Involvement of cytokines in the skin-to-lymph node trafficking of cells of the monocyte–macrophage lineage expressing a C-type lectin

Kyung-hee Chun, Yasuyuki Imai1, Nobuaki Higashi and Tatsuro Irimura

Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
1 Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan

Correspondence to: T. Irimura


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mechanism by which dermal cells expressing a macrophage calcium-type lectin (MGL) trafficked to regional lymph nodes was investigated. Conditioned medium prepared from organ cultures of mouse skin sensitized with a mixture of acetone and dibutylphthalate was shown to decrease the number of MGL+ cells in the dermis in ex vivo organ culture assays. In in vitro culture of sensitized skin, the loss of MGL+ cells was abrogated by the addition to the culture medium of mAb against IL-1ß, while addition of recombinant IL-1ß to the medium in which untreated skin was cultured induced loss of MGL+ cells. Intradermal injection of recombinant IL-1ß also resulted in a transient increase of MGL+ cells in the T cell area of draining lymph nodes in vivo, indicating that IL-1ß is central in the entire process of MGL+ cell trafficking to the lymph nodes. Supporting this is that cells producing IL-1ß were detected in the epidermis of cultured skin even early after sensitization. The possibility that IL-1ß simply down-regulates MGL expression was eliminated by Western blotting experiments with isolated MGL+ cells treated with or without IL-1ß. IL-1{alpha} and tumor necrosis factor (TNF)-{alpha} were also able to induce migration of MGL+ cells in the ex vivo assay in a manner akin to IL-1ß, and antibodies against them abrogated this. Isolated MGL+ cells from skin cultured in type I collagen matrix in vitro displayed morphological changes upon exposure to IL-1ß, IL-1{alpha} or TNF-{alpha}, indicating that these cytokines exert a direct effect on these cells. Thus, pro-inflammatory cytokines, particularly IL-1ß, are produced at the site of skin sensitization and are involved in at least initiating the trafficking of cells expressing MGL to the lymph nodes.

Keywords: cell trafficking, delayed-type hypersensitivity, IL-1, skin sensitization


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the sensitization phase of a delayed-type hypersensitivity (DTH) reaction, epidermal Langerhans cells (LC) sequester epicutaneously applied antigen or immunogenic hapten (after conjugation with host proteins) and carry them to the regional lymph nodes. Here antigen presentation to naive T cells in association with MHC class II molecules takes place (1,2). Dermal macrophages (and possibly dermal dendritic cells) have also been suggested from various artificial experimental systems to play a role in the induction of hapten-specific contact hypersensitivity (35). However, little is understood regarding the significance of differentiation, heterogeneity and cellular trafficking of dermal cells of the monocyte–macrophage lineage during the sensitization and elicitation steps of DTH.

A subpopulation of dermal cells of the monocyte–macrophage lineage can be defined by antibodies against a macrophage calcium-type lectin (MGL) (6,7). This marker is not expressed on LC. We have previously demonstrated that the efficacy of contact sensitization is greatly influenced by sensitization conditions such as the nature of the solvent wherein immunogenic haptens such as FITC are dissolved. The efficacy is positively correlated with an increase in MGL+ cells in the boundary of the T cell area of draining lymph nodes after sensitization (8). Cell transfer experiments have shown that this increase is most likely to be due to the trafficking of these cells from the dermis (8). Solvents such as acetone/dibutylphthalate (AD) mixture appear to act as an adjuvant. Such adjuvant activity is due, at least in part, to their ability to induce cell migration. We have shown that MGL+ cells in the dermis decrease in number after epicutaneous application of AD (9).

At present, however, the mechanism(s) causing the MGL+ cells to migrate in response to substances like AD are not clear. For example, the role of cytokines in this process was poorly understood. Pro-inflammatory cytokines have been reported to be involved in LC trafficking. Intradermal injection of recombinant mouse IL-1ß or tumor necrosis factor (TNF)-{alpha} increases the numbers of interdigitating dendritic cells, which are considered to be LC, in the draining lymph nodes (10). When anti-IL-1ß or anti-TNF-{alpha} antibodies are injected, LC migration into draining lymph nodes is inhibited (1113). IL-1{alpha} has also been suggested to participate in LC migration (14,15).

We have shown that trafficking of dermal MGL+ cells may be controlled by different mechanisms to those involved in LC trafficking because their initial localization and destination seemed to be different (8). When we compared the effect of various sensitizing vehicles using FITC as a contact sensitizer, we found that while SDS significantly increased the number of MGL+ cells in lymph nodes, LC migration did not occur as shown by the paucity of cells carrying FITC in the lymph nodes (8).

