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Both Pimecrolimus and Corticosteroids Deplete Plasmacytoid Dendritic Cells in Patients with Atopic Dermatitis

2006; Elsevier BV; Volume: 126; Issue: 9 Linguagem: Inglês

10.1038/sj.jid.5700368

1523-1747

Wolfram Hoetzenecker, Simone Meindl, Anton Stuetz, Georg Stingl, Adelheid Elbe‐Bürger,

Food Allergy and Anaphylaxis Research

atopic dermatitis beta-methasone-17-valerate corticosteroids dendritic cell mean fluorescence intensity plasmacytoid DC Atopic dermatitis (AD) is one of the most common inflammatory skin disorders characterized by pruritus, a chronically relapsing course and typically distributed lesions with dry skin, excoriations, and lichenification (Leung et al., 2004Leung D.Y. Boguniewicz M. Howell M.D. Nomura I. Hamid Q.A. New insights into atopic dermatitis.J Clin Invest. 2004; 113: 651-657Crossref PubMed Scopus (1189) Google Scholar). Topical treatment of acute lesions with corticosteroids (CS) is a mainstay in the therapy of the AD; however, long-term application is limited by CS-related side effects (e.g., skin atrophy) (Stoppolino et al., 1983Stoppolino G. Prisco F. Santinelli R. Sicuranza G. Giordano C. Potential hazards of topical steroid therapy.Am J Dis Child. 1983; 137: 1130-1131Google Scholar). The calcineurin inhibitors tacrolimus and pimecrolimus are now available as ointment/cream preparations and have proven to be a novel option for the topical treatment of AD (Ruzicka et al., 1997Ruzicka T. Bieber T. Schöpf E. et al.A short-term trial of tacrolimus ointment for atopic dermatitis. European Tacrolimus Multicenter Atopic Dermatitis Study Group.N Engl J Med. 1997; 337: 816-821Crossref PubMed Scopus (510) Google Scholar; Luger et al., 2001Luger T. van Leent E.J.M. Graeber M. Hedgecock S. Thurston M. Kandra A. et al.SDZ ASM 981: an emerging safe and effective treatment for atopic dermatitis.Br J Dermatol. 2001; 144: 788-794Crossref PubMed Scopus (225) Google Scholar). AD skin lesions harbor a variety of inflammatory cells, of which dendritic cells (DCs) represent a major fraction. Besides resident Langerhans cells and inflammatory dendritic epidermal cells, the presence of plasmacytoid DC (pDC) has recently been demonstrated in the dermis and less abundant in the epidermis of AD lesions (Wollenberg et al., 2002Wollenberg A. Wagner M. Günther S. Towarowski A. Tuma E. Moderer M. et al.Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases.J Invest Dermatol. 2002; 119: 1096-1102Crossref PubMed Scopus (375) Google Scholar; Hashizume et al., 2005Hashizume H. Horibe T. Yagi H. Seo N. Takigawa M. Compartmental imbalance and aberrant immune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid) dendritic cells in atopic dermatitis.J Immunol. 2005; 174: 2396-2403Crossref PubMed Scopus (43) Google Scholar). pDC are characterized by a plasma-cell-like morphology, a unique surface phenotype (HLA-DR+CD1a−CD123+BDCA-2+FcεRI+), and the ability to produce large amounts of IFN-α, which assigns them a key role in innate antiviral immunity (Cella et al., 1999Cella M. Jarrossay D. Facchetti F. Alebardi O. Nakajima H. Lanzavecchia A. et al.Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon.Nat Med. 1999; 5: 919-923Crossref PubMed Scopus (1415) Google Scholar; Siegal et al., 1999Siegal F.P. Kadowaki N. Shodell M. Fitzgerald-Bocarsly P.A. Shah K. Ho S. et al.The nature of the principal type 1 interferon-producing cells in human blood.Science. 1999; 284: 1835-1837Crossref PubMed Scopus (1889) Google Scholar). In prior studies, we have investigated the impact and mode of action of CS and pimecrolimus on the DC population in murine and healthy human and lesional AD skin and have reported divergent effects of these compounds on Langerhans cell viability and function (Hoetzenecker et al., 2004Hoetzenecker W. Meingassner J.G. Ecker R. Stingl G. Stuetz A. Elbe-Bürger A. Corticosteroids but not pimecrolimus affect viability, maturation and immune function of murine epidermal Langerhans cells.J Invest Dermatol. 2004; 122: 673-684Crossref PubMed Scopus (107) Google Scholar, Hoetzenecker et al., 2005Hoetzenecker W. Ecker R. Kopp T. Stuetz A. Stingl G. Elbe-Bürger A. Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis.J Allergy Clin Immunol. 