dermatitis
W. Ba¨umer, B. Su¨lzle, H. Weigt,* V.C. De Vries,M. Hecht,T. Tschernig and M. Kietzmann Departments of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany
*LipoNova GmbH, 30625 Hannover, Germany
Department of Functional and Applied Anatomy, Medical School of Hannover, D-30625 Hannover, Germany Fraunhofer Institute of Toxicology and Experimental Medicine, 30625 Hannover, Germany
Correspondence
allergic contact dermatitis, cilomilast, dendritic cells, matrix metalloproteinase 9, rapamycin, tacrolimus
Conflicts of interest:
None declared.
Summary
Background Cilomilast and tacrolimus as well as rapamycin are potential drugs for the treatment of allergic skin diseases like atopic dermatitis and allergic contact dermatitis.
Objectives To compare the in vitro and in vivo immunomodulatory effects of the phosphodiesterase 4 inhibitor cilomilast with those of tacrolimus and rapamycin.
Methods Thein vitroaction of cilomilast, tacrolimus and rapamycin were tested in a mixed leucocyte reaction (MLR).In vivo,the inhibitory action of the immunomod-ulatory drugs was compared in the toluene-2,4-diisocyanate (TDI)-induced aller-gic inflammatory response with particular focus on dendritic cell (DC) function.
Results Cilomilast, tacrolimus and rapamycin were all able to inhibit DC-mediated T-cell activation in a MLR. But it was demonstrated for cilomilast that the target cells are T cells rather than DC. In vivo, a combination of systemic and topical administration of each of these three substances significantly inhibited swelling in the murine ear 16 h after TDI challenge. There was also a reduction in the weight of the draining auricular lymph node, in lymphocyte cell count, and in the number of emigrated DC. The density of Langerhans cells in the epidermis was correspondingly higher in mice treated with cilomilast, tacrolimus and rapa-mycin than in those treated with vehicle. All three substances were found to inhibit DC migrationex vivo in a skin DC migration assay performed on ear tissue after TDI challenge.
Conclusions DC migration into the draining lymph node also takes place in the eli-citation phase of allergic contact dermatitis and this migration can be influenced by tacrolimus and rapamycin, and, to a lesser extent, by cilomilast.
The immunosuppressive agents tacrolimus and rapamycin are macrolide antibiotics, which are used clinically as immunosuppressive drugs for the treatment of transplant rejection and autoimmune diseases. Tacrolimus is also regis-tered for the topical treatment of atopic dermatitis. There is controversy over the inhibitory action of rapamycin and tacrolimus in models of allergic contact dermatitis. Both substances show inhibitory action in a murine contact sensitivity reaction provoked by trinitrochlorobenzene.1 Tacrolimus has also been shown to be effective in dinitro-fluorobenzene-induced contact sensitivity in domestic pigs, whereas rapamycin was not effective by either topical or systemic administration.2 Similar results were obtained by
Duncan3 in a comparable model of a delayed-type hyper-sensitivity in guinea pigs.
The phosphodiesterase 4 inhibitor cilomilast is currently being evaluated for the treatment of asthma and chronic obstructive pulmonary disease,4 and has been shown to have inhibitory effects in models of allergic contact dermatitis.5–8 Both Langerhans cells and dermal dendritic cells (DC) trans-port haptens like toluene-2,4-diisocyanate (TDI) from the skin through afferent lymphatic vessels to draining lymph nodes, where these haptens, now bound to peptides, are presented to T cells, which then become specific T cells (induction phase).
When the hapten is painted a second time, it diffuses through the skin and can be loaded to Langerhans cells or dermal DC,
which present it to specific T cells which are then activated and respond with cytokine production and activation of fur-ther inflammatory cells (elicitation phase9).
The present study was performed to compare the anti-inflammatory and immunomodulatory action of cilomilast, tacrolimus and rapamycin in a model of allergic contact der-matitis induced by TDI with particular focus on DC migration and activation.