To further investigate the mechanisms driving MGL+ cell trafficking, we have established an ex vivo culture system of skin explants. In our previous paper, we showed that soluble factors are secreted into the medium of cultures of AD-treated skin (9). These soluble factors could induce the emigration of MGL+ cells from the dermis of untreated skin explants in a manner similar to what is seen in AD-treated skin. This emigration was significantly inhibited by anti-IL-1{alpha} or anti-IL-1ß mAb but not by pertussis toxin (9). In the present report, we provide further evidence that IL-1ß and IL-1{alpha}, and also TNF-{alpha}, are directly involved in the skin-to-lymph node trafficking of MGL+ cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female, specific pathogen-free CD1 (ICR) mice (4–8 weeks old) were purchased from SLC Japan (Shizuoka, Japan). The animals were fed and housed according to guidelines set by the Ministry of Education, Science, Sports and Culture of Japan. The care and use of these animals in this experiment have been approved by the Graduate School of Pharmaceutical Sciences, University of Tokyo.

Reagents
Triton X-100, aprotinin, pepstatin A, leupeptin, poly-L-lysine, saponin and PMSF were purchased from Sigma (St Louis, MO); acetone, dibutylphthalate and collagenase (Clostridium histolyticum) from Wako Pure Chemical (Tokyo, Japan); DMEM and Ham's F-12 medium from Nissui Pharmaceutical (Tokyo, Japan); FCS from BioWhittaker (Walkersville, MD); Cellmatrix derived from porcine skin from Nitta Gelatin (Osaka, Japan); DNase I (grade II, bovine pancreas) from Boehringer Mannheim (Mannheim, Germany); paraformaldehyde and glutaraldehyde from Nacalai Tesque (Kyoto, Japan); BSA (fraction V) from Seikagaku (Tokyo, Japan); and SDS–PAGE protein reference standards (phosphorylase b, BSA, aldolase and carbonic anhydrase) from Daiichi Pure Chemicals (Tokyo, Japan).

Cytokines, antibodies and immunodetection kits were purchased as follows. Recombinant murine IL-1{alpha}, IL-1ß and TNF-{alpha} came from Genzyme (Cambridge, MA), as did hamster neutralizing antibodies against IL-1{alpha}, IL-1ß, TNF-{alpha} and hamster IgG; biotin-conjugated mouse mAb against rat {kappa} and {lambda} light chains (anti-{kappa}/{lambda}) from Sigma; purified rat IgG and alkaline phosphatase-conjugated streptavidin from Zymed (South San Francisco, CA); FITC-conjugated goat anti-hamster IgG (H + L) from Southern Biotechnology Associates (Birmingham, AL); DAB substrate kit from Vector (Burlingame, CA); HistoMark Red from Kirkegaard & Perry (Gaithersburg, MD); and goat anti-rat IgG (H + L) microbeads from Miltenyi Biotec (Bergisch Gladbach, Germany). mAb against MGL (mAb LOM-14; IgG2b and mAb LOM-8.7; IgG2a) were prepared in our laboratory as described previously (6).

Preparation of conditioned medium
Conditioned medium of skin fragments was prepared as described previously (9). In brief, mouse abdominal skin was shaved using a small animal clipper and 200 µl of AD was epicutaneously applied (A:D = 1:1). Thirty minutes after application, a skin sample (typically 1 g) taken from the application site was cut into small pieces and placed in a cell culture dish (Falcon; Becton Dickinson, Franklin Lakes, NJ) containing 5 ml of FCS-free ASF 104 medium (Ajinomoto, Tokyo, Japan) with penicillin and streptomycin (100 µg/ml each) After incubation for 24 h at 37°C in a humidified atmosphere of 5% CO2/95% air, the supernatant was collected, centrifuged at 1000 r.p.m. for 10 min, sterilized by filtration through a membrane filter (0.2 µm pore size; Millipore, Bedford, MA) and stored at 4°C.

Skin organ culture
Skin explants were cultured in DMEM/Ham's F-12 medium supplemented with 10% FCS (culture medium) by a short-term Trowell-type organ culture method (ex vivo method) (16). Shaven mouse abdominal skin, sensitized or untreated, was excised and cut into skin explants (typically 1 cmx1 cm in size) with scissors under sterile conditions. Each 1 cm2 skin sample was placed on a polyethylene terephthalate membrane (pore size 1.0 µm; Falcon Cell Culture Inserts; Becton Dickinson) with the epidermal side facing up. The membrane bearing the skin sample was then placed in a well of a six-well cell culture plate (Falcon 3046; Becton Dickinson) and enough culture medium containing 50 ng/ml of recombinant cytokine was added until the skin sample was at the air/liquid interface. The sample was then cultured in a humidified atmosphere of 5% CO2/95% air at 37°C.

