Materials and methods

Media and chemicals

MgCl₂ hexahydrate, NaCl, NH₄SCN, Trisma maleat, and sucrose were supplied from Sigma (St Louis, MO). Acetone, HCl, NaOH, and NaHCO₃ were obtained from Merck (Darmstadt, Germany). HEPES buffer solution, phosphate-buffered saline (PBS) and Hanks balanced salts solution (HBSS) w/o Ca²⁺/Mg²⁺ were purchased from Life Technologies (Paisley, Scotland). DNAse I, dispase, and adenosine triphosphate (ATP) were obtained from Boehringer (Mannheim, Germany). Trypsin was obtained from Gibco (Eggenstein, Germany). Complete RPMI 1640 (c-RPMI) was supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin (all from Gibco). Formaldehyde 36.5%, MgSO₄, and Pb(NO₃)₂ were supplied from Riedel-de-Haen (Seelze, Germany). Cacodylate acid (NH₄)₂S and Tween were obtained from Fluka (Buchs, Switzerland). Tris was purchased from Paesel & Lorei (Frankfurt, Germany).

Monoclonal antibodies

(MoAb) Anti-HLA-DR (clone HB 145) and anti-CD11b MoAb (clone OKT-6) (ATCC, Rockville, MD) were used for negative selection enrichment of T cells. Sheep anti-mouse IgG MoAb coupled with Dynal beads were from Dynal (Hamburg, Germany). Mouse anti-human HLA-DR MoAb (-chain, clone TAL.1B5) and the labeled streptavidin–biotin staining kit system 500 (Dako, Glostrup, Denmark) were used for in vivo staining of Langerhans cells. For in vitro staining of Langerhans cells, the following MoAb and isotype controls were used: phycoerythrin (PE) -conjugated anti-HLA-DR (clone L243, mouse IgG2a) and PE-conjugated IgG2a control MoAb; fluorescein isothiocyanate (FITC) -conjugated anti-B7–1 (clone BB1, mouse IgM) and anti-B7–2 (clone 2331, mouse IgG1) and FITC-conjugated IgM and IgG1 control MoAb (all from Pharmingen, San Diego, CA).

Irradiation

UV radiation between 270 and 400 nm, peaking at 310 nm was delivered from 10 fluorescent UVB lamps, Philips TL20W/12 (Philips GmbH, Hamburg, Germany), housed in a UV 800 unit (Waldmann GmbH, VS-Schwenningen, Germany). UVB irradiance (280–320 nm) at the surface of the test areas was measured with a calibrated radiometer equipped with a SCS 280 photodetector (International Light, Newburyport, MA) and was 2.5 mW per cm² at a tube to target distance of 40 cm. For determination of the minimal erythema dose (MED) UVB irradiation was administered in gradually increasing doses (15, 30, 60, 90, 115, and 145 mJ per cm²). Erythema was determined by visual assessment 24 h after irradiation as described (^{Schempp et al. 1999}).

Subjects

The protocol of the controlled prospective study was approved by the local ethics committee and written informed consent was obtained from all subjects who participated in the study. In part 1 of the study (enumeration of epidermal Langerhans cells in epidermal sheets) 12 healthy volunteers (age range 18–56 y; skin types II and III) with no history of skin disease or photosensitivity were enrolled. Six subjects (age range 20–37 y; skin types II and III) participated in part 2 of the study (mixed epidermal cell lymphocyte reaction, MECLR). The skin type was determined according to the classification of sun reactive skin types by^{Fitzpatrick 1988}). Split skin from dermatologic surgery was used for the in vitro experiments [antigen-presenting assays, fluorescence-activated cell sorter (FACS) analysis, cytokine release].

