Corticosteroids But Not Pimecrolimus Affect Viability, Maturation and Immune Function of Murine Epidermal Langerhans Cells
2004; Elsevier BV; Volume: 122; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.22324.x
ISSN1523-1747
AutoresWolfram Hoetzenecker, Josef G. Meingassner, Rupert Ecker, Georg Stingl, Anton Stuetz, Adelheid Elbe‐Bürger,
Tópico(s)Dermatology and Skin Diseases
ResumoGiven the importance of dendritic cells in the immune response, we investigated the effect of corticosteroids (CS) on the integrity, survival, and function of murine Langerhans cells (LC) in comparison with pimecrolimus, a novel anti-inflammatory drug for the topical treatment of atopic dermatitis. BALB/c mice were treated twice on one day with ethanolic solutions of the compounds. At 24–72 h after the last application, we observed fragmented DNA, caspase-3 activity, and an upregulation of CD95 expression in LC from mice treated with CS but not in LC of pimecrolimus- or vehicle-treated animals. CS-epidermal cell (EC) supernatants but not pimecrolimus-EC supernatants contained significantly lower amounts of soluble factors (GM-CSF, TNF-α, IL-1α) required for LC survival and maturation than EC supernatants from vehicle-treated mice. With regard to LC maturation, CS but not pimecrolimus inhibited the expression of CD25, CD205, and costimulatory molecules. In line with this, LC from pimecrolimus-treated mice were similar to LC from vehicle-treated mice in their capacity to stimulate antigen-presenting function and migration, whereas LC from CS-treated mice were greatly impaired in these abilities. In summary, our data show for the first time that CS but not pimecrolimus induce apoptosis in LC in situ, implying that the prolonged use of CS could have adverse effects on the skin immune system. Given the importance of dendritic cells in the immune response, we investigated the effect of corticosteroids (CS) on the integrity, survival, and function of murine Langerhans cells (LC) in comparison with pimecrolimus, a novel anti-inflammatory drug for the topical treatment of atopic dermatitis. BALB/c mice were treated twice on one day with ethanolic solutions of the compounds. At 24–72 h after the last application, we observed fragmented DNA, caspase-3 activity, and an upregulation of CD95 expression in LC from mice treated with CS but not in LC of pimecrolimus- or vehicle-treated animals. CS-epidermal cell (EC) supernatants but not pimecrolimus-EC supernatants contained significantly lower amounts of soluble factors (GM-CSF, TNF-α, IL-1α) required for LC survival and maturation than EC supernatants from vehicle-treated mice. With regard to LC maturation, CS but not pimecrolimus inhibited the expression of CD25, CD205, and costimulatory molecules. In line with this, LC from pimecrolimus-treated mice were similar to LC from vehicle-treated mice in their capacity to stimulate antigen-presenting function and migration, whereas LC from CS-treated mice were greatly impaired in these abilities. In summary, our data show for the first time that CS but not pimecrolimus induce apoptosis in LC in situ, implying that the prolonged use of CS could have adverse effects on the skin immune system. antigen antigen-presenting cell contact hypersensitivity confocal laser scanning microscope clobetasol propionate corticosteroids dendritic cells epidermal cells hydrocortisone hen egg lysozyme Langerhans cells monoclonal antibody mean fluorescence intensity ovalbumin terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling Corticosteroids (CS) are potent immunomodulatory hormones, which are widely applied in the clinic for their immunosuppressive properties. Their effect is mediated by the cytosolic glucocorticoid receptor which, upon ligand binding, translocates into the nucleus and regulates gene expression (Danielsen et al., 1987Danielsen M. Northrop J.P. Jonklaas J. Ringold G.M. Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement.Mol Endocrinol. 1987; 1: 816-822Crossref PubMed Scopus (127) Google Scholar;Hollenberg et al., 1987Hollenberg S.M. Giguere V. Segui P. Evans R.M. Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.Cell. 1987; 49: 39-46Abstract Full Text PDF PubMed Scopus (326) Google Scholar). Most cells of the immune system are adversely affected by CS (e.g., T cells, B cells, natural killer cells, macrophages/monocytes, and dendritic cells (DC)) (Schwartzman and Cidlowski, 1994Schwartzman R.A. Cidlowski J.A. Glucocorticoid-induced apoptosis of lymphoid cells.Int Arch Allergy Immunol. 1994; 105: 347-354Crossref PubMed Scopus (112) Google Scholar;Moser et al., 1995Moser M. De Smedt T. Sornasse T. et al.Glucocorticoids down-regulate dendritic cell function in vitro and in vivo.Eur J Immunol. 1995; 25: 2818-2824Crossref PubMed Scopus (205) Google Scholar;Schmidt et al., 1999Schmidt M. Pauels H.G. Lugering N. Lugering A. Domschke W. Kucharzik T. Glucocorticoids induce apoptosis in human monocytes: Potential role of IL-1β.J Immunol. 1999; 163: 3484-3490PubMed Google Scholar). In the epidermis, the main targets of CS are thought to be basal keratinocytes and Langerhans cells (LC). This assumption is supported by the fact that human basal keratinocytes and LC strongly express the glucocorticoid receptor (Serres et al., 1996Serres M. Vaic J. Schmitt D. Glucocorticoid receptor localization in human epidermal cells.Arch Dermatol Res. 1996; 288: 140-146Crossref PubMed Scopus (31) Google Scholar). With regard to LC, a decrease of MHC class II+ epidermal LC after topical treatment with CS has been observed by several investigators, but the mechanism underlying this phenomenon has not yet been elucidated. For short treatment periods, oral and topical formulations of CS are usually well tolerated, while long-term application is limited by local and systemic side-effects (e.g., skin atrophy, growth retardation, etc.) (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). As a consequence, many efforts have been made to develop therapeutic alternatives. Recently, a promising class of anti-inflammatory substances (e.g., pimecrolimus (SDZ ASM 981, Elidel), tacrolimus (FK 506, Protopic)) has been identified for the treatment of inflammatory skin diseases (Ruzicka et al., 1997Ruzicka T. Bieber T. Schof 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 (486) Google Scholar;Van Leent et al., 1998Van Leent E.J.M. Greber M. Thurston M. Wagenaar A. Spuls P.I. Bos J.D. Effectiveness of the ascomycin macrolactam SDZ ASM 981 in the topical treatment of atopic dermatitis.Arch Dermatol. 1998; 134: 805-809Crossref PubMed Scopus (204) Google Scholar;Luger et al., 2001Luger T. van Leent E.J.M. Graeber M. et al.SDZ ASM 981: An emerging safe and effective treatment for atopic dermatitis.Br J Dermatol. 2001; 144: 788-794Crossref PubMed Scopus (220) Google Scholar). Unlike CS, these compounds have no atrophogenic potential (Meingassner et al., 1997Meingassner J.G. Grassberger M. Fahrngruber H. Moore H.D. Schuurman H. Stütz A. A novel anti-inflammatory drug, SDZ ASM 981, for the topical and oral treatment of skin diseases: In vivo pharmacology.Br J Dermatol. 1997; 137: 568-576Crossref PubMed Scopus (178) Google Scholar;Reitamo et al., 1998Reitamo S. Rissanen J. Remitz A. et al.Tacrolimus ointment does not affect collagen synthesis: Results of a single-center randomized trial.J Invest Dermatol. 1998; 111: 396-398Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar;Queille-Roussel et al., 2001Queille-Roussel C. Paul C. Duteil L. Lefebvre M.C. Rapatz G. Zagula M. Ortonne J.P. The new topical ascomycin derivative SDZ ASM 981 does not induce skin atrophy when applied to normal skin for 4 weeks: A randomized, double-blind controlled study.Br J Dermatol. 2001; 144: 507-513Crossref PubMed Scopus (198) Google Scholar). Pimecrolimus and tacrolimus have a similar mode of action, which is mediated mainly by specific intracellular ligands, the immunophilins, among which macrophilin-12 binds to both compounds. The macrophilin-12 complex inhibits calcineurin and causes a downregulation of important T cell-derived cytokines (e.g., IL-2, IL-4, IL-5) (reviewed inNghiem et al., 2002Nghiem P. Pearson G. Langley R.G. Tacrolimus and pimecrolimus: From clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis.J Am Acad Dermatol. 