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Immunomodulatory effects of tretinoin in combination with clindamycin
Journal of the European Academy of Dermatology and Venereology 11 (Suppl. 1) (1998) S2–S7
Immunomodulatory effects of tretinoin in combination with clindamycin Paulette J.J. Wauben-Penris*, Dirk P. Cerneus, Wil E. van den Hoven, Peter J.M.J. Leuven, Jan H.A.M. den Brok, David W.R. Hall Yamanouchi Europe B.V. Research Laboratories, P.O. Box 108, 2350 AC Leiderdorp, The Netherlands
Abstract Background Since acne is a multifactorial skin disease, therapies affecting several etiologic factors can have a higher than expected effectiveness. A combination of the antibiotic clindamycin phosphate and the retinoic acid tretinoin was developed. Objective Anti-inflammatory and immunomodulatory effects of tretinoin in vitro were studied on human keratinocytes and peripheral blood mononuclear cells (PBMCs). Effects of clindamycin phosphate on tretinoin effects were studied. Methods Anti-inflammatory effects on keratinocytes were assessed using an in vitro model with PMA (phorbol ester)stimulated A431 cells (human epidermoid carcinoma). Immunomodulatory effects were measured on superantigen (SEB) stimulated PBMCs. Results Tretinoin showed very potent inhibition of PMA-stimulated IL-6 (interleukin 6) release by A431 cells. The addition of clindamycin phosphate did not interfere with this effect. Tretinoin very potently stimulated IL-5 release, and inhibited IFNg release by SEB-stimulated human PBMCs. This indicates an immunomodulatory effect, stimulating Th2, and inhibiting Th1 dominated responses. These features have been related to the healing of acne lesions. The addition of clindamycin phosphate did not interfere with the immunomodulatory effects of tretinoin. Conclusion The combination of tretinoin and clindamycin phosphate can be expected to be very effective in acne therapy. 1998 Elsevier Science B.V. All rights reserved Keywords: Acne vulgaris; Tretinoin; Immunology
1. Introduction Acne vulgaris is a very common skin disorder, affecting up to 40% of the teenage population [1]. It is characterized by hypercornification and comedone formation, increased sebum production, infection with Propionibacterium acnes, release of inflammatory mediators and infiltration with leukocytes. The cellular infiltrate in the acne lesions consists mainly of CD4 + T-helper lymphocytes [2]. * Corresponding author. Tel.: +31 71 5455533; fax: +31 71 5455265; e-mail: [email protected]
T-Helper cells can be divided in two subtypes based on their cytokine profiles [3]: Th1 cells primarily produce IL-2 and IFNg, and are involved in the cellular immune response and in delayed type hypersensitivity responses. Th2 cells primarily produce IL-4, IL-5 and IL-10, and play a role in B-cell help for humoral immune responses. They are involved in allergic reactions. Various diseases have been associated with either a predominant Th1 (e.g. psoriasis) or Th2 (e.g. atopic dermatitis) response [3]. Recently it has been postulated, that Th1 responses are involved in the etiology of acne, while Th2 responses could help in the healing of acne lesions
0926-9959/98/$- see front matter 1998 Elsevier Science B.V. All rights reserved PII S0926-9959 (98 )0 0087-7
P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
[4]. Certain P. acnes polypeptides were found to act as an antigen in acne patients, but not in normal humans [5]. Although there are many reasonably effective topical acne therapies available, there is a need for preparations that act on more than one factor involved in acne, e.g. an antibiotic combined with tretinoin. Clindamycin is an antibiotic, effective against P. acnes, but not widely used systemically. Tretinoin was originally developed for its beneficial effect in acne, but has also been used in a vast range of other skin diseases like lentigines, keloidal scarring, striae, dysplastic nevi, flat warts, post-inflammatory hyperpigmentation and skin aging. The effectiveness of tretinoin in acne consists of several aspects: tretinoin is known to act as a comedolytic agent, and to decrease the cornification process. Furthermore, tretinoin has anti-inflammatory and immunomodulatory effects and has recently been recognized to have immunosuppressive [6], immuno-stimulatory [7,8], immunomodulatory [9,10], pro-inflammatory [11] and antiinflammatory [12] effects. During tretinoin therapy, the amounts of the pro-inflammatory cytokine IL-1 decrease in lesional and non-lesional psoriatic skin, and remain unchanged in normal skin [13,14]. This indicates that tretinoin appears to have anti-inflammatory effects in inflamed skin, but does not affect the normal immune function of the skin. The aim of these experiments was to investigate the anti-inflammatory and immuno-modulatory effects of tretinoin, and to investigate whether the addition of clindamycin to tretinoin influences the tretinoin effects. The combination of clindamycin and tretinoin could be expected to be very effective for treatment of acne. We investigated whether an inhibition of tretinoin effects by clindamycin could be excluded. Assays were developed in our laboratory to determine the anti-inflammatory effects of compounds on cytokine release by human keratinocytes and cell lines (e.g. A431). We found, that phorbol 12-myristate 13acetate (PMA)-stimulated IL-6 release by A431 cells provides a very predictive model for compounds effective in inflammatory skin diseases: corticosteroids and cyclosporin A inhibit PMA-stimulated cytokine release in a dose- and potency-dependent way, while cyclo-oxygenase and lipoxygenase inhibitors, known not to be very effective in inflammatory skin diseases, do not affect cytokine release by A431 cells [15,16].
