In vitro effects of a spa water on the migratory and stimulatory capacities of human epidermal Langerhans cells M.J. Staquet, J. Peguet-Navarro, F. Latourre, A. Richard, A. Rougier,

ARTICLE

The properties of a defined thermal water (Source Mélusine, La Roche Posay, France) have long been associated with an improvement in inflammatory skin diseases such as psoriasis and atopic dermatitis [1]. Only a limited number of studies have been undertaken however, to determine the mechanism(s) responsible for the satisfactory clinical results. In vitro experiments have shown an inhibitory effect of selenium and strontium salts present in the thermal water from La Roche Posay on keratinocyte-derived, inflammatory cytokine production [2], and a suppressive activity of this thermal water on the allostimulatory capacity of epidermal Langerhans cells (LC) [3]. With regard to the crucial function of Langerhans cells in the skin immune responses, we investigated the in vitro effect of La Roche Posay spa water on the modulation of their phenotype, as well as their migratory and stimulatory capacities. The thermal water did not significantly affect the allostimulatory activity of human LC. Furthermore, we showed that a 2 day thermal water treatment did not alter the ability of LC to mount a hapten-dependent T cell response.

Material and methods

Preparation of media

All experiments were carried out in RPMI 1640 medium (Gibco BRL, France) supplemented with 10% FCS, 200 mM L-glutamine, 500 U/ml penicillin, 100 µg/ml streptomycin. Lyophilized RPMI reconstituted with Millipore water was referred to as the control medium (RPMI-C) in comparison with lyophilized RPMI reconstituted with La Roche Posay thermal water which was referred to as RPMI-RP. Media were filtered using a pore size of 0.22 µm, stored at 4° C, and filtered again before used.

Preparation of Langerhans cell-enriched epidermal cell suspensions

Isolated normal human epidermal cells were obtained by trypsin digestion (0.05% trypsin, 18 h, + 4° C) of skin removed during reconstructive plastic surgery. Langerhans cell enrichment was performed as described [4, 5]. A purity of 75-90% viable LC was regularly achieved.

In vitro hapten sensitization

LC-enriched cell suspensions were treated with 5 mM TNBS (2,4,6-trinitrobenzenesulfonic acid) in HBSS for 10 min at 37° C [6, 7]. After several washings, cells were then used for the migration or the T cell proliferation assays.

Migration assay

LC migration capacity was assessed by using polycarbonate filters (8 µm pore size, Falcon inserts) coated with 50 µg/cm² of growth factor-reduced Matrigel (Collaborative Research Corp.). Cells were suspended in RPMI-C or RPMI-RP supplemented with 1% BSA, 200 mM L-glutamine and seeded into the upper compartment of the chamber. Conditioned medium prepared from the culture of human dermal fibroblasts was used to fill the lower compartment of the chamber [6]. Chambers were incubated at 37° C for 18 h. The Langerhans cells recovered in the lower compartment were counted. All experiments were performed in duplicate. Results were expressed as the percentage of migrating LC per 1,000 LC introduced into the upper compartment.

Proliferation assay

The T cell proliferative response to allogeneic LC was assessed as previously described [4]. Briefly, mixed epidermal cell-lymphocyte reactions (MELR) were performed by adding 10⁵ T lymphocytes to 10³ epidermal LC in 96-well, U-bottom microtiter plates. Cultures were initiated in parallel in RPMI-C and RPMI-RP. After 5 days at 37° C, cells were pulsed for 18 h with 1 µCi/well [³H] methylthymidine (2 Ci/mmol, Amersham) and harvested onto glass microfiber strips. Results were expressed as the mean counts per min ± SD of triplicate wells.

In another set of experiments, we investigated the effect of the spa water on the capacity of cultured LC to induce a primary T cell response to TNBS [7, 8]. To this end, enriched LC suspensions were cultured for 2 days in either RPMI-C or RPMI-RP, in the presence of granulocyte-macrophage stimulating factor (GM-CSF, 200 U/ml). Viable LC were recovered, treated or not with TNBS, and added to autologous T cells, as previously described [7, 8]. T cell proliferation was assessed by incorporation of ^[3H]thymidine during the last 18 h of culture.

Flow cytometry analysis

LC-enriched epidermal cell suspensions were prepared for FACS analysis by the standard procedure [5, 9]. LC were cultured for 72 h in RPMI-C and in RPMI-RP and then stained with HLA-DR (IOT2a, Immunotech, France), B7-2 (IT2-2, Pharmingen, San Diego CA, USA), and ICAM-1 (Immunotech, France) mAbs. Negative controls used the IgG2a isotype-matched primary mAb (Sigma Chemical CO, St Louis MO, USA). Fluorescence analysis was performed on a FACScan cytometer (Becton Dickinson).

Results

Effect of the thermal water on the migratory capacity of LC

As previously reported [6], the migratory capacity of LC, freshly isolated from skin, was very low and no significant difference was observed between cells suspended in RPMI-C and those incubated in RPMI-RP. Cell viability was not affected in the presence of the thermal water. Treatment with haptens such as TNBS and FITC has been shown to increase the epidermal LC migratory property [6]. After treatment with 5 mM TNBS, we found that the number of migrating LC, resuspended in RPMI-RP, was significantly lower than that of migrating LC resuspended in RPMI-C. Results from 8 independent experiments are shown in Figure 1. Although individual variations were observed from donor to donor, the statistical analysis was significant (p = 0.028, determined with Student's two-tailed t test) when the 8 experiments were considered. The number of LC introduced into the upper chamber (3 x 10⁴ to 12.5 x 10⁴ LC) did not influence the inhibitory effect of the thermal water. Indeed, when results were expressed as a relative migration index (RMI: number of migrating LC in RPMI-RP/number of migrating LC in RPMI-C), the RMI value averaged 0.5 ± 0.3, thus confirming the inhibitory effect of RPMI-RP on epidermal LC migratory properties.