2002; 46: 228-241Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) (Grassberger et al., 1999Grassberger M. Baumruker T. Enz A. 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 (236) Google Scholar). While one can safely assume that pimecrolimus mainly acts at the level of T cells, other immunocompetent cells in the skin could also be relevant targets. Because the exact mode of action in the skin is not known for CS or pimecrolimus, we explored in detail the effects of CS on the immune functions of resident skin cells with special emphasis on LC in vivo, and compared these effects with those of pimecrolimus. We found that topical CS induce apoptosis of LC in vivo and greatly impair the maturation and antigen-presenting function of LC. In contrast, topical application of pimecrolimus did not affect viability, maturation and migration, and had no gross effects on immune functions of LC. As shown in Figure 1a and b, murine LC and keratinocytes exhibit anti-macrophilin-12 reactivity. Only a subpopulation of LC was strongly macrophilin-12 positive, whereas others expressed only moderate levels or were negative, suggesting that the epidermis contains LC with a different differentiation status. When epidermal cells (EC) were kept in culture however, macrophilin-12 expression was highly upregulated on all LC Figure 1c. This is in contrast to findings in human LC, where macrophilin-12 expression disappears during maturation (Panhans-Gross et al., 2001Panhans-Gross A. Novak N. Kraft S. Bieber T. Human epidermal Langerhans cells are targets for the immunosuppressive macrolide tacrolimus (FK506).J Allergy Clin Immunol. 2001; 107: 345-352Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The reason for this discrepancy is currently unclear. To investigate whether topically applied pimecrolimus alters macrophilin-12 expression in LC, EC suspensions were prepared at selected time points after treatment, permeabilized, stained with anti-MHC class II and anti-macrophilin-12, and analyzed by flow cytometry. After 24 h, macrophilin-12 staining in LC was reduced by ≥60% Figure 2, implying that pimecrolimus binds to macrophilin-12 in LC.Figure 2Topical treatment with pimecrolimus alters macrophilin-12 expression in LC. Mice were treated topically with pimecrolimus, and 24 and 48 h later, macrophilin-12 expression in freshly isolated LC was analyzed by flow cytometry as described in Figure 1. Dotted lines in histograms represent isotype controls. Results are representative of two independent experiments.View Large Image Figure ViewerDownload (PPT) The pharmacodynamic effect of pimecrolimus under our present treatment conditions was tested in a CHS model. In two independent experiments we found that 1% pimecrolimus, applied twice on 1 d, inhibited inflammatory ear swelling by 40% Table I.Table IPimecrolimus markedly inhibits CHS responsesaCHS in mice was elicited as described in Material and Methods. 0.5 and 8.5 h after challenge, mice (eight mice per group) were topically treated with 1% pimecrolimus or vehicle alone. Anti-inflammatory activity was calculated as the percentage inhibition of auricular weight increase (as a measure of inflammation) relative to mice treated with vehicle alone.ExperimentsGroupsDifferences in auricular weights (mg) mean±SD% inhibition mean±SEMIPimecrolimus14.8±4.539±8.4**p<0.01 versus controls.Control24.0±5.6—IIPimecrolimus16.0±2.841±4.6**p<0.01 versus controls.Control26.9±2.8—a CHS in mice was elicited as described in Material and Methods. 0.5 and 8.5 h after challenge, mice (eight mice per group) were topically treated with 1% pimecrolimus or vehicle alone. Anti-inflammatory activity was calculated as the percentage inhibition of auricular weight increase (as a measure of inflammation) relative to mice treated with vehicle alone.