The inhibition of macrophage migration by anti-cytokine antibodies was examined as follows. Skin explants from untreated abdominal skin were incubated for 24 h in conditioned medium containing 100 µg/ml of each anti-cytokine antibody. A second experiment design was to pre-incubate skin explants from AD-treated abdominal skin in culture medium containing 100 µg/ml of each anti-cytokine antibody for 2 h, after which they were removed and untreated skin explants placed into the wells. The latter explants were incubated for an additional 24 h and then kept frozen for immunohistochemical studies.

Immunohistochemical detection of MGL+ cells
MGL+ cells in skin explants or in lymph nodes were stained with mAb LOM-14 as described previously (17). In brief, cryostat sections of skin explants or lymph nodes (10 µm thickness) were mounted on poly-L-lysine-coated glass slides and incubated with mAb LOM-14 for 16 h at 4°C without any fixation. Subsequently, the sections were fixed in 2% paraformaldehyde/0.1 M sodium phosphate (pH 7.0), washed and treated with biotin-conjugated mouse anti-rat {kappa}/{lambda} mAb [1/50 dilution in 3% BSA/DPBS (Dulbecco's PBS containing 0.91 mM CaCl2 and 0.49 mM MgCl2)]. This was followed by incubation with alkaline phosphatase–streptavidin (1/100 dilution in 3% BSA/DPBS) and HistoMark Red was used for detection. Cell nuclei were counterstained with Mayer's hematoxylin solution.

Quantification of MGL+ cell migration
Migration of MGL+ cells was quantified by counting the number of LOM-14-stained cells in treated and untreated skin explants. The number of MGL+ cells stained with mAb LOM-14 was counted under a microscope at a magnification of x400 within 50 different areas (70 µmx 95 µm rectangle) randomly selected in the dermis of triplicate tissue sections. MGL+ cell numbers were determined for each area and the results were expressed as mean number ± SEM (n = 50).

Statistical significance was evaluated by the Student's t-test.

Isolation of MGL+ cells from skin
MGL+ cells were isolated from skin fragments using mAb LOM-14 as previously described (8). Briefly, shaven abdominal skin was excised and cut into 1–2 mm of pieces by scissors. The skin fragments were then digested with 50 ml of 0.1% collagenase/0.01% DNase in FCS-free sterile DMEM/Ham's F-12 medium for 2 h at 37°C with continuous stirring. The tissue digests were then passed through three layers of nylon mesh to remove tissue fragments, after which the cells were centrifuged at 1000 r.p.m. for 10 min. The cells were suspended in 0.9 ml of 0.1% BSA/DPBS and incubated with 0.1 ml of LOM-14 hybridoma culture supernatant without dilution for 30 min at 4°C. The cells were then washed twice in 0.1% BSA/DPBS, resuspended in 0.8 ml of 0.1% BSA/DPBS and incubated with 0.2 ml of goat anti-rat IgG (H + L)-conjugated microbeads for 15 min at 8°C. Positive selection was carried out using an RS+ column with a magnetic cell sorter I (Miltenyi). Cells retained in the column were recovered by washing the column outside the magnetic field. Over 90% of the obtained cells were stained with digoxigenin-labeled mAb LOM-14 plus alkaline phosphatase-labeled antidigoxigenin antibody (1/100 dilution).

Detection of MGL protein in cell lysates
MGL+ cells isolated from the skin (1x 106) were incubated in FCS-free AFS 104 medium alone or with 50 ng/ml of IL-1ß. After incubation for 24 h, the cells were lysed in 100 µl of DPBS containing 1% Triton X-100, 0.02% NaN3, 0.1 µM aprotinin, 1 µM pepstatin A, 1 µM leupeptin and 1 mM PMSF, and kept on ice for 1 h. The suspension was centrifuged at 100,000 g for 30 min and the supernatant (cell lysate) was collected. The protein concentration in the lysate was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Proteins in the lysate were separated by SDS–PAGE (10% gel) under non-reducing condition and transferred to a PVDF membrane (Millipore, Bedford, MA) using a Milli Blot-SDE system (Millipore). To block non-specific antibody binding, the membrane was treated with PBS (10 mM sodium phosphate and 0.15 M NaCl, pH 7.2) containing 2% normal goat serum and 3% BSA for 18 h at 4°C. The membrane was then incubated with mAb LOM-14 (1/10 dilution of culture supernatant in PBS containing 0.2% Tween 20) for 90 min at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rat IgG (H + L) (1/1000 dilution in PBS/0.2% Tween 20) for 90 min at room temperature. Antibody binding was visualized using a DAB substrate kit (Vector).