Staining of Langerhans cells in vivo

Circular test areas (2 cm in diameter) on the volar aspects of the forearms of 12 volunteers were treated on four consecutive days as follows (^{Schempp et al. 1997}): 1 = 5% MgCl₂ solution (15 min); 2 = 5% MgCl₂ solution (15 min) followed by UVB irradiation (1 MED); 3 = 5% NaCl solution (15 min); 4 = 5% NaCl solution (15 min) followed by UVB irradiation (1 MED); 5 = UVB irradiation only (1 MED); 6 = untreated skin. Twenty-four hours after the last treatment punch biopsies (6 mm in diameter) were obtained from the center of the test areas. Biopsies were incubated in dispase working-solution (dispase, 5.0 g; HBSS, 1000 ml; HEPES buffer, 25 ml; pH 7.0 1 M NaHCO₃) for 2 h at 37°C, the epidermis was removed and washed in ice-cold PBS for 30 min. Epidermal sheets were divided into two halves. One half was stained for adenosine triphosphatase (ATPase) and the other half for HLA-DR expression of Langerhans cells.

Adenosine triphosphatase staining for Langerhans cells

The ATPase staining of Langerhans cells was a modification of the procedure described by^{Juhlin & Shelley, (1977)}. Briefly, epidermal sheets were washed three times in Tris buffer (Trisma maleat, 23.72 g; aqua dest. 500 ml; sucrose 34.35 g; pH 7.3 NaOH) and fixed for 20 min at 4°C in 0.05 M cacodylate–formaldehyde solution (0.2 M cacodylate acid; sucrose 6.85 g; 36.5% formaldehyde, 10 ml; aqua dest., 50 ml). After fixation, samples were washed three times in Trisma buffer and incubated at 37°C for 60 min in a ATP-Pb-containing solution (ATP, 10 mg; Trisma buffer 42 ml; 5% MgSO₄, 5 ml; 2% Pb(NO₃)₂, 3 (ml). The samples were then washed again in Trisma buffer and immersed for 5 min in 22% ammonium sulfide solution. The stained specimens were mounted in Kaiser's glycerol gelatine (Merck).

HLA-DR staining for Langerhans cells

Epidermal sheets were fixed in acetone for 30 min at 4°C, were then washed at 4°C three times in 1 M PBS and incubated in Tris-buffered saline (0.05 M Tris, 100 ml; 0.85% NaCl, 900 ml) for 5 min. The specimen were then incubated for 90 min at 37°C in a 1:100 dilution of the primary anti-HLA-DR antibody. After washing three times in Tris-buffered saline the labeled streptavidin–biotin staining was performed according to the manufacturer's instructions (labeled streptavidin–biotin kit, Dako, Hamburg, Germany). The stained specimens were mounted in Kaiser's glycerol gelatine (Merck).

Enumeration of Langerhans cells

The numbers of ATPase⁺ and HLA-DR⁺ epidermal cells were determined by the same investigator using a Olympus T2 microscope (Olympus Optical Co., Tokyo, Japan). Four randomly selected fields from each specimen were counted at 200 magnification using a WHK 10  20 L-H optical grid (Olympus) to determine the number of Langerhans cells per mm². Blinded control samples were enumerated by a second investigator. This control enumeration yielded identical results when compared with the first observer (not shown). Analysis of variance (ANOVA) (BMDP statistical software, Los Angeles, CA) was performed, and single variables were compared using the Wilcoxon signed rank test with Bonferoni–Holm correction. p  0.05 were considered significant. Each point in Figure 2 represents the mean Langerhans cells frequency per mm² epidermis per volunteer.

Figure 2.

Magnesium ions reduce ATPase⁺ and HLA-DR⁺ Langerhans cells in vivo. The skin was treated and epidermal sheets were stained for HLA-DR (a) or ATPase (b) expression by epidermal Langerhans cells and the number of Langerhans cells per mm² was determined as detailed in Figure 1 and Materials and Methods. Each point represents the mean Langerhans cells frequency per mm² epidermis per volunteer (n = 12). Langerhans cells numbers are reduced by MgCl₂, UVB, MgCl₂ + UVB, and NaCl + UVB, but not by NaCl only. For statistical analysis see Table 1.