Effect of the thermal water on the expression of costimulatory molecules

We then wanted to know whether the thermal water from La Roche Posay could modulate the expression of some costimulatory molecules at the LC surface. Since HLA-DR, B7-2 and ICAM-1 play a key role in the initiation of the signal transduction cascade resulting in T cell proliferation, expression of these molecules was evaluated in the presence or absence of the thermal water. Results expressed as the ratio of the MFI (mean fluorescence intensity) after a 3 day culture in RPMI-RP/MFI in presence of RPMI-C, showed that the thermal water induced a decrease of 25% in the level of expression of HLA-DR and ICAM-1 (mean MFI ratio = 0.75 ± 0.02; 0.75 ± 0.07 respectively) and a decrease of 35% on average, of expression of B7-2 (0.64 ± 0.22) (Table I). Student's t test analysis showed that the expression of the three molecules was significantly decreased: p = 0.023 for HLA-DR, p = 0.014 for B7-2, and p = 0.001 for ICAM-1.

Effect of the thermal water on the stimulatory capacity of LC

MELR have been performed in either RPMI-C or RPMI-RP, by using epidermal cell suspensions more or less enriched with LC (7% to 90%, from different donors). In every cases, T cell proliferative responses were quite similar in RPMI-C as compared to RPMI-RP (Fig. 2). This suggests that the thermal water did not significantly affect human LC allostimulatory function.

We then asked whether a 2 day culture in the thermal water might affect the ability of LC to induce a primary T cell response to TNBS. As previously reported [7, 8], the addition of TNBS-treated LC to autologous T cells induced significant T cell proliferation which was highest when 4 x 10³ LC were added to 10⁵ T cells (Fig.3). No difference was seen whether LC had been cultured in RPMI-C or RPMI-RP.

Discussion

In this study, we analyzed the effects of the thermal water from La Roche Posay (Source Mélusine) on the migratory and stimulatory capacities of human epidermal LC. As shown in a previous study, the very low spontaneous migratory activity of freshly isolated epidermal LC was significantly increased after short contact of the cells with strong allergens such as TNBS and FITC [6]. Using the same in vitro model, the current study demonstrated a strong inhibition of migrating activity in the presence of the thermal water. The migration of antigen-bearing epidermal LC from the skin to lymph nodes draining the site of exposure is of central importance since it allows the induction of cutaneous immune responses. An early event during contact sensitization is the induction and upregulation of cytokine expression by epidermal cells [10]. The fact that the inhibitory effect was only observed with hapten-modified LC suggests an action of the thermal water upon LC activation. The inhibitory activity might be related to an indirect effect of the thermal water on the production of epidermal cytokines released during the activation stage. Indeed, an inhibitory effect of this thermal water on IL-1alpha, IL-6, and TNF-alpha production by cultured keratinocytes has been demonstrated [2]. It is therefore plausible that the thermal water induced a decrease in the secretion of IL-1 ß, which, like TNF-alpha, is known to be a stimulator of LC migration [11, 12], by epidermal LC.

A significant downregulation of both HLA-DR and costimulatory molecules has been observed on LC after a 3 day culture in the spa water. Whether this reflects a direct effect of the thermal water on LC or, alternatively, an indirect effect through inhibition of cytokine release remains to be clarified. In mice, anti-GM-CSF antibody has been shown to partially inhibit the upregulation of B7-2 expression induced on cultured LC [13]. Based on this observation, an inhibition of GM-CSF release might explain the inhibitory effect of the thermal water.

In a previous report, Wollenberg et al. [3] reported that the thermal water from La Roche Posay strongly inhibited the MELR, whereas it did not affect the response of peripheral blood mononuclear cells to mitogens. Since a detailed experimental protocol and results were not shown in their short report, we are unable to explain the discrepancy between their and our results. By using more or less enriched LC suspensions, we clearly demonstrated here that the thermal water did not significantly affect the allostimulatory activity of human LC. Furthermore, we showed that a 2 day treatment in the thermal water did not alter the ability of LC to mount a hapten-dependent T cell response.

Taken together, our results showed that the thermal water from La Roche Posay did not affect the stimulatory function of human LC, whereas it significantly decreased their in vitro migratory properties. While it is very likely that cytokines are an important element of pathogenic mechanisms in inflammatory skin diseases, present knowledge of the interactions of cytokines in human LC migration does not allow detailed interpretation. However, these results strongly suggest some, as yet undefined, link between the thermal water and the migratory function of LC and whatever the exact regulatory mechanisms are, they could explain, at least partly, the beneficial effect of the thermal water in the treatment of skin inflammatory diseases.

CONCLUSION

Acknowledgements

We thank Dr. Bouillot, Dr. Durand, and their staff for providing skin samples, G. Panaye for help in cytometry analysis.

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