Culture of MGL+ cells in type I collagen matrix
Isolated MGL+ cells (1x105) were suspended in 5 µl of FCS-free ASF 104 medium and mixed with 45 µl of Cellmatrix on ice. The mixture was dropped into a well of a 24-well cell culture plate (Falcon 3504; Becton Dickinson) and was incubated for 15 min at 37°C. After the mixture had gelled, it was incubated in FCS-free ASF 104 medium alone or with a cytokine. After incubation for 24 h, the cells were fixed with 1% paraformaldehyde/DPBS, washed twice with DPBS and stained with 0.2% crystal violet solution. After washing 3 times, the cells were observed by a microscope and photographed (Olympus, Tokyo, Japan).

Determination of IL-1ß concentrations in conditioned media
IL-1ß concentrations in conditioned media were assayed using a mouse IL-1ß ELISA Kit from R & D Systems (Minneapolis, MN).

Immunostaining of IL-1ß in skin explants
Immunostaining of intracellular IL-1ß was carried out as previously described (18,19) with some modifications. Skin explants were embedded in OCT compound (Mites, Elkhart, IN) and directly frozen in liquid nitrogen. Cryostat sections (10 µm thickness) were mounted on poly-L-lysine-coated glass slides and fixed in 4% paraformaldehyde/0.1 M sodium phosphate (pH 7.0) for 20 min at room temperature. The cell membrane was permeabilized by treating the sections with 0.1% saponin/DPBS for 30 min at room temperature. The slides were then washed 3 times in DPBS and incubated with anti-IL-1ß mAb (5 µg/ml diluted in 0.1% saponin/DPBS) for 16 h at 4°C. The slides were washed 3 times with 0.1% saponin/DPBS, incubated with normal goat serum diluted 1/100 in 0.1% saponin/DPBS for 15 min to block non-specific antibody binding and stained with FITC-conjugated goat anti-hamster IgG (H + L) (1/100 dilution in 0.1% saponin/DPBS) for 30 min at room temperature. After washing twice with 0.1% saponin/DPBS, the specimens were mounted in Vectashield (Vector) and observed with a confocal microscope (MRC-1024; BioRad, Hercules, CA) equipped with a krypton/argon laser.


    Results
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 Abstract
 Introduction
 Methods
 Results
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 References
 
Effect of recombinant IL-1ß, IL-1{alpha} or TNF-{alpha}, or antibodies against these cytokines, on loss of MGL+ cells from skin explants
In our previous papers (8,9), we showed that MGL+ cells in AD-treated dermis decrease in number and that this is the first step in the trafficking of these cells to the lymph node. We also showed that soluble factors locally available in the skin at the site of sensitization are involved in this process, as medium conditioned by incubation with AD-treated skin could induce emigration of MGL+ cells from naive skin samples. The study also suggested that these factors are inflammatory cytokines but not chemokines. Here we assessed whether IL-1{alpha}, IL-1ß or TNF-{alpha} are some of these soluble factors. Explants from AD-untreated skin were subjected to organ culture in the presence of 50 ng/ml of recombinant IL-1ß, IL-1{alpha} or TNF-{alpha}. The density of MGL+ cells in the dermis of the explants was immunohistochemically identified and quantified. This has been the most quantitative and reproducible method to assess the emigration of MGL+ cells from skin in vivo and ex vivo. As shown in Figs 1Go(a–c) and 2Go, MGL+ cells are abundant in the dermis before organ culture. The density starts to decrease at 4 h (data not shown) and then continues to decrease over the period of the organ culture. However, after 24 h culture of untreated skin explants in the presence of 50 ng/ml of either IL-1ß, IL-1{alpha} or TNF-{alpha}, the density of MGL+ cells in the dermis of skin explants is significantly decreased as compared to the explants cultured in the absence of these cytokines (Fig. 2aGo). Furthermore, when skin that had been treated with AD before excision was cultured in the presence of neutralizing antibodies to IL-1ß, IL-1{alpha} or TNF-{alpha}, the reduction in MGL+ cells caused by AD sensitization was abrogated (Fig. 2bGo). Finally, when untreated skin was cultured in conditioned medium obtained from skin treated in vivo with AD, the loss of MGL+ cells was eliminated if the antibodies to IL-1ß, IL-1{alpha}, or TNF-{alpha} were added (Fig. 2Go). These experiments show that these three cytokines are likely to be involved in MGL+ cell emigration from sensitized skin.