2005; 115: 1276-1283Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In light of this finding, we asked whether these compounds also affect pDC. For this purpose, skin biopsies from a previous clinical trial were utilized (Hoetzenecker et al., 2005Hoetzenecker W. Ecker R. Kopp T. Stuetz A. Stingl G. Elbe-Bürger A. Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis.J Allergy Clin Immunol. 2005; 115: 1276-1283Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Briefly, 22 patients suffering from AD according to the diagnostic criteria of Hanifin and Rajka (Hanifin and Rajka, 1980Hanifin J.M. Rajka G. Diagnostic features of atopic dermatitis.Acta Derm Venereol Suppl (Stockh). 1980; 92: 44-47Google Scholar; Rajka and Langeland, 1989Rajka G. Langeland T. Grading of the severity of atopic dermatitis.Acta Derm Venereol Suppl (Stockh). 1989; 144: 13-14PubMed Google Scholar) were assigned to treatment with 1% pimecrolimus cream (eight patients), matching vehicle cream (six patients), or 0.1% beta-methasone-17-valerate ((BMV), eight patients) in a 3-week, randomized, double-blind, vehicle-controlled parallel-group clinical trial. Creams were applied twice daily to all affected areas of the skin (except face) and punch biopsies (4 mm) were taken from acute lesions before treatment (day 0; begin of study) and on day 21 (end of study) after initiation of therapy. The study was conducted according to the guidelines of the Declaration of Helsinki Principles, approved by the local ethics committee of the Medical University of Vienna (code numbers: EK303/2003 and EK355/2005) and a written informed consent was obtained from all patients. Immunoperoxidase staining against the pDC-specific marker BDCA-2 revealed substantial pDC numbers in the dermis of untreated AD skin lesions (33±8 cells/mm2 dermis; n=22), whereas pDC in normal healthy control skin were essentially absent (data not shown; n=5). As shown in Figure 1, topical treatment with pimecrolimus and BMV for 21 days caused an almost complete disappearance of skin-infiltrating BDCA-2+ cells by 94 and 95%, respectively, in comparison to vehicle-treated skin. The disappearance of BDCA-2+ cells can be due to either the loss/downregulation of this marker on the responder cell or due to actual deletion/destruction of pDC. In order to address this issue, pDC were isolated from peripheral blood of healthy adult volunteers (Figure 2a) and cultured with pimecrolimus and BMV at a concentration of 10−6 mol/l (Figure 2b). We have chosen this concentration intending to use the highest, non-toxic, biologically relevant dose for pimecrolimus to see an effect (Grassberger et al., 1999Grassberger M. Baumruker T. Enz A. Hiestand P. Hultsch T. Kalthoff F. et al.A novel anti-inflammatory drug, SDZ ASM 981, for the treatment of skin diseases: in vitro pharmacology.Br J Dermatol. 1999; 141: 264-273Crossref PubMed Scopus (240) Google Scholar; Kalthoff et al., 2003Kalthoff E.S. Chung J. Musser P. Stuetz A. Pimecrolimus does not affect the differentiation, maturation and function of human monocyte-derived dendritic cells, in contrast to corticosteroids.Clin Exp Immunol. 2003; 133: 350-359Crossref PubMed Scopus (54) Google Scholar). At selected time points, cells were analyzed for marker expression and viability. Neither pimecrolimus nor BMV downmodulated the expression of BDCA-2 on pDC at any time point investigated (Figure 2b). However, we observed a marked reduction of BDCA-2+ cells on BMV (92%), but not pimecrolimus (24%) culture already after 2 days when compared to vehicle (21%) (Figure 2c). After 7 days, pimecrolimus did not alter the viability of pDC when compared to vehicle (Figure 2c). In contrast, pDC on BMV culture were all dead at this time point (not shown). From these data, we conclude that the disappearance of BDCA-2+ cells in AD lesions upon CS treatment most likely results from a depletion of pDC. The mechanism responsible for their depletion in vivo remains to be investigated. In keeping with recent reports from others (Shodell et al., 2003Shodell M. Shah K. Siegal F.P. Circulating human plasmacytoid dendritic cells are highly sensitive to corticosteroid administration.Lupus. 2003; 12: 222-230Crossref PubMed Scopus (81) Google Scholar; Suda et al., 2003Suda T. Chida K. Matsuda H. Hashizume H. Ide K. Yokomura K. et al.High-dose intravenous glucocorticoid therapy abrogates circulating dendritic cells.J Allergy Clin Immunol. 