Materials and methods
Mice
Female BALB⁄c-mice were obtained at the age of 8 weeks (20 g body weight) from Charles River (Sulzfeld, Germany).
The mice were housed in groups of six mice per cage at 22C with a 12-h light⁄dark cycle. Water and a standard diet (Altromin, Lage⁄Lippe, Germany) were availablead libitum. The animal experiment was approved by the regional government of Hannover, Germany (Reference no. 509.6-42502-03⁄711).
Dendritic cell generation from bone marrow cultures
High-purity bone marrow-derived DC were generated accord-ing to the protocol of Lutzet al.10 with slight modifications as previously described.11 Briefly, bone marrow was cultivated with RPMI 1640 (Biochrom, Berlin, Germany), 10% fetal calf serum (Biochrom), and 50lmol L)12-mercaptoethanol (Sig-ma, Deisenhofen, Germany). The medium contained 20 ng mL)1 granulocyte–macrophage colony-stimulating fac-tor (GM-CSF) (Sigma). Fresh medium supplemented with GM-CSF was added on days 3, 6 and 8. Fluorescence-activated cell sorting (FACS) analysis of the day-10 cell suspension demonstrated a high yield of CD11c and major histocompati-bility complex (MHC) class II-positive cells.11
Incubation with cilomilast, tacrolimus and rapamycin
After 9 days of cultivation the cells were placed in a 12-well plate (4·105cells well)1) and incubated for 24 h with 10lmol L)1 cilomilast (elbion AG, Radebeul, Germany), 100 nmol L)1 tacrolimus (Calbiochem, Darmstadt, Germany) or 1lmol L)1 rapamycin (Wyeth-Ayerst, Princeton, NJ, U.S.A.). These concentrations did not reduce cell viability as
determined by CellTiter AQueous One Solution cell prolifer-ation assay (Promega, Mannheim, Germany, data not shown).
At day 10 the cells were placed in a 96-well plate (5·104cells well)1), again incubated with the substances as indicated above, and stimulated by the addition of 5lL lipo-polysaccharide (LPS) (E. coli, O127:B8; Sigma, Deisenhofen, Germany) working solution (1lg mL)1 LPS, final concentra-tion). The LPS-positive control was treated with the vehicle (DMSO) in the highest final concentration used here. The supernatant was collected 24 h later and stored at )20C until determination of tumour necrosis factor (TNF)-a and interleukin (IL) 12p70 by enzyme-linked immunosorbent assay (ELISA, DuoSet and Quantikine, respectively; R & D Sys-tems, Wiesbaden, Germany). Three independent assays were performed. FACS analysis was performed on day 11 of culture (FACSCalibur; Becton Dickinson, Mountain View, CA, U.S.A.).
Cells were harvested and stained for MHC-II (M5⁄114) and CD11c (HL3; Pharmingen, Hamburg, Germany).
Mixed leucocyte reaction
T cells were isolated from the spleen of female NMRI mice by a method adapted from Gunzeret al.12 but without separation of CD4+ from CD8+ T cells. In short, spleen cells were incu-bated in an erythrocyte lysis buffer for 5 min followed by a nylon wool enrichment of T lymphocytes achieved by rinsing the cell suspension through a sterilized nylon wool column.
This leads to a purity of approximately 70% T cells.12 The cells (1·105well)1) were seeded in a U-bottomed 96-well plate. Different amounts of DC (day 10 of culture) were added (Fig. 1) and incubated with cilomilast (10lmol L)1), tacrolimus (100 nmol L)1) and rapamycin (1lmol L)1). An additional study was performed for cilomilast, in which only DC or T cells were preincubated for 1 h with 10lmol L)1 cilomilast and then washed thoroughly before the mixed leu-cocyte reaction (MLR). After 6 days, proliferation of T cells was determined by incorporation of 3H-thymidine (1lCi well)1; Hartmann Analytics, Braunschweig, Germany) during the final 18 h of culture. The cells were subsequently harvested on filter mats (Canberra-Packard, Dreieich, Germany). After drying, 20lL of liquid scintillator (Canberra-Packard) was added and the plates were sealed. Counts per minute were determined on a Topcount Microplate Scintillation Counter
a b
Fig 1. (a) Suppression of mixed leucocyte reaction by coincubation with tacrolimus (100 nmol L)1), rapamycin (1lmol L)1) and cilomilast (10lmol L)1) in different ratios of dendritic cells (DC) : T cells. (b) Comparison of preincubation of T cells with preincubation of DC with 10lmol L)1cilomilast for 1 h.