 

Antigen-presenting cell assay

Epidermal cell suspensions

Epidermal cell suspensions were generated by limited trypsinization of epidermal sheets as described (^{Weiss et al. 1995;Schempp et al. 2000}). Trypsin was used in a concentration of 0.25% in PBS supplemented with 80 U per ml DNAse to dissociate the epidermis of suction blisters. Epidermal cell were washed twice in PBS (4°C, 1200 U per min) and were further cultured in c-RPMI.

Preparation of T cells

Allogeneic resting T cells were enriched from heparinized blood by plastic adherence and immunomagnetic depletion with anti-HLA-DR and CD11b primary antibodies and second step sheep anti-mouse IgG MoAb coupled to Dynal beads (^{Dittmar et al. 1999}). The resulting T cells were >85% CD3⁺ as determined by FACS analysis. T cells were cultured in c-RPMI.

MECLR

Epidermal cells (5  10⁴) were cocultured in triplicates with allogeneic T cells (1  10⁵) in c-RPMI in 96-well round-bottom microtiter plates (Costar, Cambridge, MA) for 6 d (5% CO₂, 37°C, 168 h). [³H]-thymidine (1 Ci) was added to each well for the final 16 h of coculture. Plates were harvested with a Canberra Packard Filter Mate (Canberra Packard, Frankfurt, Germany) and incorporation of [³H]thymidine was determined by liquid scintillation spectroscopy using a Top-Count (Canberra Packard).

MECLR in vivo

Circular test areas (2 cm in diameter) on the volar aspects of the forearms of six volunteers were treated on four consecutive days as follows: 1 = 5% MgCl₂ solution (15 min); 2 = 5% MgCl₂ solution (15 min) followed by UVB irradiation (1 MED); 3 = UVB irradiation only (1 MED); 4 = untreated skin. Twenty-four hours after the last treatment suction blisters (1.5 cm in diameter) were raised on the centers of the test areas as described (^{Kiistala & Mustakallio, 1967}). Epidermal cell suspensions were prepared and an MECLR was performed as described above. Background values (T cells only, epidermal cell only) were subtracted from epidermal cell + T cell values. The influence of variables on proliferation rates was assessed with analysis of variance (ANOVA). p  0.05 were considered significant.

MECLR in vitro

Epidermal cell suspensions were prepared from split skin by incubation in dispase working solution as described above. Epidermal cells were preincubated for 24 h in the presence or absence of 1% MgCl₂. This MgCl₂ concentration was not toxic in vitro as determined by propidium iodide staining (not shown). Epidermal cell suspensions were washed and MECLR assays were further performed as described above.

Immunostaining and flow cytometry

Epidermal cell suspensions were incubated in the presence or absence of 1% MgCl₂ for 24 h. After washing two times in PBS (4°C, 1200 U per min), the cells were stained for two color FACS analysis in PBS (4°C) with the following MoAb: PE-labeled anti-HLA-DR MoAb, FITC-labeled anti-B7–1 or B7–2 MoAb, and appropriate isotype control MoAb. Epidermal cell suspensions (5  10⁴ cells per sample) were analyzed by FACScan using the CellQuest software (Becton Dickinson). The percentage of gated HLA-DR⁺/B7⁺ cells was determined with the CellQuest software.

Cytokine detection

Epidermal cell suspensions (1  10⁶ cells per ml) were incubated in the presence or absence of 1% MgCl₂ for 24 h. Supernatants were collected and were stored at -80°C. Specific cytokine enzyme-linked immunosorbent assays with recombinant human primary MoAb for TNF-, interleukin (IL) -1, IL-10, and IL-12 were performed according to the manufacturer's instructions (R&D Systems, Wiesbaden, Germany).