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As a model to investigate immunomodulatory effects of compounds, normal human peripheral blood mononuclear cells (PBMCs) were stimulated with Staphylococcus Enterotoxin B (SEB). In this model the monocytes present the superantigen to the T-cells (both cell types are enriched in the PBMCfraction). This model is dependent on co-stimulatory molecules and cytokines. The Th-1 cytokine IFNg and the Th2 cytokine IL-5 is determined. In this model we found dose- and potency dependent effects of corticosteroids, and also differential effects of PDE-inhibitors on IFNg and IL-5 [17].
2. Materials and methods 2.1. Materials Stock solutions were prepared of Tretinoin (BASF) in dimethyl sulfoxide (5 mg/ml) and Clindamycin (Biochimica Opos) in medium (20 mg/ml). Further dilutions were made in medium. Combinations of tretinoin and clindamycin phosphate were tested in the ratio 1:50, the same ratio that is present in the Velac gel. 2.2. Anti-inflammatory effects Human epidermoid carcinoma A431 cells (ATCC) were cultured in DMEM (Bio Whittaker) with penicillin and streptomycin and 5% fetal calf serum (Gibco). At confluency, they were stimulated in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 1:1 (Biowhittaker) with 10 ng/ml PMA (Sigma) in the presence or absence of test compounds. After 40–42 h, the supernatants were harvested, and the number of cells was determined using an MTT assay to assess possible toxic effects of the compounds. IL-6 levels in the supernatant were determined using ELISAs (CLB, Amsterdam). 2.3. Immunomodulatory effects Human peripheral blood mononuclear cells (containing both monocytes and lymphocytes) were isolated after informed consent of normal volunteers using Histopaque-1077 (Sigma). Cells were incubated
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P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
in DMEM with 10% normal human serum (Bio Whittaker), b-mercapto-ethanol (Merck) and antibiotics (Pen/Strep; BioWhittaker) with 100 ng/ml SEB (Sigma) and 50 ng/ml recombinant human IL-2 (R&D systems) in the presence or absence of test compounds. For the determination of IL-5 release, 20 ng/ml IL-4 was added. After 3 days the number of cells was checked with an MTT assay and IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs (Medgenix 350B10G6 and 67F12A8 for IFNg and Pharmingen TRFK5 and JESI-5A10 for IL-5).
3. Results 3.1. Anti-inflammatory effects Tretinoin (with and without clindamycin) reduced the number of A431 cells at 5 mg/ml. The other treatments did not affect the total number of cells. As can be seen in Fig. 1, at the lowest concentration tested (0.1 mg/ml) tretinoin greatly reduced IL-6 release by A431 cells. The addition of clindamycin did not influence this anti-inflammatory effect of tretinoin. 3.2. Immuno-modulatory effects As shown in Fig. 2, PBMC from different donors produced very different levels of IFNg and IL-5; tre-
tinoin reproducibly reduced increased IL-5 release in this did not influence the tretinoin IL-5 release by SEB stimulated
IFNg release and model. Clindamycin effect on IFNg and PBMCs (see Fig. 3).