Data representative of three independent experiments;n¼5 for each ratio; *P <0Æ05.
2005 British Association of Dermatologists•British Journal of Dermatology2005153,pp136–144
Cilomilast, tacrolimus and rapamycin in dermatitis, W. Ba¨umeret al. 137
(Canberra-Packard). Three independent experiments were per-formed with each substance.
Mouse ear swelling test
Sensitization was performed as previously described.7After the mice had settled in for 1 week, the skin of their abdomens was shaved and depilated with Veet (Reckitt Benckiser, Mannheim, Germany). The abdominal skin was then stripped with adhesive tape. For active sensitization, 100lL 5% TDI in acetone was administered to the abdominal epidermis on three consecutive days. The allergic reaction was elicited 21 days later by administration of 20lL 0Æ5% TDI in acetone on both the inner and outer surfaces of the left ears to determine the sensitization status. The amount of swelling was determined 24 h after challenge by comparing the thickness of the ear at that time with the individual basal value (230lm) deter-mined earlier. A cutimeter (Mitutoyo, Neuss, Germany) was used for these measurements. Animals with a mean swelling difference less than 20% of their basal value 24 h after chal-lenge were then excluded as being not sensitized; this applied to less than 5% of all mice in this study. The mice included in the study were distributed to the treatment groups (n¼6) equally according to the degree of swelling, so that each group contained animals responding to varying extents. The mice were allowed to rest until 7 days after the ear thickness returned to almost normal level. To exclude contamination by residues of the allergen on the ears, the untreated right ears were used for the main experiment as follows.
Drug administration
For oral administration 20 mg kg)1 cilomilast, 2Æ5 mg kg)1 tacrolimus and 20 mg kg)1 rapamycin were dissolved in 10%
ethanol and olive oil.3The drugs were administered 20 h and 30 min before TDI challenge.
The substances were administered intraperitoneally in 50%
polyethylene glycol 300, 2Æ5% Tween 80 (Sigma) and 10%
ethanol (VWR, Darmstadt, Germany) in the same doses and at the same times as indicated above.13 The concentrations for systemic administration of cilomilast were adopted from Gris-wold et al.5 and from our own studies;7 those for rapamycin and tacrolimus, from Duncan.3
For topical administration, the substances were dissolved in acetone⁄DMSO (1 : 9) with addition of ethanol (10%) in the following concentrations: 3% cilomilast (600lg ear)1), 1% rapamycin (200lg ear)1), and 0Æ5% tacrolimus (100 lg ear)1); each ear was treated with 20lL. The concentration for cilomilast was selected according to Griswold et al.5 and Ba¨umer et al.7,8 As the concentrations reported for tacrolimus and rapamycin range from 0Æ01% to 2%,2,3the concentrations used here were chosen after pilot experiments. Although tacro-limus is registered as a 0Æ1% ointment, it was decided to enhance the concentration, as there was a dose-dependent inhi-bition of the inflammatory reaction in experimental studies.2,3 The combination of topical and intraperitoneal administration
was performed at least twice for each substance (n¼6 for each group and experiment) with comparable results.
Skin dendritic cell migration assay
The mice were sacrificed by cervical dislocation 16 h after challenge and measurement of ear thickness. The cartilage-free dorsal halves of split mouse ear skin were cultured in 24-well microtitre plates based on the method of Ortner et al.14 Directly before cultivation, the ear halves were again treated with cilomilast, tacrolimus, rapamycin or vehicle (10lL). The halves were laid epidermal side up onto racks made of sieves.11 Macrophage inflammatory protein-3b was added (50 ng mL)1; R&D Systems) as a chemotactic factor for DC.