Results

MgCl₂ but not NaCl reduces the expression by Langerhans cells of ATPase and HLA-DR in vivo

We investigated if the number of epidermal ATPase⁺ and HLA-DR⁺ Langerhans cells was influenced by in vivo application of a 5% MgCl₂ solution. For comparison the skin was treated with a 5% NaCl solution, with UVB irradiation and with salt solutions followed by UVB irradiation. An example of HLA-DR staining for Langerhans cells in epidermal sheets is shown in Figure 1. The results of all volunteers are illustrated in (Figure 2a) (ATPase) and in (Figure 2b) (HLA-DR). Pairwise comparison of variables with the Wilcoxon test (Bonferoni-Holm corrected) revealed a significant reduction of both ATPase⁺ and HLA-DR⁺ Langerhans cells by UVB alone or MgCl₂ alone, whereas NaCl had no effect (Table 1). The combination of MgCl₂ + UVB or NaCl + UVB also significantly reduced the number of epidermal Langerhans cells, but only MgCl₂ + UVB had an additive effect when compared with UVB alone (Table 1).

Figure 1.

Effects of magnesium ions and UVB on the expression by Langerhans cells of HLA-DR molecules in vivo. The skin was treated on four consecutive days with a 5% MgCl₂, 5% NaCl solution (15 min), UVB irradiation (1 MED) alone or with salt solutions followed by UVB irradiation. The epidermis was separated from punch biopsies by dispase treatment and the sheets were stained for HLA-DR expression on epidermal Langerhans cells. The number of Langerhans cells is similar in untreated (a) and NaCl-treated (b) skin. By contrast, HLA-DR⁺ Langerhans cells are reduced by MgCl₂ (c), UVB (d), MgCl₂ + UVB (e), and NaCl + UVB (f). Scale bar: 20 m.

 

Magnesium ions inhibit the allostimulatory capacity of Langerhans cells in vivo

To investigate whether the depletion of Langerhans cells by in vivo MgCl₂ application was related to effects on the antigen-presenting cell function of Langerhans cells we applied 5% MgCl₂ with or without subsequent UVB irradiation (1 MED) to the sun-protected forearm of six volunteers on four consecutive days. Untreated skin and skin treated with UVB alone served as controls. Twenty-four hours after the last application, epidermal cell suspensions were prepared from suction blisters and were analyzed for their capacity to stimulate the proliferation of alloreactive naive T cells. Epidermal cell suspensions from untreated skin were fully capable of stimulating the proliferation of allogeneic T cells. By contrast, epidermal cells showed a significantly reduced capacity to stimulate alloreactive T cell responses following MgCl₂ (p = 0.006), UVB (p = 0.0001), and MgCl₂ + UVB (p = 0.0001) treatment (ANOVA) (Table 2).

Table 2 - In vivo application of magnesium ions inhibits the alloantigen-presenting function of epidermal cells .

Subject no.	Untreated	MgCl2 (p = 0.006) *	UVB (p = 0.0001) *	MgCl2 + UVB (p = 0.0001) *
1	90.363  1.920	58.619  6.026	23.799  3.584	8.089  1.174
2	62.717  7.978	29.753  170	41.014  5.588	24.414  5.043
3	68.783  916	60.660  10.904	46.961  8.724	2.603  1.263
4	50.687  3.621	32.395  646	27.161  3.369	17.405  6.451
5	71.903  5.761	75.381  3.312	7.341  1.259	3.999  1.054
6	38.418  9.903	24.466  5.249	27.871  2.296	24.284  5.587

^a Six subjects were treated on their volar forearms, epidermal cell suspensions were prepared and a mixed epidermal cell leukocyte reaction (MECLR) was performed as detailed in the Methods. Background values (epidermal cell alone, T cells alone) were subtracted from epidermal cell + T cells values and the mean  SD of triplicate measurements is indicated..

^* p-value compared with untreated control (ANOVA).