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Fig. 1. (a–c) Immunostaining of MGL+ cells in skin explants cultured ex vivo in media containing IL-1ß. Excised skin explants were cultured ex vivo in a culture medium containing IL-1ß (50 ng/ml) for 0 (a), 8 (b) or 12 (c) h. Frozen sections of the skin explants were stained with mAb LOM-14 (red) and counterstained with hematoxylin (purple). MGL+ cells present in the upper dermis at 0 h (a) disappeared after the culture for 8–12 h (b and c). Each scale bar indicates 20 µm. (d–h) Effect of IL-1{alpha}, IL-1ß and TNF-{alpha} on the morphology of isolated MGL+ cells cultured in a collagen matrix. MGL+ cells (1x 105) in a type I collagen matrix gel were incubated in FCS-free ASF 104 medium containing IL-1{alpha}, IL-1ß or TNF-{alpha} for 24 h at 37°C and stained with 0.2% crystal violet solution. Micrographs of the cells not cultured (d), cultured in the medium alone (e) or incubated in the medium containing 50 ng/ml of IL-1ß (f), 50 ng/ml of IL-1{alpha} (g) and 50 ng/ml of TNF-{alpha} (h) are shown. The cytokines tested induced morphological changes of MGL+ cells. Each scale bar indicates 20 µm.

 


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Fig. 2. Loss of MGL+ cells in skin explants. (a) Effect of IL-1{alpha}, IL-1ß or TNF-{alpha} on untreated skin explants. Excised skin explants were incubated in culture media containing 50 ng/ml of IL-1{alpha}, IL-1ß or TNF-{alpha} for 24 h. Data are presented as MGL+ cell numbers in the skin sections. The bar represents the mean ± SEM of MGL+ cells relative to the number of nucleated cells. The number of MGL+ cells in the dermis significantly decreased after incubation with IL-1ß, IL-1{alpha} or TNF-{alpha} (*P < 0.005). (b) Inhibitory effects of anti-cytokine antibodies on the migration of MGL+ cells from AD-treated skin explants. Skin explants excised from AD-treated skin were pre-incubated in culture medium alone (shaded bar), or media containing 100 µg/ml of anti-IL-1ß mAb (diagonally hatched bar), anti-IL-1{alpha} mAb (vertically hatched bar), anti-TNF-{alpha} mAb (horizontally hatched bar) or hamster IgG as a control (cross-hatched bar) for 2 h. After washing, the explants were cultured for 24 h. As a control, explants of untreated skin were excised from the same mouse and incubated in culture medium alone (open bar). The bar represents the mean ± SEM of MGL+ cells. The anti-cytokine antibodies had significant inhibitory effects (*P < 0.005). (c) Inhibitory effects of anti-cytokine antibodies on MGL+ cell migration induced by media conditioned by culturing AD-treated skin. Untreated skin explants were incubated in unconditioned medium alone (open bar), in conditioned medium alone (shaded bar), or in conditioned medium containing 100 µg/ml of anti-IL-1ß mAb (diagonally hatched bar), anti-IL-1{alpha} mAb (vertically hatched bar), anti-TNF-{alpha} mAb (horizontally hatched bar) or hamster IgG as a control (cross-hatched bar) for 24 h. Data are presented as MGL+ cell numbers in the skin sections. The bar represents the mean ± SEM of MGL+ cells. Inhibitory effects of the anti-cytokine antibodies were significant (*P < 0.005).

 
Detection of IL-1ß in conditioned medium prepared from AD-sensitized skin
As more evidence exists for a role of IL-1ß in trafficking of cells from skin, we further studied the role of IL-1ß in our system. To determine whether IL-1ß is present in the conditioned medium of AD-sensitized skin, we took advantage of the fact that AD treatment sensitizes only when applied onto skin as being a part of the live animal but not if applied in vitro onto skin samples after taken from a mouse (8). This phenomenon was also observed with activity of the conditioned medium to induce migration of MGL+ cells. As measured by ELISA, immunoreactive IL-1ß could not be detected in conditioned medium prepared from either control untreated skin or skin treated with AD in vitro, while 4.5 ng/ml of IL-1ß was present in conditioned medium from skin treated with AD in vivo (Table 1Go). The amount of IL-1ß in the conditioned media correlated with the extent of loss of MGL+ cells from the explants (data not shown)