** p<0.01 versus controls. Open table in a new tab Contact hypersensitivity (CS) exert their biological activity by binding to their receptor (Reichardt et al., 2000Reichardt H.M. Tronche F. Berger S. Kellendonk C. Schutz G. New insights into glucocorticoid and mineralcorticoid signaling: Lessons from gene targeting.Adv Pharmacol. 2000; 47: 1-21Crossref PubMed Scopus (55) Google Scholar). FACS analysis revealed that approximately a quarter of the freshly isolated LC population strongly expresses the receptor (Figure 3a, left panel), suggesting that LC are heterogeneous in terms of sensitivity to CS. Furthermore, we found that one-third of MHC class II- EC, presumably keratinocytes, express the glucocorticoid receptor (Figure 3a, right panel). To determine the effects of pimecrolimus and CS on the fate and phenotype of EC, single-cell suspensions from treated mice were prepared and analyzed at selected time points. While the percentage of dead EC was essentially equal in vehicle (5%)- and pimecrolimus (6%)-treated skin areas, the percentage of dead EC was significantly increased (p<0.001) up to 13% and 14% in hydrocostisone (HC) and clobetasol propionate (CL)-treated mice, respectively. As shown in Figure 3b, neither CS nor pimecrolimus had changed the numbers and MHC class II expression levels of LC when assessed at and compared with controls 24 h after cessation of treatment. Two days later, however, treatment with CS but not with pimecrolimus had resulted in a decrease of MHC class II+, CD24+, CD32+, CD11b+, and F4/80+ cells (Figure 3b, supplementary data (Fig S3C) online). Furthermore, mean fluorescence intensity (MFI) of surface MHC class II molecules of LC was reduced Figure 3b, while the expression intensity of the other markers was not affected on the remaining cells. Effects on LC phenotype were also not seen when mice were treated with pimecrolimus twice/day for 5 consecutive days (supplementary data (Fig S3D, E) online). To determine whether the CS-induced numerical reduction of LC is attributable to an altered migration pattern of LC, we used the skin explant culture model described byLarsen et al., 1990Larsen C.P. Steinman R.M. Witmer-Pack M. Hankins D.F. Morris P.J. Austyn J.M. Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (569) Google Scholar. Figure 4a shows that after 72 h of organ culture CD74+ LC in the epidermis of CL-treated mice were decreased compared to controls. A dramatic reduction of cords and cells within these cords in CL-treated dermis indicates that the decrease of LC observed in the epidermis was not due to an altered migration pattern of LC (Figure 4a, supplementary data (Fig S4) online). The few MHC class II+ cells within the cords were negative for maturation markers (e.g., CD80 (CD86, CD40 see supplementary data Fig S4 online)). In line with this, the emigrated DC/LC in the CL-treated group were highly diminished and showed a low expression of MHC II and CD86 Figure 4b, c. In contrast, LC in epidermal/dermal sheets and emigrated DC/LC in control- and pimecrolimus-treated skin were comparable in numbers and maturation marker pattern Figure 4a, b and c. To test whether LC depletion is due to apoptosis, epidermal sheets were double-stained with the (TUNEL) kit and an LC-specific MoAb and examined with a CLM. Morphologically, LC in the CS-treated groups became round in shape, with scanty or no dendrites as compared with LC of the control mice. While in the control- and pimecrolimus-treated epidermis 2%–3% MHC class II+ EC were TUNEL+, indicating the natural turnover of LC, the percentage of apoptotic LC significantly increased to 13% and 24% in the HC and CL group, respectively Figure 5a, b. In addition, TUNEL+/MHC class II- cells were also present in the CS-treated groups, most of which were probably keratinocytes Figure 5a. These findings show that topically applied CS but not pimecrolimus induce apoptosis in LC and keratinocytes in situ. Because caspase-3 is a key effector caspase in various forms of apoptosis and degrades several cellular proteins, we investigated its involvement in CS-induced LC apoptosis. Indeed, CS- but not pimecrolimus- or vehicle-treatment induced caspase-3 activity in LC Figure 6a. Recent in vitro and in vivo studies have suggested a susceptibility of LC to CD95/CD95L-mediated apoptosis (Kawamura et al., 1999Kawamura T. Azuma M. Kayagaki N. Shimada S. Yagita H. Okumura K. Fas/Fas ligand-mediated elimination of antigen-bearing Langerhans cells in draining lymph nodes.Br J Dermatol. 