 

Magnesium ions inhibit the allostimulatory capacity of Langerhans cells in vitro

Next we questioned whether the reduced allostimulatory capacity of epidermal cell suspensions after in vivo MgCl₂ application was primarily caused by the emigration of Langerhans cells from the epidermis. To address this issue we preincubated epidermal cells with MgCl₂in vitro before coculture with allogeneic T cells – a model that does not allow the Langerhans cells to emigrate. Again, epidermal cell suspensions from untreated skin stimulated the proliferation of alloreactive T cells. In contrast, epidermal cells pretreated with MgCl₂ showed a significantly reduced capacity to stimulate T cell proliferation (Figure 3).

Figure 3.

Magnesium ions inhibit the alloantigen-presenting function of Langerhans cells in vitro. Epidermal cells were generated from split skin and cells from the same cell preparation were preincubated in the presence or absence of 1% MgCl₂in vitro for 24 h. Subsequently, the cells were washed and epidermal cell (5  10⁴) were cocultured with allogeneic T cells (1  10⁵) for 6 d. Cell proliferation was determined by [³H]thymidine incorporation. The mean  SD of three independent experiments is shown.

 

Magnesium ions inhibit the expression by Langerhans cells of HLA-DR and B7 molecules in vitro

To investigate whether the reduced allostimulatory capacity of epidermal cell suspensions was associated with an altered expression by Langerhans cells of HLA-DR or costimulatory B7 molecules in vitro, we incubated epidermal cell suspensions in the presence or absence of 1% MgCl₂ for 24 h. Two color FACS analysis with PE-labeled anti-HLA-DR MoAb and FITC-labeled anti-B7–1 or B7–2 MoAb of a representative experiment is shown in Figure 4. Untreated epidermal cell suspensions contained 0.8% HLA-DR⁺ Langerhans cells (Figure 4b). These HLA-DR⁺ cells additionally expressed B7–1 (Figure 4c) and B7–2 (Figure 4d) molecules. Preincubation of epidermal cells with 1% MgCl₂ almost completely suppressed the expression of HLA-DR and B7 molecules on Langerhans cells (Figure 4e,f).

Figure 4.

Magnesium ions inhibit the expression by Langerhans cells of HLA-DR and B7 molecules in vitro. Epidermal cells from the same cell preparation were preincubated in the absence (a–d) or presence (e, f) of 1% MgCl₂in vitro for 24 h. Subsequently, the cells were washed and were labeled with MoAb against HLA-DR (PE) (b–f), IgG (a, b), B7–1 (FITC) (c, e), or B7–2 (FITC) (d, f). 5  10⁴ cells per sample were analyzed by FACScan. The percentage of HLA-DR⁺/B7⁺ cells is displayed in the upper right corner. One of three independent experiments is shown.

 

Effects of magnesium ions on cytokine production of epidermal cell suspensions in vitro

To elucidate further the mechanism by which MgCl₂ inhibits the allostimulatory capacity of epidermal cells we assessed the constitutive production of several cytokines by epidermal cells. Epidermal cell suspensions were incubated in the presence or absence of 1% MgCl₂ for 24 h. Subsequently, the supernatants were collected and analyzed for tumor necrosis factor (TNF)-, IL-1, IL-10, and IL-12 by specific enzyme-linked immunosorbent assays. MgCl₂ almost completely suppressed the production of TNF- and decreased the concentration of IL-12 (Figure 5). IL-1 and IL-10 were not detectable in this in vitro model.

Figure 5.

The constitutive secretion of TNF- and IL-12 by epidermal cells is reduced by magnesium ions. Epidermal cells (1  10⁶ cells per ml) were incubated in the presence or absence of 1% MgCl₂ for 24 h. Supernatants were collected and specific cytokine enzyme-linked immunosorbent assays with recombinant human primary MoAb for TNF-, IL-1, IL-10, and IL-12 were performed. The mean  SD of triplicate measurements is shown. The experiment was repeated with similar results.

