L Chen

Early up‐regulation of Th2 cytokines and late surge of Th1 cytokines in an atopic dermatitis model

We investigated cytokine profiles in interleukin (IL)‐4 transgenic (Tg) mice with a skin inflammatory disease resembling human atopic dermatitis. cDNA microarray revealed that the mRNAs encoding IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, IL‐12p40, IL‐13, tumour necrosis factor (TNF)‐α, TNF‐β and interferon (IFN)‐γ were up‐regulated in the skin of late lesion Tg mice and to a lesser degree in non‐lesion Tg mice when compared to those of non‐Tg mice. Real time reverse transcription–polymerase chain reaction (RT‐PCR) analyses indicated that the cDNA copy numbers of IL‐1β, IL‐4, IL‐6, IL‐10, TNF‐α and IFN‐γ from the skin of late, early and non‐lesions increased significantly compared to non‐Tg mice. IL‐2 and IL‐12p40 cDNA copy numbers were increased significantly in early, but not late, lesions. Interestingly, IL‐1β, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, TNF‐α, and IFN‐γ cDNAs were increased significantly the skin of before‐onset and/or non‐lesion mice. Flow cytometry analyses demonstrated an increased percentage of keratinocytes producing IL‐4 as the disease progressed. The percentage of IL‐2, IL‐4, IL‐10 and IFN‐γ‐producing T cells and IL‐12‐producing antigen‐presenting cells in skin‐draining lymph nodes and inflammatory skin also increased, particularly in mice with late lesion. These results suggest that disease induction is primarily triggered by Th2 cytokines and that Th1, Th2 and non‐Th proinflammatory cytokines are all involved in the disease process.

The Progression of Inflammation Parallels the Dermal Angiogenesis in a Keratin 14 IL-4-Transgenic Model of Atopic Dermatitis

The role angiogenesis plays in atopic dermatitis is not well understood. The authors previously demonstrated ultrastructurally dermal microvascular angiogenesis in the IL‐4‐transgenic mouse model of atopic dermatitis. Here, they determine the angiogenic factors involved in dermal microvascular angiogenesis, regulatory function of inflammatory cytokines on the VEGF‐A production, and microvascular permeability in this model. Computer‐assisted photometric analyses for immunofluorescence‐labeled CD31 demonstrated a progressive increase in blood vessel number, diameter, and percent dermal areas occupied by CD31+ vessels as the disease evolves in transgenic mice from before disease onset through early and late skin lesions. Similar findings were documented for VEGR2+ vessels. Quantification of skin angiogenic factor mRNAs showed progressive increase of transcripts of VEGF‐A, but not VEGF‐B, VEGF‐C, or VEGF‐D. ELISA showed a similar increase of VEGF‐A in the serum and skin of transgenic mice. IL‐6 and IFN‐γ stimulated VEGF‐A mRNA production in the skin and in primary keratinocytes of transgenic mice. Other skin angiogenic factors that increased included Ang‐1, Ang‐2, GBP‐1, and VE‐cadherin. Microvascular leakage began in the transgenic mouse skin before disease onset and peaked in the late stage. In conclusion, IL‐6 and IFN‐γ may play important roles in upregulation of VEGF‐A, along with other pro‐angiogenic factors, to induce dermal microvascular angiogenesis.

The disease progression in the keratin 14 IL-4-transgenic mouse model of atopic dermatitis parallels the up-regulation of B cell activation molecules, proliferation and surface and serum IgE

We have previously characterized the keratin 14 interleukin‐4‐transgenic (IL‐4‐Tg) mouse model of atopic dermatitis as a chronic pruritic inflammatory skin disease typified by skin infiltration of inflammatory cells and early up‐regulation of Th2 cytokines and late surge of Th1 cytokines. In the present study, we examined the involvement of B cells. Systematic examinations of the following immunological parameters on B cells were carried out in non‐Tg control mice and in IL‐4‐Tg mice at before disease onset and early and late disease stages so that we could determine the immunological sequence of events leading to the disease development: surface expressions of IA/IE, activation and costimulatory molecules, proliferation under LPS or IgM stimulation, quantification of cell surface and serum IgE, IgG1, and IgG2a. Our results showed that as the disease progresses from before onset to early disease and to late disease, there is a parallel increase in surface markers of B cell activation (IA/IE, CD44, CD69, CD80 and CD86), in B cell proliferation, and in cell surface and serum IgE. Significant increases of Th2‐driven serum IgG1 and IgE in early disease was followed by significant increase of Th1‐driven IgG2a in late disease. Importantly the significant increases of activation molecule (IA/IE), proliferation (to LPS), and surface IgE on B cells of the IL‐4‐Tg mice precedes the up‐regulation of serum IgE and disease onset. These data suggest that activated B cells may play a role in atopic dermatitis disease development by up‐regulating serum IgE concentration, which serves as a marker of disease onset.

The development of atopic dermatitis is independent of Immunoglobulin E up‐regulation in the K14‐IL‐4 SKH1 transgenic mouse model

Background We have successfully generated an IgE‐associated (extrinsic/allergic) mouse model of atopic dermatitis in K14‐IL‐4‐Tg/CByB6 mice. The newly described subset of non‐IgE‐associated (intrinsic/non‐allergic) atopic dermatitis in human patients raises the question on the role of IgE in the pathogenesis.

Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

Epidermolysis bullosa acquisita is an autoimmune blistering disease characterized by circulating and skin basement membrane‐bound IgG autoantibodies to type VII collagen, a major structural protein of the dermal–epidermal junction. Regulatory T cells (Treg) suppress self antigen‐mediated autoimmune responses. To investigate the role of Treg in the the autoimmune response to type VII collagen in a mouse model, a monoclonal antibody against mouse CD25u2003was used to deplete Treg. A recombinant mouse type VII collagen NC1 domain protein and mouse albumin were used as antigens. SKH1 mice were used as a testing host. Group 1 mice received NC1 immunization and were functionally depleted of Treg; group 2 mice received NC1 immunization and rat isotype control; and group 3 mice received albumin immunization and were functionally depleted of Treg. Results demonstrated that anti‐NC1 IgG autoantibodies with high titres, as determined by enzyme‐linked immunosorbent assay and Western blotting, developed in all mice immunized with NC1 (groups 1 and 2), but were undetected in group 3 mice. The predominant subclasses of anti‐NC1 autoantibodies were IgG1, IgG2a and IgG2b; furthermore, these antibodies carried only the kappa light chain. IgG autoantibodies in the sera of NC1‐immunized mice reacted with mouse skin basement membrane in vitro and deposited in skin basement membrane in vivo as detected by indirect and direct immunofluorescence microscopy, respectively. Our data suggest that the development of autoimmunity against type VII collagen in mice is independent of Treg function and the autoimmune response is mediated by both Th1 and Th2 cells. We speculate that the basement membrane deposition of IgG may eventually lead to blister development.