Langerhans Cells in Feline Foetal Epidermis-Immunohistochemical Study of Spatial Distribution

Gorošová A., E. Matalová, I. Kociánová, F. Tichý: Langerhans Cells in Feline Foetal Epidermis Immunohistochemical Study of Spatial Distribution. Acta Vet. Brno 2008, 77:

In general, skin represents an important anatomical and physiological barrier but also a communication means between organisms and their external environment.Moreover, skin is an integral part of the immune system.Different cell types present in the skin such as Langerhans cells (LC), dermal dendritic cells, epidermotropic T-lymphocytes together with skin draining lymph nodes belong to an integrated tissue system mediating skin immune protection and forming so called skin-associated lymphoid tissue (SALT) (Streilein et al. 1999).
Both innate and acquired immunity operate in close relationship open exposure of the skin to various antigens.LC stimulate proliferation of important T-helper lymphocytes by antigen presentation on their surface, thus inducing also cytotoxic T-lymphocytes to moderate the allogeneic reaction.LC produce interleukin 1 and other cytokines, moreover, they are capable of phagocytosis and contain many different enzymes (Goodell et al. 1985).
LC stem cells originate from pluripotent hematopoietic progenitor cells CD34+ in the bone marrow.Recent results identified monocytes with a high expression of Gr-1(Ly-6c/G) marker as the direct precursors for LC in vivo and showed the importance of the colony-stimulating factor 1 (CSF-1) in this process (Ginhoux et al. 2006).As immature cells, LC migrate to non-lymphatic tissues, such as the skin, where they finish their maturation by expression of specific molecules (ATPase, CD45, MHC II, langerin/CD207) and formation of the Birbeck granules (Elbe et al. 1989;Foster and Holbrook 1989;Koch et al. 2006).The Birbeck granula, characteristic structures in LC, were described as para-crystallic, membrane-bound, disc-shaped bodies, and are located particularly between the cell membrane and the Golgi apparatus (Holibka 1998).
Langerhans cells in the cat skin were first described by Tsagarakis et al. (1994).LC were detected in adult skin using TEM and immunohistochemistry, and were found in the epidermis and in the oral, vaginal, rectal and oesophageal mucosa.Later immunophenotypic characterisations of feline Langerhans cells were performed (Marchal et al. 1997) and gene expression in Langerhans cells analyzed (Rust et al. 2006).
The biology of LC in adults has been recently reviewed (Bechan et al. 2006) and interactions of different cells in pathophysiology of Langerhans cell histiocytosis discussed.However, there is insufficient information on foetal migration and location of Langerhans cells in the skin.Therefore, this study aims to demonstrate the distribution and specific locations of Langerhans cells in feline foetal skin in order to better understand the involvement of Langerhans cells in the skin development with possible further contributions to veterinary treatments.

Specimens
Investigated tissues were collected from 3 foetuses on day 40 of ontogenesis (DO).The foetuses were obtained from the Small Animal Clinic, University of Veterinary and Pharmaceutical Sciences, Brno.The foetuses were euthanized using T-61 (Intervet, Germany) after umbilical cord cut.Nine selected skin areas were harvested immediately after euthanasia and immersed into liquid nitrogen.Serial sections (10 m) were split over 2 slides and prepared for further analyses.

Antibodies
Specific mouse monoclonal antibodies for CD antigen detections in cats were obtained from the School of Veterinary Medicine, University of California, Department of Pathology, Davis, California, USA.To precise the localization of Langerhans cells, two epitopes of the CD molecule were detected -CD1a (Fe1.5F4).The monoclonal antibody was used at a 1 : 10 dilution.

Immunohistochemistry
Monoclonal antibody (MAb) was applied for 1 h at the room temperature.Secondary antibody against mouse IgG labelled by peroxidase (Vectastain ABC-Peroxidase kit mouse IgG, VC-PK-4002-KI01, Diagenes) was applied for 30 min.This reagent was a 1 : 400 dilution of antibody diluent (S 3022, Dako).Endogenous peroxidase was quenched in 3.0% hydrogen peroxide in PBS for 5 min at room temperature.The colour reaction mediated by diaminobenzidine (Liquid DAB+, K 3468, Dako) as the substrate enabled visualisation of Langerhans cells in the skin.Positive cells were brown; the slides were counterstained by Mayer's haematoxylin.

Regio intermandibularis
LC were found scattered in small clusters in stratum germinativum of the epidermis and in the epithelial part of the hair follicle infundibulum, close to sebaceous glands (Plate I, Fig. 1).
Regio axilaris LC were scattered among keratinocytes of stratum germinativum, some individual CD1a positive cells were found also in the dermis around hair follicles (Plate I, Fig. 2).

Regio prepubica
LC were detected in clusters among cells of stratum germinativum of epidermis.None or only few positive cells were found in the dermis (Plate II, Fig. 3).

Regio inguinalis
Only few positive cells were found in this region, particularly scattered in stratum germinativum and stratum granulosum, in the dermis, positive cells were found exclusively around hair follicles (Plate II, Fig. 4).

Regio parietalis
LC were found individually or clustered in stratum germinativum of the epidermis and also in the dermis in the region of the hair follicle infundibulum close to the ostium of sebaceous glands (Plate III, Fig. 5).