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Table 1. Detection of IL-1ß production in conditioned media
 
Detection of cells that produce local IL-1ß after AD application
To investigate which are the cells producing IL-1ß in the skin after sensitization, the presence of cell-associated IL-1ß was immunohistologically assessed under a confocal microscope using cryostat sections of AD-treated skin explants permeabilized with saponin. This technique allows IL-1 production at a single-cell level to be detected (20). Skin was treated with AD, and cultured for 0, 2, 4 and 8 h. Cells containing detectable levels of IL-1ß were found in the epidermis of untreated skin and by 4 h after treatment the number of such cells was markedly increased (Fig. 3AGo). It is noteworthy that the epidermis was not uniformly stained with anti-IL-1ß mAb. Rather, a scattered distribution of IL-1ß-producing cells was observed. It is also evident that cells in the dermis were not stained at this stage. After 8 h of organ culture, however, IL-1ß in the epidermis became undetectable (Fig. 3AGo).



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Fig. 3. (A) Detection of IL-1ß-producing cells in AD-treated skin. Mouse abdominal skin was epicutaneously applied with AD and excised. The skin explants were cultured for 0 (a), 2 (b), 4 (c) and 8 (d) h. Frozen sections of skin explants were stained with anti-IL-1ß plus FITC-conjugated goat anti-hamster IgG (H + L) and observed using a confocal microscope. Hamster IgG was used as a negative control (data not shown). IL-1ß-producing cells (indicated as red arrows in b and c) were detected in the epidermis at 4 h (c). Each scale bar indicates 50 µm. (B) Effect of intradermal injection of IL-1ß on appearance of MGL+ cells in draining lymph nodes. Mice were intradermally injected with mouse IL-1ß (25 ng per limb) into their forelimbs and brachial lymph nodes were dissected at 0 (a), 4 (b), 12 (c) and 24 (d) h after the injection. Frozen sections of the lymph nodes were stained with mAb LOM-14 (red). B, B cell area; T, T cell area; S, subcapsular sinus. The position of the T cell area was confirmed by staining the serial section with anti-CD4 antibody (data not shown). The injection of IL-1ß resulted in the accumulation of MGL+ cells in the lymph node, especially in its T cell area. Each scale bar indicates 20 µm.

 
Increase of MGL+ cells in regional lymph nodes after intradermal administration of IL-1ß
That IL-1ß induces trafficking of MGL+ cells was further confirmed by intradermal injection of recombinant mouse IL-1ß (50 ng protein) into forelimb skin. Regional lymph nodes were obtained at 4, 12 and 24 h after administration, and the presence of MGL+ cells was assessed by immunohistochemistry. In untreated mice, the distribution of MGL+ cells was restricted to the lymph node subcapsular sinus (Fig. 3BGo) and medulla (not shown) as described previously (8). After injection of IL-1ß, however, many MGL+ cells were seen in the T cell area of the lymph node paracortex (Fig. 3BGo). The intensity of staining with mAb LOM-14 peaked 24 h after the IL-1ß injection (Fig. 3BGo). These changes were similar to that seen in the regional lymph nodes after epicutaneous sensitization, particularly sensitization with allergen combined with AD as described previously, but more rapid and intense (8). The results also support the hypothesis that IL-1ß locally released in the skin environments can induce trafficking of MGL+ cells to regional lymph nodes.

IL-1ß does not down-regulate expression of MGL in cells isolated from skin
The results described both above and in our previous paper (9) suggest that the decrease of MGL+ cells from the dermis at the site of AD application may be due to trafficking of MGL+ cells to draining lymph nodes, as previously proposed (8). However, it is still possible to argue that the decrease of MGL signals in the dermal cells could be due to down-regulation of MGL expression induced by soluble factors such as IL-1ß. To rule out this possibility, MGL+ cells were purified from untreated skin and cultured in the presence or absence of 50 ng/ml of IL-1ß. The expression of MGL in the cell lysates was assessed by immunoblot analysis using the LOM-14 mAb specific for the MGL marker. The 42 kDa band intensity for cell lysates cultured in the presence of IL-1ß for 24 h was equivalent to those of freshly isolated cell lysates and cell lysates from control cultures (Fig. 4Go). These results indicate that IL-1ß does not down-regulate MGL expression in MGL+ cells.