4. Discussion Interleukin 6 is a pleiotropic cytokine with numerous biological activities (for a review see [18]). It is produced by normal constituents of the skin, including keratinocytes, fibroblasts and dermal endothelial cells. It is also synthesized by inflammatory cells infiltrating the skin in various pathological conditions. IL6 plays a crucial role in the pathogenesis of both local and systemic inflammation, tumor development and auto-immune diseases. In synergy with IL-1, IL-6 represents an essential factor that controls the initial steps in peripheral T cell activation and proliferation. It also, by an autocrine mechanism stimulates keratinocyte proliferation. The inhibition by retinoic acid of cytokine release by keratinocytes, could explain its anti-inflammatory effects in cutaneous pathology. The inhibition of cytokine release from activated human keratinocytes by retinoic acid could affect chemoattraction of haematopoietic inflammatory cells, their recruitment into skin lesions, and accumulation of their pro-inflammatory mediators. Furthermore, an inhibition of IL-6 release could inhibit the hyperproliferation of kerati-
Fig. 1. Effects of tretinoin, clindamycin, and the combination on IL-6 release by PMA-stimulated human epidermoid carcinoma cells A431. At confluency, A431 cells were stimulated in the absence of serum with 10 ng/ml PMA, in the presence or absence of different concentrations of tretinoin, clindamycin or the combination in a ratio 1:50. After 40–42 h supernatants were harvested, and IL-6 levels were determined using an ELISA.
P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
nocytes, thus adding to the direct effect of tretinoin as a comedolytic agent. We investigated the immuno-modulatory effects of tretinoin on SEB-activated PBMCs. In this model, lymphocytes are activated by the superantigen SEB, presented by monocytes [17]. A decrease in IFNg release, and a concomitant increase in IL-5 release strongly suggests a switch towards a Th2-dominated response after tretinoin treatment. Two lines of evi-
S5
dence exist in the literature giving evidence for the influence of tretinoin on Th1/Th2 balance. Firstly, increased IFNg levels, and as a consequence reduced antibody responses were reported in vitamin A deficient mice ([9,19], see [20] for review). The second evidence can be found in the beneficial effects of tretinoin in experimental allergic encephalomyelitis, even when administered after disease onset [10]. This effect is mediated by the development of T cells of the
Fig. 2. Effects of tretinoin on release of (A) IL-5 and (B) IFNg by PBMC from different normal human volunteers. Human peripheral blood mononuclear cells were isolated. Cells were stimulated with 100 ng/ml SEB and 50 ng/ml recombinant human IL-2 in the presence or absence of 10 − 7 M tretinoin. For the determination of IL-5 release also 20 ng/ml IL-4 was added. After 3 days IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs.
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P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
Fig. 3. Effects of tretinoin, clindamycin and the combination on release of (A) IL-5 and (B) IFNg by normal human PBMC. Human peripheral blood mononuclear cells were stimulated with 100 ng/ml SEB and 50 ng/ml recombinant human IL-2 in the presence or absence of test compounds. For the determination of IL-5 release also 20 ng/ml IL-4 was added. After 3 days IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs.
Th2 phenotype, which, in turn, might be responsible for the decrease in the encephalitogenicity of allergenspecific T cells. We found effects of tretinoin in the SEB-PBMCmodel, while we did not find tretinoin effects on pure lymphocyte cytokine release. This is in accordance with the conclusions of Dupuy et al. [6], that retinoids primarily affect antigen presenting cells and Cantorna et al. [21], that the tretinoin induced down-regulation of IFNg synthesis depends on the CD28 co-stimulatory pathway. In conclusion, we found evidence of anti-inflammatory and immuno-modulatory effects of tretinoin. Furthermore, the addition of clindamycin phosphate did not interfere with the tretinoin effectiveness. In addition we also found no effects of tretinoin addition on the anti-P. acnes effects of clindamycin (Yamanou-
chi Europe, data on file). Because clindamycin and tretinoin have very different mechanisms of action in acne, and because they do not negatively influence each others activity, the combination of both compounds in one preparation is expected to add to the anti-acne effectiveness of the product.
References [1] Ebling FJG, Cunliffe WJ. Disorders of the sebaceous glands. In: Champion RH, Burton JL, Ebling FJG, editors. Textbook of Dermatology. Oxford: Blackwell, 1993, pp. 1699–1744. [2] Wilcox HE, Ashbee HR, Cunliffe WJ, Ingham E. Characterisation of the human peripheral blood T-cell response to P. acnes. Sebaceous gland, acne and related disorders. Basic and Clinical Research, Clinical Entities and Treatment. Berlin, 11–13th April, 1997, Abstract 68.