Discussion

This study investigated the effects of magnesium ions and low-dose UVB irradiation on epidermal Langerhans cells. In line with published reports (^{Aberer et al. 1981;Lynch et al. 1981;Koulu et al. 1985;Alcalay et al. 1989;Murphy et al. 1993}), in vivo UVB irradiation (1 MED) resulted in a decrease of epidermal ATPase⁺ and HLA-DR⁺ Langerhans cells and a reduced allostimulatory capacity of epidermal cell suspensions. Similarly to UVB, the topical application of MgCl₂ but not of NaCl reduced the number of epidermal Langerhans cells. This reduced expression by Langerhans cells of ATPase and HLA-DR molecules in vivo was of functional relevance as demonstrated in vivo and in vitro: epidermal cell suspensions showed a reduced capacity to stimulate allogeneic T cell proliferation. The in vitro results suggest that the inhibitory effects of magnesium ions on epidermal cell suspensions are unlikely due to an emigration of Langerhans cells from the epidermis as Langerhans cells could not emigrate in this model. The impaired antigen-presenting function of Langerhans cells was associated with a reduced expression by Langerhans cells of class II and costimulatory B7 molecules, and with a suppression of the constitutive TNF- production by epidermal cells in vitro.

Epidermal Langerhans cells are highly sensitive to the effects of UV radiation. In vivo low-dose UVB irradiation results in a decrease of Langerhans cells surface markers, i.e., ATPase, HLA-DR, and CD1a molecules (^{Aberer et al. 1981;Lynch et al. 1981;Koulu et al. 1985;Alcalay et al. 1989;Murphy et al. 1993}). In parallel, the function of Langerhans cells isolated from UVB-exposed skin is impaired: epidermal cell suspensions from UVB-exposed skin show a reduced capacity to stimulate allogeneic or antigen-specific T cell proliferation in vitro (^{Cooper et al. 1985;Kremer et al. 1997;Dittmar et al. 1999}), and in UVB-exposed skin, contact hypersensitivity responses are suppressed (^{Toews et al. 1980;Lynch et al. 1981}). Also, epidermal cells irradiated in vitro with UVB have a reduced capacity to stimulate allogeneic or antigen-specific T cell responses (^{Stingl et al. 1981;Aberer et al. 1982}).

UVB-exposed murine Langerhans cells have been shown to preferentially activate cells of the Th2 subset (^{Simon et al. 1990}) and to induce specific clonal anergy in Th1 cells (^{Simon et al. 1991}). These disturbed functions of UVB-exposed Langerhans cells may result from: (i) the appearance of T6^–DR⁺ antigen-presenting cells in skin and subsequent activation of CD8⁺ suppressor lymphocytes (^{Cooper et al. 1985;Baadsgaard et al. 1987, 1988}); (ii) the increased production of suppressive mediators, such as IL-10 (^{Enk et al. 1993;Peguet-Navarro et al. 1994;Ullrich, 1994;Kang et al. 1998}); or (iii) a disturbed expression of costimulatory molecules, such as B7–1 and B7–2, that are involved in T cell activation (^{Weiss et al. 1995;Rattis et al. 1996;Denfeld et al. 1998;Dittmar et al. 1999}).

Most of the therapies used to treat psoriasis suppress cellular immune function and inflammation. These include UV irradiation, psoralen + UVA, cyclosporine, corticosteroids, methotrexate, anthralin, and retinoids. Most of these therapies have been shown to reduce Langerhans cell numbers and Langerhans cell function (^{Koulu & Jansen, 1982;Haftek et al. 1983;Morhenn et al. 1983;Koulu et al. 1985;Gottlieb, 1988;Alcalay et al. 1989}). There is evidence for cell-mediated immune mechanisms in the pathogenesis of psoriasis, and it has been proposed that Langerhans cells in psoriasis plaques could activate dermal T cells in an autologous MECLR (^{Gottlieb, 1988}). Alternatively, Langerhans cells could present an unknown autologous or exogenous antigen to T lymphocytes. T cell activation would then lead to the release of mediators of inflammation, and possibly of epidermal growth factors (^{Gottlieb, 1988;Bieber & Braun-Falco, 1989;Streilein, 1990}).