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Fig. 4. Detection of MGL protein on isolated MGL+ cells after IL-1ß treatment by Western blotting. MGL+ cells isolated from mouse abdominal skin by MACS were incubated in FCS-free ASF 104 medium alone or with IL-1ß (50 ng/ml). Cell lysates were separated by SDS–PAGE (10% gel) under non-reducing conditions, transferred to PVDF membranes and stained with mAb LOM-14. Lane 1, isolated cells before culture; lane 2, cells cultured in ASF 104 medium for 24 h; lane 3, cells cultured for 24 h in ASF 104 medium containing 50 ng/ml of IL-1ß. The addition of IL-1ß did not alter the expression level of MGL. The position of a marker (aldolase, 42 kDa) is shown on the left (arrowhead). Lysates from equal numbers of cells were loaded in each lane.

 
IL-1{alpha}, IL-1ß and TNF-{alpha} induce morphological changes in MGL+ cells
When skin explants are cultured in the presence of IL-1{alpha}, IL-1ß or TNF-{alpha}, morphological changes are induced in MGL+ cells in the dermis and s.c. tissue in that the cells adopted a more extended form after 12 h of culture (data not shown). In our previous paper, we observed that AD treatment induced similar morphological changes in MGL+ cells in the dermis in vivo (9). To determine if IL-1{alpha}, IL-1ß or TNF-{alpha} affect the morphology of MGL+ cells directly, we placed isolated MGL+ cells in an artificial connective tissue-like environment represented by a type I collagen matrix gel. Thus, MGL+ cells isolated from untreated skin were cultured in the collagen matrix in the absence or the presence of 50 ng/ml of either IL-1ß, IL-1{alpha} or TNF-{alpha}. Some cells migrated out off the collagen gel matrix droplet in the presence of cytokines (Fig. 1f–hGo) while few cells were found outside the matrix in the absence of cytokine (Fig. 1eGo). Those MGL+ cells migrating out of the matrix in the presence of IL-1ß, IL-1{alpha} or TNF-{alpha} (Fig. 1f–hGo respectively) all displayed a dendritic shape with lamellipodia. In contrast, cells cultured without cytokines retained a round shape (Fig. 1eGo), similar to freshly isolated MGL+ cells (Fig. 1dGo).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In our previous paper, we found a correlation between the initiation of MGL+ cell trafficking during the sensitization phase of DTH by directly observing the skin site (9). Namely, the number of cells expressing MGL in the skin is markedly decreased upon treatment with AD, a strong adjuvant for DTH sensitization. The MGL+ cells were likely to be histiocytic macrophages. In this study, we also established a skin organ culture system to allow us to detect the decrease of MGL+ dermal cells in vitro, and by doing so found that IL-1ß and IL-1{alpha} might act as the cytokines initiating the migration of these cells.

In the present paper, we have shown that IL-1ß and IL-1{alpha} are indeed involved in MGL+ cell migration, and that TNF-{alpha} also acts similarly. We first demonstrated that the presence of recombinant IL-1ß, IL-1{alpha} or TNF-{alpha} in the medium, in which an untreated skin explant is cultured, induces the loss of MGL+ cells from the dermis (Fig. 2aGo). TNF-{alpha} appears to be less potent than IL-1ß at equivalent protein concentrations. In addition, when AD-treated skin explants are cultured in the presence of mAb to these three cytokines, the loss of MGL+ cells from the dermis is abrogated (Fig. 2bGo). As a final demonstration of the involvement of these cytokines, we cultured untreated skin explants in medium that had been conditioned by preculture with treated skin. Normally, this conditioned medium induces loss of MGL+ cells from the dermis of the untreated skin, but when mAb to IL-1ß, IL-1{alpha} or TNF-{alpha} were included, the loss was inhibited (Fig. 2cGo). These observations thus strongly suggest that IL-1ß, IL-1{alpha} and TNF-{alpha} are all involved in the disappearance of MGL+ cells from the dermis after sensitization for DTH.

Subsequently we focused particularly on the role of IL-1ß because of published evidence suggesting that this cytokine might play a central role in initiating cell trafficking from the skin. Thus, IL-1ß is known to be increased in skin sites upon exposure to allergen (21), mRNA corresponding to IL-1ß is detectable as early as 15 min after allergen exposure (22) and IL-1ß is known to play a critical role in the migration of LC (10,11,13). The evidence implicating TNF-{alpha} and IL-1{alpha} is somewhat weaker. TNF-{alpha} (10,12,23) and IL-1{alpha} (11,1315) are both suggested to induce LC migration, although the role of IL-1{alpha} is still controversial. Both cytokines are mainly produced by keratinocytes during sensitization of DTH (22).

