Induction of oxazolone-mediated features of atopic dermatitis in NOD-scid IL2Rγ(null) mice engrafted with human peripheral blood mononuclear cells

Abstract

Animal models mimicking human diseases have been used extensively to study the pathogenesis of autoimmune diseases and the efficacy of potential therapeutics. They are, however, limited with regard to their similarity to the human disease and cannot be used if the antagonist and its cognate receptor require high similarity in structure or binding. Here, we examine the induction of oxazolone-mediated features of atopic dermatitis (AD) in NOD-scid IL2Rγ(null) mice engrafted with human peripheral blood mononuclear cells (PBMC). The mice developed the same symptoms as immunocompetent BALB/c mice. Histological alterations induced by oxazolone were characterized by keratosis, epithelial hyperplasia and influx of inflammatory cells into the dermis and epidermis. The cellular infiltrate was identified as human leukocytes, with T cells being the major constituent. In addition, oxazolone increased human serum IgE levels. The response, however, required the engraftment of PBMC derived from patients suffering from AD, which suggests that this model reflects the immunological status of the donor. Taken together, the model described here has the potential to evaluate the efficacy of therapeutics targeting human lymphocytes in vivo and, in addition, might be developed further to elucidate molecular mechanisms inducing and sustaining flares of the disease.

Fig. 1.

Challenge with oxazolone results in epithelial hyperplasia and influx of inflammatory cells into the epidermis and dermis and keratosis as observed in immunocompetent mice. (A–C) HE-stained photomicrographs of skin sections from BALB/C mice: (A) no treatment, (B) challenged with ethanol and (C) challenged with oxazolone. (D–F) HE-stained skin sections from NOD-scid IL2Rγ^null mice: (D) non-engrafted no treatment, (E) non-engrafted challenged with oxazolone and (F) engrafted challenged with oxazolone. Arrows indicate epithelial hyperplasia. Scale bars: 50 μm.

Fig. 2.

Upon challenge with oxazolone, human leukocytes (mainly consisting of T cells) infiltrate the dermis and epidermis. (A,B) Photomicrographs of immunohistochemically stained paraffin sections of the skin: (A) stained with anti-hCD45 antibody and (B) stained with anti-CD3 antibody. Skin samples for both A and B were taken from the following mice: BALB/c mouse challenged with ethanol (a); non-engrafted NOD-scid IL2Rγ^null mouse (b); BALB/c mouse challenged with oxazolone (c); and engrafted NOD-scid IL2Rγ^null mouse treated with oxazolone (d). Arrows indicate invaded human leukocytes. Scale bars: 100 μm; 10 μm (insets).

Fig. 3.

Challenge with oxazolone results in histological changes in NOD-scid IL2Rγ^null mice engrafted with PMBCs derived from patients with AD. Histological changes were classified according to a histological score and depicted in a boxplot diagram. Sample sizes: non-engrafted n=4, non-AD n=9 and AD n=12. P<0.01 for AD vs non-AD treated with oxazolone. P=0.22 for AD versus non-AD treated with ethanol (Kruskal-Wallis test followed by multiple comparisons).

Fig. 4.

Treatment-dependent human IgG and IgE secretion. (A,B) PBMC derived from three different patients suffering from AD and from two healthy volunteers (non-AD) were analyzed in vitro and in vivo with respect to their capacity to secrete human IgG (A) and IgE (B). Sample sizes: in vitro AD n=3; in vitro non-AD n=2; in vivo AD after treatment with IL-4 n=5, NaCl n=4, ethanol n=13 or oxazolone n=12; in vivo non-AD after treatment with IL-4 n=8, NaCl n=4, ethanol n=8 or oxazolone n=9. (A) hIgG secretion. IL-4 had moderate effects on IgG levels in vitro and in vivo, whereas ethanol and oxazolone induced the secretion of hIgG. (B) hIgE secretion. IL-4 induced IgE secretion in vitro in the AD group and in one donor of the non-AD group, whereas in vivo IL-4 failed to induce hIgE in both groups. Ethanol and oxazolone induced hIgE secretion in the AD group but not the non-AD group. IgE levels in the four different groups were significantly different. Kruskal-Wallis test followed by multiple comparisons revealed that the oxazolone- and ethanol-treated groups were significantly different to the IL-4-treated group (P=0.01 and P=0.01, respectively).

Fig. 5.

Immunological background of the donor determines the CD4:CD8 ratio in organs of engrafted NOD-scid IL2R γ ^null mice. (A,B) CD4:CD8 ratio of human T cells in the spleen, blood and skin of mice engrafted with PBMC derived from a patient suffering from AD and two healthy volunteers (non-AD) was compared with the CD4:CD8 ratio in whole blood from the respective donors prior to engraftment. (A) Representative flow cytometric analysis of human T cells stained with anti-human CD4 and CD8. (B) Quantitative analysis of CD4:CD8 ratio. Sample sizes: AD n=4, non-AD1 n=4 and non-AD2 n=7.