Regio interdigitalis
LC were scattered among cells of stratum germinativum, located around hair follicles, and rarely also in the superficial dermis (Plate III, Fig. 6).

Regio vertebralis thoracis
LC formed cell clusters in the epidermis located in areas of hair germs, very few LC were found in the superficial dermis close to the hair follicle infundibulum (Plate IV, Fig. 7).

Regio pelvina
LC were present among keratinocytes in stratum germinativum of the epidermis, mostly forming clusters.Positive cells in the dermis were detected close to the hair follicle isthmus (Plate IV, Fig. 8).

Regio radicis caudae
LC in stratum germinativum were present in cell clusters rather than individually.LC in the dermis were found among cells of the outer epithelial sheath in the area of the infundibulum of hair follicles (Plate V, Fig. 9).
Comparison of different body surface regions of feline foetuses at 40 DO showed the LC distributed particularly in the dorsal body parts of the foetuses, in the areas of regio parietalis, vertebralis thoracis, pelvina and radicis caudae.In the epidermis, LC were located particularly among keratinocytes of stratum germinativum (stratum basale and stratum spinosum), scattered or clustered among epidermal cells closing the hair canal in the region around the hair follicle.In the dermis, LC were found particularly among cells of outer root sheath in the region of the hair follicle infundibulum close to the ostium of sebaceous gland ductus, some were found also in the upper part of the hair follicle isthmus.

Discussion
Compared to much data obtained in vitro, only few observations point to the function of Langerhans cells (LC) in vivo (Romani et al. 2006).There is also a lack of evidence about the timing of LC appearance during prenatal development and their spatial distribution.
LC were demonstrated in an 18-week-old human foetus in the tonsilla palatina epithelium (Holibka 1998).In a marsupial (Dasyurus hallucatus), LC were found in epidermis only after 23 days post partum (Pralomkarn et al. 1990).In both cases, the appearance of LC seems to correlate with the timing of hair formation.A higher density of LC was reported in the hair canal before opening in the human foetal dermis (Chang et al. 2005).To obtain evidence about foetal distribution of LC in pets at the stage of hair follicle formation, feline foetal skin on day 40 of ontogenesis (40 DO) was investigated in our study.On 30 DO the formation of protective hair starts in the feline foetal dermis and on 39 DO macroscopically distinguishable white spots created by growing protective hair can be seen in the epidermis.The protective hair reaches the body surface around 50 DO and the furring continues up to 3 mm long hair.The hair density differs in different parts of the feline foetus, with more distant hair in the lateral regions and in limbs and higher hair density in the dorsal regions of the body.
The results yielded from the comparison of nine different body surface regions of feline foetuses on 40 DO showed the LC distributed particularly in the dorsal body parts of the foetus, in the areas of regio parietalis, vertebralis thoracis, pelvina and radicis caudae.These findings support the idea of a relationship between LC density and hair follicles distribution because in all these regions the thick type epidermis and distinct furring can be found.The described observations must be considered also in the view of Langerhans cells as sentinel cells whose prime function is the surveillance of the epidermal environment and initiation of immune response against microbial threats (Romani et al. 2006).The hair follicles constitute an easy way for microbes to gain entry into the skin, particularly during the microbial colonization of the skin after birth (Marchini et al. 2005).Therefore, LC homing seems to correlate with these protection desiring regions.Moreover, clusters of LC in the interfollicular epidermis and among keratinocytes in the region of the hair follicle infundibulum may correlate with sites called EPU (epidermal proliferative unit).The EPU serves as an important source of stem cells and contributes to the tissue stability maintenance and cell proliferation control (Kaur 2006).
Langerhans cells seem to be involved in the pathogenesis of skin disorders evoked by hypersensitivity to local and systemic allergens (Toews et al. 1980;Streilein et al. 1982), including atopic dermatitis (Roosje at al. 1997).Increased numbers of Langerhans cells in the epidermis and dendritic cells in the dermis were demonstrated in atopic skin lesions of experimental animals.Moreover, an increased number of LC in the epidermis was found also during some immune diseases, e. g. lupus erythematosus, hypersensitive dermatitis, and pyodermia (Day 1996).Neoplastic transformations of LC exist in dogs, e.g.canine cutaneous histiocytoma (CCH), a malignant histiocytosis (Affolter andMoore 2000, 2002).Symptoms and immunosuppressive mediated response correspond to disorders in the immune system regulations.Thus, LC and dendritic cells are in focus in tumour research and therapy (Yoshida et al. 2003).
An active role in epidermal immunity is also played by epidermal keratinocytes.These cells produce interleukin 1, interleukin 3, prostaglandins, leukotriens and interferons.Keratinocytes may fulfil phagocytosis and thus present antigens connected with lymphocyte-mediated skin diseases (probably as a result of -interferon secretion by activated lymphocytes).LC make an adhesive contact with keratinocytes by molecules of E-cadherin (Teunissen et al. 1994).Moreover, LC and lymphocytes seem to communicate by soluble and membrane-bound molecules to make both cell types more active (Rotsztejn et al. 2006).Therefore, skin immunity, and particularly keratinocytes and Langerhans cells are of major research interest in human as well as veterinary medicine of pets (Scott et al. 1995).Moreover, due to the immunophenotypic and ultrastructural similarity of human and feline Langerhans cells, some applications may be extrapolated among the species (Marchal et al. 1997).