The racks were changed daily to new wells and new media (including macrophage inflammatory protein-3b). Migrated cells from each ear (days 2 and 3) were pooled and counted with a haemocytometer (Neubauer, VWR). The viability of the cells was assessed by trypan blue exclusion.
Determination of dendritic cells in lymph nodes
The auricular lymph nodes were sampled and single-cell sus-pensions were prepared in phosphate-buffered saline by means of a glass potter (VWR). The cells were counted (Casy plus, Scha¨rfe System GmbH, Reutlingen, Germany) and stained for CD11c+ cells.
Preparation of epidermal sheets for immunohistochemistry
The epidermal sheets were prepared and evaluated as previ-ously described.11 In short, skin was floated on 0Æ5 mol L)1 ammonium thiocyanate (Riedel de Hae¨n, Hannover, Germany) for 10 min at 37C. The epidermis was separated from the dermis and immediately fixed in cold acetone. DC were detec-ted with monoclonal antimouse MHC class II (I-A⁄I-E, rat IgG2b; Beckton Dickinson, Heidelberg, Germany). Labelling of the antibodies was visualized using biotinylated rabbit-antirat immunoglobulin G (Dako, Hamburg, Germany) and streptavi-dine–fluorochrome (carbocyanin 3; Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.). Analyses were per-formed using the Kontron KS 400 image analysis system. The density of Langerhans cells was analysed (at 40· magnifica-tion on a calibrated grid). Sixteen randomly chosen areas were counted per ear. Six ears, each treated with vehicle, cilomilast, tacrolimus or rapamycin were analysed.
Zymography
Ear halves were cultivated for 3 days (for the migration assay) and separated into dermal and epidermal sheets as described above and homogenized in liquid nitrogen. Each sample was diluted to a concentration of 200lg mL)1 of protein, and 25lL of each sample was subjected to electrophoresis on a 3Æ9% acrylamid stacking gel⁄7Æ5% acrylamid separating gel 138 Cilomilast, tacrolimus and rapamycin in dermatitis, W. Ba¨umeret al.
(Pro Sieve 50; Biozym, Hessisch Oldendorf, Germany) contain-ing 1 mg mL)1gelatine in the presence of sodium dodecyl sul-phate under nonreducing conditions. After electrophoresis, gels were washed twice with 2Æ5% Triton X-100, rinsed with water, and incubated at 37C overnight in 50 mmol L)1Tris, 5 mmol L)1CaCl2and 200 mmol L)1NaCl. Gels were stained with Coomassie Brilliant Blue (VWR) and de-stained in a tion of 30% methanol and 10% acetic acid (all salts and solu-tions from VWR). Gelatinase activities appeared as clear bands against a blue background. Molecular weights of gelatinolytic bands were estimated using recombinant protein molecular weight markers (10–250 kDa; Bio-Rad, Munich, Germany).
Six samples of epidermis and dermis were evaluated from each treatment group. Quantification of the gelatinolytic bands was performed by densitometry. The image acquisition was carried out with an HP Scan Jet 7400c scanner using the program Pho-toImpact 4.0 (Ulead Systems, Kaarst, Germany). The densito-metric evaluation was made using Scion Image software for PC (Scion Corporation, Frederick, MD, U.S.A.). Grey values of the background and of the gelatinolytic band were transformed into optical densities [OD¼log10 (255⁄grey value)]. The OD of the background was subtracted from the OD of the gelatino-lytic band. Each gel consisted of three vehicle controls, which were compared with three ears treated with cilomilast, tacroli-mus or rapamycin. Two gels from each treatment group were performed, resulting inn¼6 per group. To compare the gela-tinolytic bands of the different gels, it was decided to set the gelatinolytic bands of the vehicle controls as 100% and to pre-sent the inhibitory action as the percent of inhibition (Table 1).
