The human skin transplanted onto the nude mouse

 1. The nude mouse  

The nude mouse is a hairless mutant discovered in 1962 by N.R. Grist, but that was described in detail only in 1966 by S. Flanagan that gave it the name “nude” due to its lack of body hair.

In addition to the absence of hairs, the nude mice had a low fecundity and a high mortality in both male and female. It is only in 1968 that E Pantelouris has reported that these mice had a rudimentary thymus or no thymus. It is this unique characteristic that made the athymic nude mouse very popular as a tool in immunology and cancerology.

Pathogen-free congenitally athymic nude mouse, Swiss nu/nu.

The interest raised by this new mutation was linked to the key role played by the thymus in the generation of mature T lymphocytes. The lack of thymus and of mature T lymphocytes lead to the unability to develop a large number of immune responses, such as antibody production, cell- mediated immune responses, delayed type hypersensitivity reactions, killing of virus-infected or malignant cells, and of major interest for our studies, graft rejection.

 2. Transplantation of human skin onto the nude mouse

J. Rygaard, in 1969, was the first one to show that rat skin grafted onto the nude mouse was not rejected and survived till the death of the host. Afterwards, it has been estabished that nude mice maintain lifetime skin grafts from a wide variety of mammals, including man, and from birds.

In 1973, N.D. Reed and D.D. Manning (Reed and Manning,1973) reported, for the first time, that human skin could be grafted onto nude mice and retained all the morphological and ultrastructural features associated with normal skin.

Moreover, skin grafts from patients with psoriasis or lamellar ichthyosis onto nude mice show excellent preservation of most of the gross and histological features of the disease (Krueger et al., 1975; Briggaman and Lazarus, 1976; Briggaman and Wheeler, 1980; Haftek et al., 1981). Grafted human skin can preserve and develop markers of psoriatic skin, e.g. histological appearance, increased labelling index and increased level of plasminogen activator (Fräki et al. 1983).

Using pig skin, pig skin grafts and human skin grafts, it has also been shown that the grafted skin retains donor responsive features to agents which induce (TPA) and inhibit (betamethasone valerate) a proliferative response (Krueger et al., 1980; Krueger and Shelby, 1981).Similarly, human sebaceous glands of human facial skin grafted onto nude mice respond to stimulation by androgens (Petersen et al., 1984).

In the 1980^th, it clearly appeared that , at this time, the validation of the technique was mainly based on histological criteria. It was not very clear whether the graft cells were only of the donor origin or whether they were partly contaminated by host cells, transforming the transplant in a chimaeric tissue; this would make the model less interesting for clinical research.

Therefore, to further validate this model, it was necessary to define the species origin (mouse or human) of the cells and extracellular matrix components of the grafted skin. For this purpose, we have used the technique of immunofluorescence staining with mouse or human specific antibodies directed against cells or extracellular matrix constituents of the skin to characterize the species origin of the major constituents of the human skin transplanted onto the nude mouse.

  2.1 The model

In fact, it was difficult to graft full-thickness human skin and as figured below, it is a simplified split thickness skin (0.4 to 0.7 mm) that we have used for our studies. The deeper part of the epidermal appendages and of the dermis were absent.
Pathogen-free congenitally athymic nude mice, Swiss nu/nu were grafted with 1 cm diameter biopsies of split thickness skin (0.4 to 0.7 mm) from discarded mammary skin of healthy human subjects undergoing mammary reduction.

  2.2 The epidermis   of the transplanted human skin

Because of the delay necessary for the complete revascularization of the graft, the grafted epidermis suffers and nearly completely degenerate. Histologically, only the basal layer seems to survive while the suprabasal cells show signs of degenerescence and constitute eventually a dead layer. When the nutrient blood bed is reconstituted, a new pluristratified epidermis is reconstructed from the surviving basal cells and the classically described epidermal layers are observed once again (see figure below) This regeneration seems to be a centripetal process, occurring from the border to the centre of the graft in time correlation with the gradual revascularization. At the periphery of the graft where the human skin is in contact with the mouse skin, the human epidermis appears thicker than the mouse epidermis (see figure below).

With an antibody directed against HLA-ABC antigens, we can observe that the grafted epidermis and cells in the human dermis are labeled while the mouse epidermis is negative (see figure below).

The grafted human epidermis (in yellow) and cells in the human dermis (in green) is labeled by the anti-HLA-ABC while there is no staining in the mouse skin (in red because of the Blue Evans counterstaining). Human involucrin (in yellow) is detected in the upper layers of the stratum spinosum and in the stratum granulosum of the human skin.

In the median zone of the graft, the keratinization pattern as revealed by the different monoclonal antibodies, BC1, BC16, and KL1, and the involucrin   distribution are identical to that observed in normal human skin. BC1 antibody stains predominantly basals cells and a few cells above the basal layer, BC16 antibody reacts with the entire epidermis, and KL antibody labeled preferentially suprabasal keratinocytes. Involucrin was detected in the upper layers of the stratum spinosum and in the stratum granulosum. In the area adjacent to the mouse epidermis, the staining observed with KL1 is suprabasal and the involucrin can be observed in the outer two third of the human epidermis.

Transversal section of transplanted human skin, 1 month after transplantation, at the center of the graft (fig.1), or at the junction between human skin and mouse skin in histology (fig.2), or after immunolabeling with an anti-HLA-ABC antibody (fig.3), BC 16 antibody (fig. 4), anti-human involucrin antibody (fig.5) or BC1 antibody.

At the junction between human skin and mouse skin, we can see that only the thicker human epidermis is labeled by all the human-specific antibodies.

Transversal section of the transplanted human epidermis, at the center of the graft, at two months after grafting. Immunolabeling with KL1 antibody.