2003; 112: 1237-1239Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; Abe and Thomson, 2006Abe M. Thomson A.W. Dexamethasone preferentially suppresses plasmacytoid dendritic cell differentiation and enhances their apoptotic death.Clin Immunol. 2006; 118: 300-306Crossref PubMed Scopus (42) Google Scholar), our data suggest that the reduction of pDC after topical treatment with CS may be owing to apoptosis. With regard to pimecrolimus, we have no evidence for direct effect on apoptosis in pDC in contrast to CS treatment. Theoretical possibilities are that indirect mechanisms such as cell migration and/or withdrawal of cytokines owing to an apoptosis-induced depletion of T cells upon pimecrolimus treatment (Hoetzenecker et al., 2005Hoetzenecker W. Ecker R. Kopp T. Stuetz A. Stingl G. Elbe-Bürger A. Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis.J Allergy Clin Immunol. 2005; 115: 1276-1283Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) may be responsible for their depletion.Figure 2CS but not pimecrolimus strongly reduce pDC numbers in vitro. Buffy coats from peripheral blood of healthy adult volunteers were purchased from the local transfusion service (Rotes Kreuz, Vienna, Austria). Peripheral blood monocytes were isolated as interface cells after density gradient centrifugation (Lymphoprep; Axis-Shield, Rodelokka, Norway) and erythrocytes were removed with ammonium chloride (0.8% NH4Cl/0.1 mm EDTA). pDC (mean purity: 93.88%±5.21 (n=4)) were isolated using a negative isolation kit according to the manufacturer's manual (Miltenyi Biotec Inc.). (a) Freshly isolated cells were stained for the indicated markers and 30,000 events/sample were acquired. pDC were cultured (5 × 106/ml) for the indicated time periods (day 2: n=3; day 7: n=1) in RPMI 1640 medium (GIBCO Life Technologies, Carlsbad, CA) supplemented with 10% FCS, 2 mm l-glutamine, antibiotics, and 10 ng/ml recombinant human IL-3 (Pepro Tech, Rocky Hill, NJ) in the presence of vehicle, pimecrolimus, or BMV (all 10−6 mol/l). Fluorescence was measured with a FACSCalibur flow cytometer, and data were analyzed using Cell Quest software (both Becton Dickinson, San Jose, CA). (b) Mean fluorescence intensity (MFI) was determined for BDCA-2 expression and (c) dead cells were identified by 7-amino-actinomycin D uptake (Sigma Chemicals Co., St Louis, MO).View Large Image Figure ViewerDownload (PPT) It is well known that patients with AD have recurrent bacterial or viral skin infections. The reason for this particular susceptibility is not entirely understood. Recent evidence exists that these individuals have a defect in the production of natural antimicrobial peptides (Ong et al., 2002Ong P.Y. Ohtake T. Brandt C. Strickland I. Boguniewicz M. Ganz T. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1634) Google Scholar; Rieg et al., 2005Rieg S. Steffen H. Seeber S. Humeny A. Kalbacher H. Dietz K. et al.Deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo.J Immunol. 2005; 174: 8003-8010Crossref PubMed Scopus (230) Google Scholar). Another reason could be a defect in other elements of the innate immune system. Indeed, pDC are less abundant in the infiltrate of AD lesions as compared to allergic contact eczema (Wollenberg et al., 2002Wollenberg A. Wagner M. Günther S. Towarowski A. Tuma E. Moderer M. et al.Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases.J Invest Dermatol. 2002; 119: 1096-1102Crossref PubMed Scopus (375) Google Scholar; Bangert et al., 2003Bangert C. Friedl J. Stary G. Stingl G. Kopp T. Immunopathologic features of allergic contact dermatitis in humans: participation of plasmacytoid dendritic cells in the pathogenesis of the disease?.J Invest Dermatol. 2003; 121: 1409-1418Crossref PubMed Scopus (90) Google Scholar; Stary et al., 2005Stary G. Bangert C. Stingl G. Kopp T. Dendritic cells in atopic dermatitis: expression of FcεRI on two distinct inflammation-associated subsets.Int Arch Allergy Immunol. 2005; 138: 278-290Crossref PubMed Scopus (44) Google Scholar). It will be interesting to see whether a functional defect of pDC may contribute to the high susceptibility of these patients to skin infections. We thank Drs G. Stary and C. Bangert for their kind and helpful advice. Financial support is acknowledged from Novartis Pharma AG, Basel.