Statistical evaluation
Figures are presented as mean (± SEM). Statistically significant differences between the drug treatments and controls were assessed by a one-way ANOVA followed by a post hoc test (Dunnett’s test).
Results
Mixed leucocyte reaction
Incubation of the MLR with tacrolimus (100 nmol L)1), rapa-mycin (1lmol L)1) and cilomilast (10lmol L)1) inhibited
the DC-induced T-cell proliferation (Fig. 1a). A dose–response experiment revealed a significant inhibition of T-cell prolifer-ation by cilomilast only at 10lmol L)1, whereas proliferation was inhibited by tacrolimus and rapamycin even at the lowest doses tested (10 pmol L)1 and 100 pmol L)1, respectively, data not shown). Preincubation of T cells with cilomilast gave results comparable to those of coincubation, whereas preincu-bation of DC with cilomilast inhibited T-cell proliferation only moderately (Fig. 1b).
Lipopolysaccharide-induced cytokine secretion
LPS induced a significant increase in TNF-a and IL-12 secre-tion in bone marrow-derived DC. The increase in TNF-awas inhibited significantly by cilomilast, whereas only a moderate inhibition was caused by tacrolimus and rapamycin. Cilomilast also inhibited the IL-12 production in three independent Table 1 Effects of rapamycin, tacrolimus and cilomilast pretreatment
on matrix metalloproteinase-9 activity in toluene-2,4-diisocyanate-challenged ear skin 3 days after organ culture (migration assay). Per cent inhibition (mean ± SD) compared with vehicle-treated skin (epidermis and dermis for rapamycin),n¼6 each group
Rapamycin
aP< 0Æ05 compared with vehicle-treated ears.
a
b
Fig 2.Concentration of tumour necrosis factor (TNF)-a(a) and interleukin (IL)-12 (p70) (b) in supernatant of bone marrow-derived dendritic cells (DC) 24 h after lipopolysaccharide (LPS) treatment (1lg mL)1). DC were preincubated with cilomilast (10lmol L)1), tacrolinus (100 nmol L)1) and rapamycin (1lmol L)1) 24 h and 30 min before LPS stimulation. Data representative of three independent experiments;n¼5 in each group; *P <0Æ05 compared with cells stimulated with LPS (+ vehicle).
2005 British Association of Dermatologists•British Journal of Dermatology2005153,pp136–144
Cilomilast, tacrolimus and rapamycin in dermatitis, W. Ba¨umeret al. 139
experiments, but results with tacrolimus and rapamycin also displayed only moderate inhibition (Fig. 2).
Mouse ear swelling test, skin dendritic cell migration assay, matrix metalloproteinase-9activity
A pilot study demonstrated that only an inhibition of ear swelling (i.e. inflammatory reaction) was accompanied by a
decrease in skin DC migration (Table 2). Therefore, for the main experiments it was decided to administer high doses sys-temically and topically. TDI induced a strong increase in ear swelling, which was nearly eliminated by cilomilast and signi-ficantly reduced by tacrolimus and rapamycin (Table 3).
Directly after determination of the degree of ear swelling, the dorsal ear halves were cultivated for 72 h and the ventral halves were prepared for the determination of Langerhans cell density. Significantly fewer cells migrated from those treated with cilomilast, tacrolimus or rapamycin than from those trea-ted with vehicle (Table 3). The matrix metalloproteinase (MMP)-9 activity in the epidermis and dermis of mouse ears treated with vehicle was higher than in those treated with ra-pamycin (Fig. 3 and Table 1). Although it was also possible to detect MMP-9 activity in the epidermis in the vehicle used for cilomilast and tacrolimus, this activity was weak, and no bands were detected in epidermis treated with cilomilast and tacrolimus. Therefore it was decided to determine MMP-9 activity for whole skin for cilomilast and tacrolimus; this was significantly reduced in ear skin treated with cilomilast and tacrolimus (Table 1).