P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7 [3] Romagnani S. Human Th1 and Th2: doubt no more, Immunol Today 1991;12:256–257. [4] Holland DB, Roberts SG, Cunliffe WJ. Localisation of keratin 17 mRNA in acne. Sebaceous gland, acne and related disorders. Basic and Clinical Research, Clinical Entities and Treatment. Berlin, 11–13th April, 1997, Abstract 75. [5] Webster GF. Is acne a hypersensitivity disease? Sebaceous gland, acne and related disorders. Basic and Clinical Research, Clinical Entities and Treatment. Berlin, 11–13th April, 1997, Abstract 59. [6] Dupuy P, Bagot M, Heslan M, Dubertret L. Synthetic retinoids inhibit the antigen presenting properties of epidermal cells in vitro, J Invest Dermatol 1989;93:455–459. [7] Jiang X, Dillehay DL, Everson MP, Tilden AB, Lamon EW. Potentiation of IL-2 induced T-cell proliferation by retinoids, Int J Immunopharmacol 1992;14:195–204. [8] Meunier L, Bohjanen K, Voorhees JJ, Cooper KD Retinoic acid upregulates human Langerhans cell antigen presentation and surface expression of HLA-DR and CD 11c, a b2 integrin critically involved in T-cell activation, J Invest Dermatol 1994;103:775–779. [9] Cantorna MT. In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function, J Immunol 1994;152:1515–1522. [10] Racke M. Retinoid treatment of experimental allergic encephalomyelitis: IL-4 production correlates with improved disease course, J Immunol 1995;54:450–458. [11] Fisher GJ, Esman J, Griffiths CEM, Talwar HS, Duell EA, Hammerberg C, Elder JT, Karabin GD, Nickoloff BJ, Cooper KD, Voorhees JJ. Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin, J Invest Dermatol 1991;96:699–707. [12] Gille J, Paxton LLL, Lawley TJ, Caugham SW, Swerlick RA.
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
[21]
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Retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells, J Clin Invest 1997;99:492–500. Gruaz D, Didierjean L, Grassi J, Frobert Y, Dayer JM, Saurat JH Interleukin 1 alpha and beta in psoriatic skin: enzymoimmunoassay, immunoblot studies and effect of systemic retinoids, Dermatologica 1989;179:202–206. Chatellard-Gruaz D, Didierjean L, Gumowski-Sunek D, Saurat JH. Effect of topical retinoic acid on the interleukin 1a and b immunoreactive pool in normal human epidermis, Br J Dermatol 1990;123:283–289. Wauben-Penris PJJ, den Brok JHAM, Leuven PJMJ. Human keratinocyte cultures for in vitro testing of possible antiinflammatory drugs, 3rd Congress of the EADV. 1993;B017:338. Wauben-Penris PJJ, den Brok JHAM, Leuven PJMJ. Keratinocyte cultures for in vitro testing of possible anti-inflammatory drugs. Dermatology 2000 1993;774:285. Wauben-Penris PJJ, den Brok JHAM, Cerneus DP. Immunomodulatory effects of tretinoin: promotion of Th2 responses, Immunol Lett 1997;56:170–171. Paquet P, Pierard GE Interleukin-6 and the skin, Int Arch Allergy Immunol 1996;109(4):308–317. Cantorna MT, Nashold FE, Hayes CE. Vitamin A deficiency results in a priming environment conductive for TH1 cell development, Eur J Immunol 1995;25:1673–1679. Hayes CE, Nashold F, Chun TY, Catorna M. Vitamin A: regulator of immune function. In: Livrea MA, Vidali G, editors. Retinoids: From Basic Science To Clinical Applications. Basel: Birkhauser, 1994:215–229. Cantorna MT, Nashold FE, Chun TY, Hayes CE. Vitamin A down-regulation of IFNg synthesis in cloned mouse Th1 lymphocytes depends on the CD 28 costimulatory pathway, J Immunol 1996;156:2674–2679.