All the suprabasal keratinocytes and few basal keratinocytes are labeled in yellow while the other basal keratinocytes and the dermis is counterstained in red with Blue Evans.

These data indicate that, at two months after transplantation, most of the transplanted epidermis demonstrate a normal keratin pattern and involucrin distribution.

In agreement with the data of Krueger et al. (1983) and by using the techniques of histology, immunohistology and transmission electron microscopy  , we could observe that the human Langerhans cells   are maintained in the grafted human skin. While their number decreases during the first weeks after grafting, it returns to normal, at one month after transplantation.

Unlike the keratinocytes and the Langerhans cells for which we had species-specific antibodies, we had not species specific anti-melanocyte antibodies. Observations with transmission electron microscopy show the presence of melanocytes in the basal layer of the grafted human epidermis and the graft is rapidly strongly repigmented. Melanocytes contain vimentin. At this stage, no epidermal cells are labeled with an antibody directed against mouse vimentin. On the other hand, cells, a part of them being in basal position in the grafted human epidermis, are positive for human vimentin. It is therefore likely that the melanocytes observed in transmission electron microscopy are human.

  2.3 The dermal epidermal junction of the transplanted human skin

Two months after grafting, the dermal epidermal junction of the tranplanted human skin shows the same organisation than the one observed in normal human skin. From the epidermis to the dermis, it consists of the plasma membrane with hemidesmosomes, a lamina lucida with anchoring filaments, a lamina densa, and the sublamina densa area with anchoring fibrils, microfibrillar bundles, and collagen fibers.

By indirect immunofluorescence, two months after transplantation, the dermal epidermal junction of the grafted skin is linearly labeled with antibodies directed against laminin, BP antigen, and human type IV collagen, discretely stained with anti-fibronectin antibody, and is negative with the anti-fibrinogen antibody. Mouse type IV collagen was not detected in the dermal epidermal junction of the grafted skin but was observed in blood vessels of the graft and was linearly distributed at the dermal epidermal junction of the mouse skin.

We can see that the anti-human type IV antibody stained the dermal epidermal junction of the transplanted human skin and the blood vessels of the grafted dermis. The anti-mouse type IV collagen labeled the dermal-epidermal junction of the mouse skin and the blood vessels in the mouse dermis but also in the grafted human dermis. There is an area of overlapping of the two types of type IW collagen under the human epidermis close to the junction between the human and the mouse skin. We can assume that in this specific zone, human type IV collagen is produced by the human keratinocytes and mouse type collagen is produced by mouse dermal fibroblasts.

  2.4 The dermis of the transplanted human skin

The dermis of human skin is composed of a very well developed extracellular matrix which surrounds a relatively small number of cells, mainly fibroblasts. The intermediate filaments of fibroblasts are made of vimentin. With an antibody directed against calf vimentin that has been shown to react with human vimentin and not with mouse vimentin, we can observe that human cells are present in the dermis of grafted skin. On the other hand, cells labeled for mouse vimentin are only observed at the level of blood vessels.

Moreover, the major components of the dermal extracellular matrix, i.e. type I, III, V, or VI collagen are also of human origin.

In figs 2, 3, and 4, the mouse skin has been folded over the human skin before cutting. The stained areas appear in yellow/green and the unlabeled tissue is counterstained in red (figs 1, 3, and 4) with Blue Evans or the nucleus are counterstained in orange (fig. 2) with propidium iodide. We can see that the human dermis is stained with the human specific antibodies directed against vimentin, elastin, and type I collagen while the anti-mouse type I collagen labels only the mouse dermis.

 Conclusion

In summary, we can conclude that human skin transplanted onto the nude mouse preserves its own immunological markers and its own different constituents with the exception of the blood vessels that are made of mouse endothelial cells lying on a mixed basement membrane (#Démarchez M et al. 1987).

In several others articles, we have used this model to study major processes occurring in human skin, such as:

• The revascularization process of normal human skin and of human skin reconstructed in vitro (article in preparation and #Demarchez M et al., 1987)

• The self-reproducing capacity of human Langerhans cells (#Czernielewski et al., 1987)

• The localization of the in vivo activity of epidermal transglutaminase  (#Michel and Demarchez, 1988)

• The wound healing process of human skin (Wound healing of human skin grafted onto the nude mouse)

• The migration of Langerhans cells into human epidermis of “reconstructed” skin, normal skin, or healing skin, after grafting onto the nude mouse (#Démarchez et al., 1992)

 References

Czernielewski JM, Demarchez M. Further evidence for the self-reproducing
capacity of Langerhans cells in human skin. J Invest Dermatol. 1987
Jan;88(1):17-20.

Demarchez M, Hartmann DJ, Prunieras M. An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse. Transplantation. 1987 Jun;43(6):896-903.

Demarchez M, Hartmann DJ, Regnier M, Asselineau D. The role of fibroblasts in dermal vascularization and remodeling of reconstructed human skin after transplantation onto the nude mouse. Transplantation. 1992 Aug;54(2):317-26.

Démarchez M, Asselineau D, Régnier M, Czernielewski J. Migration of Langerhans cells into the epidermis of human skin grafted into nude mice. J Invest Dermatol. 1992 Nov;99(5):54S-55S. Review.

Démarchez M, Asselineau D, Czernielewski J. Migration of Langerhans cells into human epidermis of “reconstructed” skin, normal skin, or healing skin, after grafting onto the nude mouse. J Invest Dermatol. 1993 May;100(5):648-52.

Krueger GG, Daynes RA, Emam M. Biology of Langerhans cells: selective migration of Langerhans cells into allogeneic and xenogeneic grafts on nude mice. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1650-4.

Michel S, Démarchez M. Localization and in vivo activity of epidermal
transglutaminase. J Invest Dermatol. 1988 Apr;90(4):472-4.