Lymph node weight, cell count and CD11c+ cells
The weights and cell counts of draining lymph nodes of mice treated with TDI were significantly increased. Pretreatment with cilomilast caused a slight reduction in lymph node weight, while both tacrolimus and rapamycin induced a signi-ficant reduction. The cell count was also signisigni-ficantly reduced by tacrolimus. The number of DC per lymph node was also reduced by these immunomodulators, although cilomilast failed to induce a significant inhibition (Fig. 4a–c).
Table 2 Pilot study concerning different routes of administration
Drug and route of
Cilomilast and rapamycin failed to inhibit the toluene-2,4-diiso-cyanate-induced ear swelling and skin dendritic cell migration, whereas tacrolimus caused a slight inhibition (values are mean±SD of n¼3–4 each group). This pilot study is of a descriptive character only; therefore no statistics were performed.
Table 3Ear swelling 16 h after toluene-2,4-diisocyanate challenge
Endpoint
Untreated
control Vehicle Cilomilast Rapamycin Tacrolimus Ear swelling (lm) 0 ± 2a 131 ± 34 5 ± 3a 10 ± 4a 18 ± 4a Migrated skin DC nd 1539 ± 337 233 ± 106a 139 ± 72a 400 ± 77a After determination of the ear thickness the mice were sacrificed to determine skin den-dritic cell (DC) migration from mouse ear explants. Ears were again treated topically with the substances directly before cultivation as indicated in Materials and methods. n¼6 in each group.aP <0Æ05 compared with vehicle control; nd, not determined.
A B C D
Fig 3. Gelatine zymogram for matrix metalloproteinase (MMP)-9 activity. Lanes A and B are for epidermis, lanes C and D, dermis, of mouse ears treated with vehicle (A⁄C) or rapamycin (B⁄D) 3 days after cultivation (migration assay). The pro-MMP-9 (92 kDa) and the active MMP-9 (~80 kDa) bands are seen on the right. The kDa specifications indicate the molecular weight standard (left lane).
140 Cilomilast, tacrolimus and rapamycin in dermatitis, W. Ba¨umeret al.
Major histocompatibility complex II-positive cells in the epidermis
The MHC II+ cell counts in the epidermis of mice challenged with TDI were significantly higher 16 h after treatment with
cilomilast, tacrolimus and rapamycin than in those treated with vehicle (Fig. 5, Table 4).
Discussion
The present study was performed to compare the immuno-modulatory action of cilomilast, tacrolimus and rapamycin in the elicitation phase of allergic contact dermatitis. In addition to thein vivo study we also conductedin vitro experiments on the influence of DC activation by the immunomodulators on the T-cell response. There has been intense research on this subject.15–17 Our results on the inhibitory action of cilomi-last, tacrolimus and rapamycin in the MLR can also be explained by the inhibition of the T-cell response. It cannot be excluded that the DC function was also affected by coin-cubation, as was shown for cilomilast. Preincubation of T cells with cilomilast led to results comparable to those after coincubation, whereas preincubation of DC resulted in only a moderate inhibition of the MLR (Fig. 1b). Essayan et al.18 obtained similar results for the PDE4 inhibitor rolipram in a model of antigen-driven proliferation. The question of the influence of cilomilast, tacrolimus and rapamycin on DC
The present study was performed to compare the immuno-modulatory action of cilomilast, tacrolimus and rapamycin in the elicitation phase of allergic contact dermatitis. In addition to thein vivo study we also conductedin vitro experiments on the influence of DC activation by the immunomodulators on the T-cell response. There has been intense research on this subject.15–17 Our results on the inhibitory action of cilomi-last, tacrolimus and rapamycin in the MLR can also be explained by the inhibition of the T-cell response. It cannot be excluded that the DC function was also affected by coin-cubation, as was shown for cilomilast. Preincubation of T cells with cilomilast led to results comparable to those after coincubation, whereas preincubation of DC resulted in only a moderate inhibition of the MLR (Fig. 1b). Essayan et al.18 obtained similar results for the PDE4 inhibitor rolipram in a model of antigen-driven proliferation. The question of the influence of cilomilast, tacrolimus and rapamycin on DC