Immunomodulatory effects of tretinoin in combination with clindamycin Paulette J.J. Wauben-Penris*, Dirk P. Cerneus, Wil E. van den Hoven, Peter J.M.J. Leuven, Jan H.A.M. den Brok, David W.R. Hall Yamanouchi Europe B.V. Research Laboratories, P.O. Box 108, 2350 AC Leiderdorp, The Netherlands
Abstract Background Since acne is a multifactorial skin disease, therapies affecting several etiologic factors can have a higher than expected effectiveness. A combination of the antibiotic clindamycin phosphate and the retinoic acid tretinoin was developed. Objective Anti-inflammatory and immunomodulatory effects of tretinoin in vitro were studied on human keratinocytes and peripheral blood mononuclear cells (PBMCs). Effects of clindamycin phosphate on tretinoin effects were studied. Methods Anti-inflammatory effects on keratinocytes were assessed using an in vitro model with PMA (phorbol ester)stimulated A431 cells (human epidermoid carcinoma). Immunomodulatory effects were measured on superantigen (SEB) stimulated PBMCs. Results Tretinoin showed very potent inhibition of PMA-stimulated IL-6 (interleukin 6) release by A431 cells. The addition of clindamycin phosphate did not interfere with this effect. Tretinoin very potently stimulated IL-5 release, and inhibited IFNg release by SEB-stimulated human PBMCs. This indicates an immunomodulatory effect, stimulating Th2, and inhibiting Th1 dominated responses. These features have been related to the healing of acne lesions. The addition of clindamycin phosphate did not interfere with the immunomodulatory effects of tretinoin. Conclusion The combination of tretinoin and clindamycin phosphate can be expected to be very effective in acne therapy. 1998 Elsevier Science B.V. All rights reserved Keywords: Acne vulgaris; Tretinoin; Immunology
1. Introduction Acne vulgaris is a very common skin disorder, affecting up to 40% of the teenage population [1]. It is characterized by hypercornification and comedone formation, increased sebum production, infection with Propionibacterium acnes, release of inflammatory mediators and infiltration with leukocytes. The cellular infiltrate in the acne lesions consists mainly of CD4 + T-helper lymphocytes [2]. * Corresponding author. Tel.: +31 71 5455533; fax: +31 71 5455265; e-mail: [email protected]
T-Helper cells can be divided in two subtypes based on their cytokine profiles [3]: Th1 cells primarily produce IL-2 and IFNg, and are involved in the cellular immune response and in delayed type hypersensitivity responses. Th2 cells primarily produce IL-4, IL-5 and IL-10, and play a role in B-cell help for humoral immune responses. They are involved in allergic reactions. Various diseases have been associated with either a predominant Th1 (e.g. psoriasis) or Th2 (e.g. atopic dermatitis) response [3]. Recently it has been postulated, that Th1 responses are involved in the etiology of acne, while Th2 responses could help in the healing of acne lesions
0926-9959/98/$- see front matter 1998 Elsevier Science B.V. All rights reserved PII S0926-9959 (98 )0 0087-7
P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
[4]. Certain P. acnes polypeptides were found to act as an antigen in acne patients, but not in normal humans [5]. Although there are many reasonably effective topical acne therapies available, there is a need for preparations that act on more than one factor involved in acne, e.g. an antibiotic combined with tretinoin. Clindamycin is an antibiotic, effective against P. acnes, but not widely used systemically. Tretinoin was originally developed for its beneficial effect in acne, but has also been used in a vast range of other skin diseases like lentigines, keloidal scarring, striae, dysplastic nevi, flat warts, post-inflammatory hyperpigmentation and skin aging. The effectiveness of tretinoin in acne consists of several aspects: tretinoin is known to act as a comedolytic agent, and to decrease the cornification process. Furthermore, tretinoin has anti-inflammatory and immunomodulatory effects and has recently been recognized to have immunosuppressive [6], immuno-stimulatory [7,8], immunomodulatory [9,10], pro-inflammatory [11] and antiinflammatory [12] effects. During tretinoin therapy, the amounts of the pro-inflammatory cytokine IL-1 decrease in lesional and non-lesional psoriatic skin, and remain unchanged in normal skin [13,14]. This indicates that tretinoin appears to have anti-inflammatory effects in inflamed skin, but does not affect the normal immune function of the skin. The aim of these experiments was to investigate the anti-inflammatory and immuno-modulatory effects of tretinoin, and to investigate whether the addition of clindamycin to tretinoin influences the tretinoin effects. The combination of clindamycin and tretinoin could be expected to be very effective for treatment of acne. We investigated whether an inhibition of tretinoin effects by clindamycin could be excluded. Assays were developed in our laboratory to determine the anti-inflammatory effects of compounds on cytokine release by human keratinocytes and cell lines (e.g. A431). We found, that phorbol 12-myristate 13acetate (PMA)-stimulated IL-6 release by A431 cells provides a very predictive model for compounds effective in inflammatory skin diseases: corticosteroids and cyclosporin A inhibit PMA-stimulated cytokine release in a dose- and potency-dependent way, while cyclo-oxygenase and lipoxygenase inhibitors, known not to be very effective in inflammatory skin diseases, do not affect cytokine release by A431 cells [15,16].