1999; 141: 201-205Crossref PubMed Scopus (17) Google Scholar,Kawamura et al., 2000Kawamura T. Azuma M. Kayagaki N. Shimada S. Yagita H. Okumura K. Fas/Fas ligand-mediated apoptosis of murine Langerhans cells.J Dermatol Sci. 2000; 22: 96-101Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar;Matsue et al., 1999Matsue H. Edelbaum D. Hartmann A.C. et al.Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells.J Immunol. 1999; 162: 5287-5298PubMed Google Scholar). Therefore, we tested a potential involvement of the CD95 system in CS-induced apoptosis and determined CD95 and CD95L expression on LC and keratinocyte surfaces. By FACS analysis of freshly isolated EC suspensions 24 h after the last application of compounds, we observed an upregulation of CD95 expression on MHC class II+ LC treated with CS but not on LC of pimecrolimus-treated mice and controls Figure 6b. In contrast, CD95L expression was upregulated neither on MHC class II+ nor on MHC class II- EC. In line with this, CS-induced LC apoptosis was not reduced by a neutralizing CD95L monoclonal antibody (MoAb) in vitro (data not shown). These results suggest that CS induce LC death directly and may involve the CD95 pathway in a CD95L-independent fashion.Figure 4Pimecrolimus has no effects on migration and maturation of LC in contrast to CS. BALB/c mice were treated twice on one day on both sides of the ears with vehicle, pimecrolimus, and CL. Forty-eight hours after the last application, dorsal ear skin was floated on medium and cultured. After 3 d, epidermal and dermal sheets were prepared, stained for the indicated markers, and analyzed using a CLM (A). Emigrated cells were harvested, stained with trypan blue to exclude dead cells and counted (B). Results represent mean±SD of DC/LC per well over six wells. Emigrated cells were analyzed by flow cytometry for the expression of the indicated markers (C). **p<0.01 versus control. Scale bars: 20 μm.View Large Image Figure ViewerDownload (PPT)Figure 5CS but not pimecrolimus induce apoptosis in LC in situ. (A) BALB/c mice were topically treated with compounds and vehicle, and 72 h later ear epidermal sheets were subjected to the TUNEL technique and then stained with a PE-conjugated anti-MHC class II MoAb. To induce DNA breaks in the nucleus (=positive control; 95% LC), epidermal sheets were incubated with 1500 U per mL DNase I (1 h, 37°C) and then subjected to double labeling. Data analysis and acquisition was performed as described in Materials and Methods. Results are shown as a dot plot. (B) Forty randomly selected fields per sheet (n=8 per group) were scanned. Experiments were repeated three times and 960 pictures per group were analyzed. ***p<0.001 versus control, *p<0.05 versus control. Scale bars: 20 μm.View Large Image Figure ViewerDownload (PPT)Figure 6CS induce caspase-3 activity and upregulate CD95 expression in LC in contrast to pimecrolimus. BALB/c mice were topically treated with compounds and vehicle, and 24 (B) and 48 (A) h later EC suspensions were prepared and analyzed for CD95 expression and caspase activity by flow cytometry. 