Magnesium ions have been shown to suppress allergic contact dermatitis (^{Greiner & Diezel, 1990}) and inhibit 5-lipoxygenase activity in human polymorphonuclear leukocytes (^{Ludwig et al. 1995}). A possible mechanism by which Mg²⁺ ions exert their anti-inflammatory properties may be the competitive displacement of calcium ions from their binding sites (^{Tsien et al. 1987;Kramer et al. 1991;Graeff et al. 1995}).

Consequently, competitive inhibition of the Ca²⁺-sensitive phospholipase A₂ may occur, resulting in the suppression of cutaneous inflammation (^{Kramer et al. 1991}). This is of importance because in psoriatic epidermis raised phospholipase A₂ activity has been demonstrated (^{Forster et al. 1985}). Lanthanides, another group of Ca²⁺-competitive elements, have been shown to inhibit Langerhans cell ATPase and contact sensitization with dinitrofluorobenzene (^{Diezel et al. 1989;Gruner et al. 1991}).

The findings from this study suggest that divalent magnesium ions may also inhibit Langerhans cell ATPase. Membrane ecto-ATPase protects Langerhans cells against the permeabilizing effects of extracellular ATP (^{Girolomoni et al. 1993}) and its downregulation is indicative of the reduction of Langerhans cell viability and antigen-presenting capacity (^{Aberer et al. 1981;Koulu et al. 1985;Gruner et al. 1991;Girolomoni et al. 1993}).

Furthermore, the inhibition of calcium-dependent mechanisms by magnesium ions may be involved in the reduced expression by Langerhans cells of HLA-DR molecules. It has been shown that interferon--induced HLA-DR molecule expression is associated with a rapid increase of calcium ions and that the induction of HLA-DR expression can be blocked by the inhibition of the interferon--induced calcium influx (^{Klein et al. 1990;Ryu et al. 1993}). Our finding of the concomitant downregulation of HLA-DR and B7 molecules by magnesium ions suggests that the regulation of B7 is also dependent on calcium-sensitive mechanisms.

Finally, the addition of magnesium ions to unstimulated epidermal cell suspensions resulted in a reduced production of TNF- and, less pronounced, IL-12 in vitro. In the epidermis, TNF- is primarily produced by human keratinocytes and not by Langerhans cells, although activated Langerhans cells may also secrete TNF- (^{Schreiber et al. 1992}; for review see^{Matsue et al. 1992;Kimber et al. 2000}).

Thus, downregulation of the constitutive TNF- production by epidermal cell suspensions suggests the involvement of keratinocytes in the modulation of the antigen-presenting function of Langerhans cells. The ability to present antigen is acquired by Langerhans cells during culture and is modulated by epidermal cytokines, that of greatest importance being TNF- (reviewed by^{Kimber et al. 2000}). TNF- seems also to be involved in the induction and maintenance by Langerhans cells of major histocompatibility complex class II antigens and B7 costimulatory molecules (reviewed by^{Kimber et al. 2000}). Thus, in addition to direct effects on Langerhans cells, i.e., blocking of ATPase and HLA-DR expression, magnesium ions may indirectly inhibit the antigen-presenting function of Langerhans cells via the reduction of keratinocyte-derived cytokines, i.e., TNF-.

Taken together, this study demonstrates specific inhibitory effects of Mg²⁺ ions on epidermal Langerhans cell function that may account for the efficacy of Dead Sea water and MgCl₂ containing topical applications in the treatment of inflammatory skin diseases.