An automated method for the quantification of immunostained human Langerhans cells
Introduction
Allergic contact dermatitis is a frequent health problem. Contact allergens are currently screened using animal models, such as the guinea pig maximisation test (GPMT) and the murine local lymph node assay (LLNA). The assessment of the sensitisation potential of a single chemical requires 24 to 32 guinea pigs or 16 to 30 mice. The accuracy of both the GPMT and the LLNA models for predicting human contact sensitisers is about 70% (Anonymous, 1999). Differences in the response of the immune system and skin morphology could account for part of the low efficiencies (Bouclier et al., 1990).
Immature dendritic cells (DCs), such as Langerhans cells (LCs) in the epidermis, take up antigen in the peripheral tissue (Shelley and Juchlin, 1976). After activation, e.g., induced by contact allergens, LCs migrate to the draining lymph node and mature (Aiba et al., 1997, Drexhage et al., 1979, Macatonia et al., 1986, Silberberg et al., 1976). Mature LCs or DCs stimulate the development of hapten-specific naive T cells leading to antigen-specific sensitisation (Inaba et al., 1986, Soeberg et al., 1978, Streilein, 1989). The subsequent application of a contact allergen on the skin elicits an allergic contact dermatitis (Roitt et al., 1998).
Migration of epidermal LCs can be studied in human organotypic skin explant cultures (hOSECs), where LCs spontaneously migrate out through lymphatic vessels (Czernielewski et al., 1984, Lukas et al., 1996, Rambukkana et al., 1995). The topical exposure of hOSEC to contact sensitisers accelerates LC migration out of the epidermis, relative to spontaneous migration and the migration induced by control chemicals. This acceleration of epidermal LC migration induced by a contact sensitizer may be used as a screening system for contact allergens (Pistoor et al., 1996, Rambukkana et al., 1996). Manual counting of epidermal LCs is labour intensive and subject to intra- and inter-personal variation. For these reasons we developed an image analysis routine using Leica QWin image analysis software which can be used to quantify LCs in immunohistochemically stained skin sections in situ.
Section snippets
Human organotypic skin explant cultures (human OSECs, hOSECs)
Dulbecco’s phosphate-buffered saline (DPBS) (BioWhittaker, Verviers, Belgium), mineral oil, nickel sulfate, potassium dichromate and sodium dodecyl sulfate (SDS) (all Sigma–Aldrich, Zwijndrecht, The Netherlands) were preheated to 37°C, prior to application onto the skin. Human breast skin was obtained as a waste product of cosmetic surgery. Sterile biopsies were cut (approx. 0.25 cm2) and these were incubated dermal-side down in Dulbecco’s Modified Eagles Medium with ultraglutamine 1, with 4.5
Visual examination of eLC stainings
LCs were stained in cryostat sections using MHC-II, CD1a, or Lag antibodies (Fig. 2A–C). MHC-II staining of human LCs was not specific as in some experiments all keratinocytes in the epidermis were MHC-II positive (data not shown). For the studies reported in this article, skin LCs were defined as Lag+ or CD1a+. In practice, all epidermal and dermal CD1a+ cells were Lag+ and vice versa (data not shown). Lag stains the Birbeck granules, which are present in the LC body, and positive cells appear
Detection of LCs
Epidermal LCs can be visualised using a number of unique markers. Adequate and accurate counting of LCs requires that these markers are stable and uniquely expressed on LCs. We stained LCs in cryostat sections with three antibodies, anti-MHC-II, CD1a and Lag (Fig. 2A–C). LCs could be visualised using CD1a or Lag staining and all epidermal and dermal LCs double stained for both CD1a and Lag markers (data not shown). Our results are in agreement with in vivo data showing that LCs emigrating from
Acknowledgements
This study was subsidised by a grant from the Dutch Platform Alternatives for Animal Experiments to G.R.E. and P.K.D. for the support of J.J.L.J. and C.L. (grant No. 96-32). P.K.D. also acknowledges grant support from the Dr. Hadwen Trust Research in Humanities, UK.
References (27)
- et al.
Dose response and time course for induction of T6− DR+ human epidermal antigen-presenting cells by in vivo ultraviolet A, B, and C irradiation
J. Am. Acad. Dermatol.
(1987) - et al.
Defined in situ enumeration of T6 and HLA-DR expressing epidermal Langerhans cells: morphologic and methadologic aspects
J. Invest. Dermatol.
(1986) - et al.
An assessment of Langerhans cell quantification in tissue sections
J. Am. Acad. Dermatol.
(1984) - et al.
A monoclonal antibody specifically reactive to human Langerhans cells
J. Invest. Dermatol.
(1986) - et al.
Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model
J. Invest. Dermatol.
(1996) - et al.
The role of Langerhans cells in allergic contact hypersensitivity. A review of findings in man and guinea pigs
J. Invest. Dermatol.
(1976) - et al.
Epidermal damage induced by irritants in man: a light and electron microscopic
J. Invest. Dermatol.
(1989) - et al.
Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules
Eur. J. Immunol.
(1997) - Anonymous, 1999. The murine local lymph node assay: a test method for assessing the allergic contact dermatitis...
- et al.
Comparison of different methods for enumeration of Langerhans cells in vertical cryosections of human skin
Br. J. Dermatol.
(1988)
Experimental models in skin pharmacology
Pharmacol. Rev.
Studies on human skin lymph containing Langerhans cells from sodium lauryl sulfate contact dermatitis
J. Invest. Dermatol.
Human Langerhans cells in epidermal cell culture, in vitro skin explants and skin grafts onto “nude” mice
Arch. Dermatol. Res.
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