cis-retinoic acid cream significantly inhibits the in vivo migration of PMN. This implies that the ant• potential of a retinoid is not indicative of its anti-inflammatory potential. Elastase is a marker enzyme for PMN. However, monocytes contain about 5% of the activity found in P M N [12]. Histological studies in LTB4-induced inflammation of the skin indicate that P M N are the dominating cell type during the first 24 h after LTB 4 application. Subsequently mononuclear ceils invade the skin. In an ultrastructural study it has been shown that the intraepidermal accumulations of P M N still possess their granules following epicutaneous application of LTB4, whereas degranulation of some P M N is sporadically restricted to the phase of extravasation [30]. Therefore, elastase can be regarded as a reliable marker for P M N in biopsies taken 24 h after epicutaneous application of LTB4.
Migration of P M N was inhibited by 13-cis-retinoic acid in vivo but apparently not in vitro [5]. An explanation for the discrepancy between the in vivo and in vitro situation might be a direct interference by retinoids with the tissue, for example as a modification of the stratum corneum. The retinoic acid treated skin might have different pharmacodynamic properties, resulting in a modified bioavailability of LTB4. An alternative explanation might be an interaction of retinoids with metabolic events in the P M N themselves which are required for the invasion of these cells into the tissues. Evidence to support this would be the observation that amongst a series of retinoids, 13-cis-retinoic acid proved to be the most efficient inhibitor of lysosomal enzyme release from the specific and azurophyllic granules of the P M N following activation in vitro [5]. Furthermore, it has been shown that elastase and cathepsin G are of importance for the passage of P M N into the tissue, and the inhibitory effect of 13-cis-retinoic acid could be explained by a decreased release of this enzyme [1]. Additionally it has been suggested that 13-cis-retinoic acid inhibits the migration of P M N through endothelial cell coated filters [36]. The exact mechanisms involved in this inhibition are still unclear. Our knowledge of the mechanism of action of topical retinoids in psoriasis and acne is still fragmentary. The ant• effect of all-trans-retinoic acid has been suggested by several uncontrolled studies [14, 15, 24]. In psoriasis it has been proposed as an adjunct therapy to topical corticosteroids [35]. However, no reduction in epidermal turnover has been demonstrated in psoriasis treated with topical retinoids [19]. Similarly in ache no change in sebum excretion rate has been reported [17]. The mechanism of action might be non-specific keratolysis [11]. The exploration of LTB4-induced P M N chemotaxis might elucidate alternative mechanisms of action of topical retinoids. Topical application of retinoids provides the possibility of bypassing the systemic side-effects of these drugs. Based on the present study it seems worthwhile to investigate the effect of topical 13-cis-retinoic acid in psoriasis.
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