Joachim Degwert

In vitro model for contact sensitization: II. Induction of IL-1β mRNA in human blood-derived dendritic cells by contact sensitizers

Epidermal mRNA for interleukin 1beta (IL-1beta) has been shown to be increased following exposure of mouse skin to sensitizing compounds. In addition, this early upregulation of IL-1beta was specific for contact sensitizers, while expression of IL-1beta was unaffected by irritants. Langerhans cells are the major source of IL-1beta within the epidermis in the induction phase of skin sensitization. Since the isolation of Langerhans cells from skin biopsies results only in low frequencies, we decided to use dendritic cells (DCs) generated from peripheral blood as Langerhans cell equivalents to investigate the ability of five contact sensitizers and one irritant to induce IL-1beta gene expression in vitro. For our studies we cultivated DCs in serum-free medium supplemented with granulocyte/macrophage-colony stimulation factor (GM-CSF) and interleukin 4 (IL-4). The DCs showed a typical dendritic morphology, a characteristic expression of surface markers and high stimulatory capacity for autologous T cells. 5-day-old DCs were incubated with subtoxic concentrations of the contact sensitizers pentadecyl-catechol, 2,4,6-trinitrobenezene sulfonic acid, 2,4-dinitrofluorobenzene, NiSO(4), K(2)Cr(2)O(7) and the irritant sodium dodecyl sulfate. IL-1beta mRNA expression was detected by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and non-radioactive hybridization procedures. For all contact sensitizers, expression of IL-1beta mRNA increased, whereas treatment with the irritant SDS had no significant effect on IL-1beta expression. Thus we developed an in vitro system, which may be useful to evaluate allergic potentials of chemicals and products.

In vitro model for contact sensitization: I. Stimulatory capacities of human blood-derived dendritic cells and their phenotypical alterations in the presence of contact sensitizers.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in many non-lymphoid tissues and a specialized form of DC-the Langerhans cell (LC)-is found in the skin. The functionality of LC as APC is crucial for the induction of an allergic contact dermatitis. For a long time LC research has been hampered by the limiting numbers of functionally active LC that could be isolated from human skin. The addition of GM-CSF and IL-4 to the non-adherent fraction of mononuclear cells from peripheral blood generated a large amount of CD1a(+) HLA-DR(+) DC. These in vitro-generated DC exhibited the morphology, phenotype and autologous T-lymphocyte stimulating capacity of the human DC/LC system. We had tested phenotypical alterations of in vitro-generated DC under the influence of subtoxic concentrations of different chemicals and contact sensitizers. In vitro stimulation with the contact sensitizers urushiol, primin, C10-and C11-primin analogues, alantolactone, isoalantolactone and NiSO(4) resulted in a decrease of HLA-DR expression on the surface of these cells if the incubation period did not exceed 3 hr. Incubation with irritants like sodium lauryl sulfate (SLS) and benzalkonium chloride did not change or increase the HLA-DR surface expression under these conditions. With regard to the adhesion molecule ICAM-1, there was no clear difference between irritants and contact sensitizers. But based on the alteration of HLA-DR expression of dendritic cells under short-term exposure conditions, there was a clear-cut difference between irritants and contact sensitizers. In summary, this system can be used to discriminate between contact sensitizers and irritants.

Phenotype and Alloactivating Capacity of Dendritic Cells Generated under Different Culture Conditions from Human Peripheral Blood

Dendritic cells (DCs) are a family of specialized cells distributed in various tissues with stimulatory capacities for primary and secondary immune responses especially by antigen presentation to T-lymphocytes. The isolation of skin associated DCs from skin biopsies is hampered by the limited numbers of DCs and tedious isolation procedures. Recently, several investigators described procedures for the in vitro generation of cells with the feature of DCs/Langerhans cells (LCs) from human peripheral blood to have sufficient numbers of cells to investigate the characteristics of the cells (Caux et al., 1992; Thomas et al., 1993; Sallusto and Lanzavecchia, 1994).

Antigen Presenting and Primary in Vitro Sensitizing Capacity of CD1a+ Dendritic Cells Generated from Human Blood

Dendritic cells (DCs) are highly specialized antigen presenting cells (APCs) initiating primary T-lymphocyte associated immune responses1. DCs are located in many non-lymphoid tissues and a specialized form of DCs - the Langerhans cells (LCs) - is found in the skin. Here they fulfill their in vivo functions by capturing antigens in the epidermis and presenting these antigens to T-lymphocytes in a HLA-restricted way2. Until now the small numbers of functional active LCs which could be isolated from human skin were the limiting factor in this field of research3.

In vitro analysis of immunoprotective effects of topical sunscreens

The aim of this study was to evaluate procedures for the assessment of the immunoprotective capacities of topical sunscreens, using cell cultures. The exposure of humans to solar or ultraviolet radiation has been shown to induce numerous changes at the level of the immunoresponsiveness of the skin, which could be described as immune suppression. In this study, a sunscreen containing the commercially available UVB filters octyl triazone, phenylbenzimidazole sulfonic acid and methylbenzylidene camphor, was tested because of its capacity to protect from the immunosuppressive effects of UVB radiation. In contrast to control suncreens with a comparable sun protector factor (SPF), but that did not contain the UVB-filter octyl triazone, the immunoprotective sunscreen protected from two forms of immune suppression as assessed in vitro. Expression of the immune-relevant cellular communication structure intercellular adhesion molecule-1 is decreased on the cell surface of epidermal cells under the influence of UVB. This effect of immune suppression is totally abolished by the immunoprotective capacity of the tested sunscreen, as monitored cytofluorometrically. The mixed-lymphocyte reaction (MLR) was also used to assess the protective capacities of sunscreens against UVB-radiation-induced suppression of the immune response. Again, this UVB-induced suppression of the MLR was prevented by the sunscreen containing octyl triazone. In summary, the results of this study demonstrate the usefulness of the above-mentioned methods for the in vitro evaluation of the immunoprotective properties of topical sunscreens.