We found that AD treatment induces IL-1ß secretion into the conditioned medium as measured by an ELISA (Table 1Go). Only conditioned medium obtained from culturing skin that had been treated with AD prior to excision contained IL-1ß. Skin treated with AD after excision failed to secrete measurable IL-1ß, as did untreated skin. These data are in accordance with the observation that AD treatment sensitizes only when applied onto skin but not when applied in vitro onto excised skin. We also found that when AD-untreated mice are injected with recombinant IL-1ß, an increase in MGL+ cell numbers in the T cell area of the draining lymph nodes could be observed (Fig. 3BGo). This is similar to what is observed in the draining lymph nodes after skin sensitization with AD (Fig. 3AGo) (8). These data strongly suggest that locally available IL-1ß not only initiates macrophage migration from dermis but is also directly responsible for the completion of the entire trafficking process that culminates with the appearance of MGL+ cells in the draining lymph nodes.

We established single-cell culture experiments to assess how IL-1ß affects MGL+ cells in the absence of effects from the tissue environment and from interactions with other types of cells. We had previously observed that in vivo AD treatment induced changes in dermal MGL+ cells in that they became more extended and appeared to have increased motility 4–8 h after sensitization (9). To assess if IL-1ß could induce similar changes, MGL+ cells were isolated from untreated skin and cultured within a collagen matrix that mimics the dermal environment. Upon application of recombinant IL-1ß to the culture, cells were found to leave the collagen matrix. These cells had altered from being round cells to having a dendritic appearance with lamellipodia, which is indicative of increased cell motility (Fig. 1fGo). IL-1{alpha} and TNF-{alpha} also induced similar changes (Fig. 1g and hGo), whereas culture in the absence of these cytokines did not cause such changes (Fig. 1eGo). It has recently been reported that dermal dendritic cells with high levels of MHC class II expression will migrate upon stimulation with IL-1ß (24). The relationship between MGL+ cells and dermal dendritic cells is an interesting issue for future studies. Our preliminary studies using bone marrow-derived mature dendritic cells have suggested, however, that MGL expression on these cells is low.

Finally, we showed that IL-1ß is available in the skin environment at an early stage after sensitization with AD, as cells containing intracellular IL-1ß could be detected in the epidermis as early as 4 h after AD treatment (Fig. 3AGo). In situ hybridization experiments to determine precisely how early IL-1ß mRNA transcription is initiated upon AD treatment are currently being planned. Notably, while IL-1ß-producing cells were detected within the epidermis at 4 h, they appeared to have migrated down to the dermis by 8 h. The initial scattered distribution of the IL-1ß-producing cells within epidermis and their subsequent migration suggest that LC may be the main producers of IL-1ß upon AD treatment. This conclusion is consistent with earlier reports indicating that LC produce IL-1ß upon skin sensitization (22).

While we have mainly focused on IL-1ß in this study, other pro-inflammatory cytokines as IL-1{alpha} and TNF-{alpha} are most likely also involved in MGL+ cell trafficking, as suggested by data presented here. Furthermore, it is possible that cytokines act in concert during sensitization because intradermal injection of IL-1ß has been reported to enhance IL-1{alpha}, IL-1ß, TNF-{alpha}, MIP-2 and IL-10 mRNA expression in skin (11). IL-1ß and TNF-{alpha} have also been reported to promote dermal dendritic cell migration in an interdependent manner (24). Further studies are needed to clarify whether these cytokines are available at the sensitization site and which cells produce them.

In conclusion, we have demonstrated that pro-inflammatory cytokines, particularly IL-1ß, are involved in initiating the trafficking of MGL+ cells during DTH sensitization with an allergen solvent. The trafficking might be occurring as a conditioned process with LC migration, which is also regulated by pro-inflammatory cytokines. Further study in this area should permit us to understand how the cytokine cascade during contact sensitization is regulated and how its outcome can be modulated with the ultimate goal being to prevent contact hypersensitivity by blocking the cytokine cascade.


    Acknowledgments
 
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (07407063, 07557154, 09254101, 11557180 and 11672162), the Research Association for Biotechnology, the Program for Promotion of Basic Research Activities for Innovative Biosciences, and the Cosmetology Research Foundation. We thank Ms Chizu Hiraiwa for her assistance in preparing this manuscript.


    Abbreviations
 
AD mixture of acetone and dibutylphthalate
DPBS Dulbecco's PBS
DTH delayed-type hypersensitivity
LC Langerhans cells
MGL macrophage calcium-type lectin
TNF tumor necrosis factor

    Notes
 
Transmitting editor: M. Miyasaka

Received 17 June 2000, accepted 28 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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