104 MHC class II+ LC were acquired and antigen expression is shown as a histogram. Data are representative of three experiments.View Large Image Figure ViewerDownload (PPT) When testing whether the topical application of CS and pimecrolimus alters the secretion of certain keratinocyte-derived cytokines which are critical for LC survival, we found that CS-EC supernatants contained significantly lower amounts of GM-CSF, TNF-α, and IL-1α than EC supernatants from vehicle-treated mice Figure 7. Surprisingly, pimecrolimus-EC supernatants contained significantly higher levels of these cytokines compared to controls. Concerning the role of GM-CSF for LC survival, we found that the addition of GM-CSF to EC suspensions prepared from CS-treated mice greatly improved LC viability Table II. These data show that CS but not pimecrolimus lead to a downregulation of pro-inflammatory cytokines in keratinocytes and, thus, may in part deliver an initial pro-apoptotic signal for LC.Table IIAddition of GM-CSF to EC suspensions prepared from CS-treated mice greatly improves LC viabilityaBALB/c mice were treated twice on 1 d on both sides of the ears with a 20 μL ethanolic solution of clobetasol propionate (0.05%). Controls were treated with ethanol (vehicle) alone. Forty-eight hours after the last application, EC suspensions were prepared from auricular skin, adjusted to equal numbers of LC, as determined by flow cytometry, and cultured in normal cell culture medium for 2 d in the presence or absence of GM-CSF (200 U per mL). Cells were counted under a light microscope and dead cells were excluded by trypan blue uptake. Data are representative of at least two experiments.TreatmentCytokinesNumbers of viable EC after 2 d of cultureNumbers of viable LC after 2 d of cultureControl—1.2×1062.2×105 (100%)Clobetasol—2.1×1060.5×105 (25%)ControlGM-CSF0.9×1062.3×105 (104%)ClobetasolGM-CSF2.7×1061.8×105 (86%)a BALB/c mice were treated twice on 1 d on both sides of the ears with a 20 μL ethanolic solution of clobetasol propionate (0.05%). Controls were treated with ethanol (vehicle) alone. Forty-eight hours after the last application, EC suspensions were prepared from auricular skin, adjusted to equal numbers of LC, as determined by flow cytometry, and cultured in normal cell culture medium for 2 d in the presence or absence of GM-CSF (200 U per mL). Cells were counted under a light microscope and dead cells were excluded by trypan blue uptake. Data are representative of at least two experiments. Open table in a new tab To investigate whether topical application of immunomodulating compounds influences the endocytic capacity of LC, auricular and tail skin were treated with pimecrolimus, CS, or vehicle. Forty-eight hours after treatment, EC suspensions were prepared, and FITC-ovalbumin (OVA) uptake, mainly by fluid phase, by LC was analyzed. As shown in Figure 8a, endocytic activity of LC isolated from CS-treated mice was similar to controls. In contrast, antigen capture was increased in LC treated with pimecrolimus. In view of this finding, we evaluated the ability of LC to process and present a soluble antigen upon topical treatment with the compounds. Freshly isolated EC from pimecrolimus-, CS- or vehicle-treated skin areas were pulsed with hen egg lysozyme (HEL) for 24 h. Subsequently, LC-enriched EC (adjusted to equal numbers of LC) were cocultured with the HEL-specific, MHC class II-restricted Tcell hybridoma 1H11.3 for 24 h. Figure 8b shows that LC-enriched EC from pimecrolimus-treated mice were superior to LC-enriched EC from vehicle-treated mice in their ability to stimulate T cells, as assessed by their IL-2 production. In contrast, LC-enriched EC from CS-treated mice showed much lower efficiency in stimulating hybridoma cells. To test whether the enhanced (pimecrolimus) or decreased (CS) stimulation of hybridoma cells is due to altered capacities of LC or to secondary effects (e.g., factors secreted from keratinocytes), LC were highly enriched, and the assay was performed as described. While LC from pimecrolimus-treated mice presented antigen as effectively as LC from vehicle-treated mice, LC from CS-treated mice again were defective in presenting antigen Figure 8c. To evaluate whether topical application of pimecrolimus and CS has any effects on the maturation and immune function of LC, 48 h EC suspension cultures from CS-, pimecrolimus-, or vehicle-treated skin were tested for viability, phenotype, and LC-dependent immune functions. While the viability of EC prepared from pimecrolimus- and vehicle-treated skin areas was greater