Correlation of disease evolution with progressive inflammatory cell activation and migration in the IL-4 transgenic mouse model of atopic dermatitis

Abstract

Atopic dermatitis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration in the skin. In order to assess the roles of inflammatory cells in this disease, we analysed the activation status and surface markers of various leucocytes in the IL-4 transgenic mouse model of atopic dermatitis, by flow cytometry, immuofluorescence microscopy, and T cell proliferation assays. The studies were performed with a nontransgenic mouse control and transgenic mice at three disease stages: before disease onset, early skin disease, and late skin disease, so that we can delineate the immunological sequence of events. As the skin disease evolves, the skin draining lymph node cells from IL-4-Tg mice show a spontaneous proliferation and a progressively enhanced proliferative response to stimulants including anti-CD3, Con A, PHA, and Staphylococcus enterotoxins A and B. As the disease evolves, the percent of lymphoid organ T cells expressing activation molecules (CD44 and CD69) and costimulatory molecules (ICOS and PD-1) are progressively increased; the percent and total number of T cells are reduced in an incremental manner in the secondary lymphoid organs while the number of T cells infiltrating the skin increases in an incremental fashion; the total number of dendritic antigen presenting cells, macrophages, and NK cells gradually increases in the lymphoid organs. Collectively, our results suggest that there is a continued and progressive migration of activated inflammatory cells from the secondary lymphoid organs into the skin where they participate in immune responses resulting in the pathology associated with inflammation.

Fig. 1

As the disease progresses T cells in the secondary lymphoid organs decrease progressively while exhibiting increased expression of activation molecules and costimulatory molecules ICOS and PD-1. Spleen and LN cells were collected from Non-Tg and Tg mice at different stages of disease, and then stained with specific Abs for the given cell surface molecules, followed by FACS analyses. (a) percentage and (b) absolute number changes of CD3+ cells; (c) and (d), the percentage of CD4+ and CD8+ cells in CD3+ cells; (e) and (f) (g) and (h) represent percentage of CD44+ and CD69+ cells in CD4+ and CD8+ cells. (i) and (j), (k) and (l), (m) and (n), represent, respectively, percentage of CD28+, ICOS+ and PD-1+ cells in CD4+ and CD8+ cells. Data were the average of three experiments. ♦ Spleen, ▪ LN, ○ Average percentage of spleen and LN (a, c–h, i–n).

Fig. 2

The highest percent and total number of CD11c+ dendritic cells, as well as the highest percent of costimulatory molecules barring dendritic cells, occurred in the Tg mice before onset. Spleen and LN cells were collected from Non-Tg and Tg mice at different stages of disease, and then stained with specific Abs against CD11a, CD11c, CD80, CD86, B7 h and B7-DC, followed by FACS analyses. (a) percentage and (b) absolute number changes of CD11c+ cells; (c-f) represent percentages of CD80+, CD86+, B7 h+ and B7-DC+ cells, respectively, in CD11c+ cells. Data were obtained from the average of three experiments. ✦ Spleen, ▪ LN, ○ Average percent of spleen and LN (a, c–f).

Fig. 3

The increases of Mac+ cell frequency and number in the secondary lymphoid organs occurred predominantly at the Tg-LL mice. Spleen and LN cells were collected from Non-Tg and Tg mice at different stages of disease, and then stained with specific Abs against CD11a and Mac3, followed by FACS analyses. (a) percentage and (b) absolute number changes of CD11a+/Mac3+ cells. Data were obtained from the average of three experiments ✦ Spleen, ▪ LN, ○ Average percent of spleen and LN (a).

Fig. 4

As the disease progresses, the frequency and absolute numbers of DX5+ cells in the lymphoid organs showed an increasing trend. Spleen and LN cells were collected from Non-Tg and Tg mice at different stages of disease, and then stained with specific Abs against CD11a and DX5, followed by FACS analyses. (a) percentage and (b) absolute number changes of DX5+ cells. Data were obtained from the average of three experiments ✦ Spleen, ▪ LN, ○ Average percent of spleen and LN (a).

Fig. 5

As the disease progresses, skin draining LN cell spontaneously proliferated in the absence of stimulant and proliferated in a progressively enhanced manner in response to mitogens and superantigens. LNs were obtained from one mouse of each group including non-Tg Tg-BO, Tg-EL and Tg-LL. A total of 1 × 10⁵ cells in single cell suspension were obtained and cultured with or without anti-CD3 (1 µg/ml), Con A (5 µg/ml), PHA (5 µg/ml), SEA (25 ng/ml) or SEB (25 ng/ml) for 72 h, and then WST-8 was added for an additional 4 h. The absorbance at 450 nm was read in a microplate reader with a reference wavelength at 600 nm. Data are expressed as mean ± SD in triplicate. *Statistical significance versus Non-Tg mice; ♯Statistical significance versus Tg-BO. Similar results were obtained in two additional experiments.

Fig. 6

As the disease progresses, inflammatory cells infiltrate progressively in diseased skin. (a) Immunofluorescence microsopic examination. Frozen sections were incubated with primary rat monoclonal Abs against CD3 (upper row), CD4 (middle row), CD8 (lower row), and MHC II (IA-IE) (not shown), followed by Alexa fluor 488-conjugated goat anti-rat IgG Abs. The sections were then examined under a fluorescence microscope. Illustrations shown are representatives of 15 samples examined. NL, nonlesional skin from diseased mice. Scale bar = 38 µm. (b–e) Bar graph presentations of the inflammatory cell number in the skin. The sections on each slide were examined and five different areas in each sample (n = 15) were counted to determine the average number of a particular cell present per HPF (at 40× objective lens). *Statistical significance versus Non-Tg mice; ♯Statistical significance versus Tg-NL. (f) FACS analyses of CD3+, CD4+, CD8+ and MHC II cells in the skin lesions. The single cell suspension from early and late lesional ears was labelled with PE- or FITC-conjugated monoclonal Abs to mouse CD11a, CD3, CD4, CD8 or MHC II and analysed. The data were expressed as the percentage of CD3+ cells in CD11a+ leucocytes, the percentage of CD4+ and CD8+ cell subsets in CD3+ cells, and the percentage of IA-IA+/CD11a+ cells. The data presented are the averages of three experiments.

Fig. 6

As the disease progresses, inflammatory cells infiltrate progressively in diseased skin. (a) Immunofluorescence microsopic examination. Frozen sections were incubated with primary rat monoclonal Abs against CD3 (upper row), CD4 (middle row), CD8 (lower row), and MHC II (IA-IE) (not shown), followed by Alexa fluor 488-conjugated goat anti-rat IgG Abs. The sections were then examined under a fluorescence microscope. Illustrations shown are representatives of 15 samples examined. NL, nonlesional skin from diseased mice. Scale bar = 38 µm. (b–e) Bar graph presentations of the inflammatory cell number in the skin. The sections on each slide were examined and five different areas in each sample (n = 15) were counted to determine the average number of a particular cell present per HPF (at 40× objective lens). *Statistical significance versus Non-Tg mice; ♯Statistical significance versus Tg-NL. (f) FACS analyses of CD3+, CD4+, CD8+ and MHC II cells in the skin lesions. The single cell suspension from early and late lesional ears was labelled with PE- or FITC-conjugated monoclonal Abs to mouse CD11a, CD3, CD4, CD8 or MHC II and analysed. The data were expressed as the percentage of CD3+ cells in CD11a+ leucocytes, the percentage of CD4+ and CD8+ cell subsets in CD3+ cells, and the percentage of IA-IA+/CD11a+ cells. The data presented are the averages of three experiments.