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As a model to investigate immunomodulatory effects of compounds, normal human peripheral blood mononuclear cells (PBMCs) were stimulated with Staphylococcus Enterotoxin B (SEB). In this model the monocytes present the superantigen to the T-cells (both cell types are enriched in the PBMCfraction). This model is dependent on co-stimulatory molecules and cytokines. The Th-1 cytokine IFNg and the Th2 cytokine IL-5 is determined. In this model we found dose- and potency dependent effects of corticosteroids, and also differential effects of PDE-inhibitors on IFNg and IL-5 [17].
2. Materials and methods 2.1. Materials Stock solutions were prepared of Tretinoin (BASF) in dimethyl sulfoxide (5 mg/ml) and Clindamycin (Biochimica Opos) in medium (20 mg/ml). Further dilutions were made in medium. Combinations of tretinoin and clindamycin phosphate were tested in the ratio 1:50, the same ratio that is present in the Velac gel. 2.2. Anti-inflammatory effects Human epidermoid carcinoma A431 cells (ATCC) were cultured in DMEM (Bio Whittaker) with penicillin and streptomycin and 5% fetal calf serum (Gibco). At confluency, they were stimulated in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 1:1 (Biowhittaker) with 10 ng/ml PMA (Sigma) in the presence or absence of test compounds. After 40–42 h, the supernatants were harvested, and the number of cells was determined using an MTT assay to assess possible toxic effects of the compounds. IL-6 levels in the supernatant were determined using ELISAs (CLB, Amsterdam). 2.3. Immunomodulatory effects Human peripheral blood mononuclear cells (containing both monocytes and lymphocytes) were isolated after informed consent of normal volunteers using Histopaque-1077 (Sigma). Cells were incubated
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P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
in DMEM with 10% normal human serum (Bio Whittaker), b-mercapto-ethanol (Merck) and antibiotics (Pen/Strep; BioWhittaker) with 100 ng/ml SEB (Sigma) and 50 ng/ml recombinant human IL-2 (R&D systems) in the presence or absence of test compounds. For the determination of IL-5 release, 20 ng/ml IL-4 was added. After 3 days the number of cells was checked with an MTT assay and IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs (Medgenix 350B10G6 and 67F12A8 for IFNg and Pharmingen TRFK5 and JESI-5A10 for IL-5).
3. Results 3.1. Anti-inflammatory effects Tretinoin (with and without clindamycin) reduced the number of A431 cells at 5 mg/ml. The other treatments did not affect the total number of cells. As can be seen in Fig. 1, at the lowest concentration tested (0.1 mg/ml) tretinoin greatly reduced IL-6 release by A431 cells. The addition of clindamycin did not influence this anti-inflammatory effect of tretinoin. 3.2. Immuno-modulatory effects As shown in Fig. 2, PBMC from different donors produced very different levels of IFNg and IL-5; tre-
tinoin reproducibly reduced increased IL-5 release in this did not influence the tretinoin IL-5 release by SEB stimulated
IFNg release and model. Clindamycin effect on IFNg and PBMCs (see Fig. 3).