than 70%, that of EC from HC- and CL-treated mice was only 55% and 39% (n=3), respectively. FACS phenotyping, while revealing comparable levels of surface-bound MHC class II molecules on LC from all groups, showed a decreased expression of CD25, CD205, and the costimulatory molecules CD40, CD54, CD80, CD86 on LC derived from CS-treated animals compared to pimecrolimus- and vehicle-treated mice Figure 9a. In line with this, LC-enriched EC (adjusted to equal numbers of viable LC) from pimecrolimus-treated mice induced a vigorous allogeneic T cell response comparable to that evoked by LC-enriched EC from vehicle-treated mice, with a peak occurring on days 4 and 5 for CD8+ and CD4+ T cells, respectively Figure 9b. In contrast, LC-enriched EC from CS-treated mice were greatly hampered in this capacity Figure 9b. To study the quality of the T cell response, CD4+ T cells, primed in the MLR by LC-enriched EC from vehicle- or pimecrolimus-treated mice, were expanded for 7 d and then restimulated with irradiated antigen-presenting cells (APC). While the analysis of cytokines revealed high levels of IFN-γ in both groups, there was a relative increase of IL-4 and IL-10 in the pimecrolimus as compared to the vehicle group, indicating that pimecrolimus-treated LC are able to skew a T cell response into a TH2 direction Figure 9c. Taken together, these results show that, in contrast to CS, pimecrolimus does not interfere with the maturation and immune function of LC and those of other EC, although the quality of the T cell response was slightly different. Besides the long-used CS, a new class of topical immunomodulators—tacrolimus and pimecrolimus, also termed calcineurin inhibitors—has recently been shown to be effective in the treatment of eczematous skin diseases. While CS and calcineurin inhibitors mediate their immunosuppressive function by affecting skin-infiltrating T cells, mast cells, and basophils, their impact on APC in the skin seems to be divergent. With regard to CS, several investigators have shown a depletion of LC and an impairment of their immune functions upon topical treatment (Nordlund et al., 1981Nordlund J.J. Ackles A.E. Lerner A.B. The effects of ultraviolet light and certain drugs on Ia-bearing Langerhans cells in murine epidermis.Cell Immunol. 1981; 60: 50-63Crossref PubMed Scopus (63) Google Scholar;Belsito et al., 1982Belsito D.V. Flotte T.J. Lim H.W. Baer R.L. Thorbecke G.J. Gigli I. Effect of glucocorticosteroids on epidermal Langerhans cells.J Exp Med. 1982; 155: 291-302Crossref PubMed Scopus (119) Google Scholar,Belsito et al., 1984aBelsito D.V. Baer R.L. Gigli I. Thorbecke G.J. Effect of combined topical glucocorticoids and ultraviolet B irradiation on epidermal Langerhans cells.J Invest Dermatol. 1984; 83: 347-351Abstract Full Text PDF PubMed Scopus (11) Google Scholar,Belsito et al., 1984bBelsito D.V. Baer R.L. Thorbecke G.J. Gigli I. Effect of glucocorticoids and gamma radiation on epidermal Langerhans cells.J Invest Dermatol. 1984; 82: 136-138Abstract Full Text PDF PubMed Scopus (23) Google Scholar;Berman et al., 1983Berman B. France D.S. Martinelli G.P. Hass A. 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Tacrolimus affects the phenotype and function of human LC in vitro, leading to a downregulation of MHC I and II, CD25, CD40 and CD80 molecules and impairing their allostimulatory activity (Panhans-Gross et al., 2001Panhans-Gross A. Novak N. Kraft S. Bieber T. Human epidermal Langerhans cells are targets for the immunosuppressive macrolide tacrolimus (FK506).J Allergy Clin Immunol. 2001; 107: 345-352Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar).Wollenberg et al., 2001Wollenberg A. Sharma S. von Bubnoff D. Geiger E. Haberstok J. Bieber T. Topical tacrolimus (FK506) leads to profound phenotypic and functional alterations of epidermal antigen-presenting dendritic cells in atopic dermatitis.J Allergy Clin Immunol. 2001; 107: 519-525Abstract Full Text Full Text PDF PubMed Scopus (158) Google Sch
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