4. Discussion Interleukin 6 is a pleiotropic cytokine with numerous biological activities (for a review see [18]). It is produced by normal constituents of the skin, including keratinocytes, fibroblasts and dermal endothelial cells. It is also synthesized by inflammatory cells infiltrating the skin in various pathological conditions. IL6 plays a crucial role in the pathogenesis of both local and systemic inflammation, tumor development and auto-immune diseases. In synergy with IL-1, IL-6 represents an essential factor that controls the initial steps in peripheral T cell activation and proliferation. It also, by an autocrine mechanism stimulates keratinocyte proliferation. The inhibition by retinoic acid of cytokine release by keratinocytes, could explain its anti-inflammatory effects in cutaneous pathology. The inhibition of cytokine release from activated human keratinocytes by retinoic acid could affect chemoattraction of haematopoietic inflammatory cells, their recruitment into skin lesions, and accumulation of their pro-inflammatory mediators. Furthermore, an inhibition of IL-6 release could inhibit the hyperproliferation of kerati-
Fig. 1. Effects of tretinoin, clindamycin, and the combination on IL-6 release by PMA-stimulated human epidermoid carcinoma cells A431. At confluency, A431 cells were stimulated in the absence of serum with 10 ng/ml PMA, in the presence or absence of different concentrations of tretinoin, clindamycin or the combination in a ratio 1:50. After 40–42 h supernatants were harvested, and IL-6 levels were determined using an ELISA.
P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
nocytes, thus adding to the direct effect of tretinoin as a comedolytic agent. We investigated the immuno-modulatory effects of tretinoin on SEB-activated PBMCs. In this model, lymphocytes are activated by the superantigen SEB, presented by monocytes [17]. A decrease in IFNg release, and a concomitant increase in IL-5 release strongly suggests a switch towards a Th2-dominated response after tretinoin treatment. Two lines of evi-
S5
dence exist in the literature giving evidence for the influence of tretinoin on Th1/Th2 balance. Firstly, increased IFNg levels, and as a consequence reduced antibody responses were reported in vitamin A deficient mice ([9,19], see [20] for review). The second evidence can be found in the beneficial effects of tretinoin in experimental allergic encephalomyelitis, even when administered after disease onset [10]. This effect is mediated by the development of T cells of the
Fig. 2. Effects of tretinoin on release of (A) IL-5 and (B) IFNg by PBMC from different normal human volunteers. Human peripheral blood mononuclear cells were isolated. Cells were stimulated with 100 ng/ml SEB and 50 ng/ml recombinant human IL-2 in the presence or absence of 10 − 7 M tretinoin. For the determination of IL-5 release also 20 ng/ml IL-4 was added. After 3 days IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs.
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P.J.J. Wauben-Penris et al. / J. Eur. Acad. Dermatol. Venereol. 11 (Suppl. 1) (1998) S2–S7
Fig. 3. Effects of tretinoin, clindamycin and the combination on release of (A) IL-5 and (B) IFNg by normal human PBMC. Human peripheral blood mononuclear cells were stimulated with 100 ng/ml SEB and 50 ng/ml recombinant human IL-2 in the presence or absence of test compounds. For the determination of IL-5 release also 20 ng/ml IL-4 was added. After 3 days IFNg and IL-5 levels in the supernatants were determined using sandwich ELISAs.
Th2 phenotype, which, in turn, might be responsible for the decrease in the encephalitogenicity of allergenspecific T cells. We found effects of tretinoin in the SEB-PBMCmodel, while we did not find tretinoin effects on pure lymphocyte cytokine release. This is in accordance with the conclusions of Dupuy et al. [6], that retinoids primarily affect antigen presenting cells and Cantorna et al. [21], that the tretinoin induced down-regulation of IFNg synthesis depends on the CD28 co-stimulatory pathway. In conclusion, we found evidence of anti-inflammatory and immuno-modulatory effects of tretinoin. Furthermore, the addition of clindamycin phosphate did not interfere with the tretinoin effectiveness. In addition we also found no effects of tretinoin addition on the anti-P. acnes effects of clindamycin (Yamanou-
chi Europe, data on file). Because clindamycin and tretinoin have very different mechanisms of action in acne, and because they do not negatively influence each others activity, the combination of both compounds in one preparation is expected to add to the anti-acne effectiveness of the product.
References [1] Ebling FJG, Cunliffe WJ. Disorders of the sebaceous glands. In: Champion RH, Burton JL, Ebling FJG, editors. Textbook of Dermatology. Oxford: Blackwell, 1993, pp. 1699–1744. [2] Wilcox HE, Ashbee HR, Cunliffe WJ, Ingham E. Characterisation of the human peripheral blood T-cell response to P. acnes. Sebaceous gland, acne and related disorders. Basic and Clinical Research, Clinical Entities and Treatment. Berlin, 11–13th April, 1997, Abstract 68.
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