**2.1 Ovalbumin (OVA)-sensitized mice**

40 Atopic Dermatitis – Disease Etiology and Clinical Management

in STAT6, a common critical transcription factor for IL-4 and IL-13 signals, exhibited skin lesions comparable to those of STAT6-positive littermates (13). Here, we summarize the results of analyses investigating the roles of Th2-type cytokines, using mice epicutaneously

Category Application Phenotypes References

No change in chemokine expression No change in infiltration of CD45+, CD4+,

Thinning of epidermis and dermis No change in chemokine expression No change in infiltration of CD45+, CD4+,

Presence of *Staphylococcus aureus* 

Degranulation of mast cells in dermis

Increased dendritic cells, macrophages, and NK cells in skin and lymphoid organs

Infiltration of macrophages, dendritic cells, eosinophils, CD4+ cells, and mast cells Increased expression of CCL2/MCP-1,

CCL5/RANTES, CCL11/Eotaxin, CCL17/TARC, CCL22/MDC, CCL27/CTACK, TSL, and VEGF

Increased serum IgE and IgG1 Increased T cell proliferation

No change in thickness of epidermis and dermis

Infiltration of mononuclear cells and eosinophils in

Increased expression of IL-5, IL-13,IL-12p40, IFN-γ,

15

15

16-19

20

sensitized with allergens and genetically engineered mice (Table 1).

or CD8+ cells

No eosinophil

or CD8+ cells

Spongiosis Acanthosis

dermis and epidermis

TNF-α, and IL-1β

Bacterial pyoderma

Dry lichenified skin lesion

Increased serum IgE and IgG1 Increased systemic immune responses

Pruritis Loss of hair Erythema Crusting Excoriation

Erosions

Table 1. Summary of effects of Th2-type cytokines in model mice

Xerosis Conjunctivitis Pruritis

Decreased eosinophils Decreased serum IgE

Ovalbuminsensitized mouse

Genetically engineered mouse

IL-4– deficient mouse

IL-5– deficient mouse

IL-4 transgenic mouse

IL-13 transgenic mouse

Geha's group established AD model mice by repeated epicutaneous sensitization to OVA (14). These mice manifest AD-like skin lesions including acanthosis, spongiosis, and infiltration of neutrophils, lymphocytes, mast cells, and eosinophils into the dermis. Expression of Th2-type (IL-4, IL-5 and IL-13) cytokines is up-regulated with little or no change of IFN-γ expression (2, 14). Furthermore, serum levels of total or OVA-specific IgE and IgG1 are elevated. This mouse model has been used to investigate involvement of various molecules associated with the pathogenesis of AD.

#### **2.1.1 Application of OVA-sensitized mouse model to IL-4–deficient mice**

The role of IL-4 in OVA-sensitized AD model mice is complex (15). The eosinophil numbers decrease in the dermis of OVA-sensitized IL-4–deficient mice showing an important role of IL-4 for infiltration of eosinophils. However, the numbers of CD45+, CD3+, CD4+, and CD8+ cells and expression of MIP-2, MIP-1β, IP-10, and RANTES increase compared to wild type mice, suggesting that IL-4 has an inhibitory role in expression of chemokines to recruit T cells. It is of note that thickness of the epidermis and dermis are not changed compared to wild type mice showing that IL-4 has a subtle effect on skin thickening. In these mice, total or OVA-specific IgE levels significantly decrease, which argues against the roles of IgE in the pathogenesis of this mouse model. Correspondingly, OVA-sensitized IgE-deficient mice show no change in histological views of the skin tissues, infiltration of CD45+, CD3+, CD4+, and CD8+ cells and expression of IL-4, IL-5 and IFN-γ compared to OVA-sensitized wild-type mice (15), suggesting that this mouse model is independent of IgE. Furthermore, the redundancy of IL-4 and IL-13 *in vivo* may be the reason why the effects of IL-4–deficiency on skin thickening are subtle in this mouse model.

#### **2.1.2 Application of OVA-sensitized mouse model to IL-5–deficient mice**

IL-5–deficient mice had virtually no eosinophil (15). These mice sensitized with OVA had thinning of the epidermis and dermis compared with wild-type mice. However, infiltration of CD45+, CD3+, CD4+, and CD8+ cells and expression of chemokines were equivalent to wild-type mice. These results may suggest that IL-5 partially contributes to generating ADlike lesions in this mouse model.

#### **2.2 Genetically engineered mice for Th2-type cytokines**

Thus far, transgenic mice that express IL-4 or IL-13 specifically in keratinocytes have mainly been generated using keratin promoters. In contrast to the IL-4-deficient mice, these gain-offunction mice manifest overt skin lesions, which indicates topical overexpression of either IL-4 or IL-13 that is sufficient to induce phenotypes similar to AD patients.

#### **2.2.1 IL-4 transgenic mice**

Chan's group established and characterized IL-4 transgenic mice in which expression of IL-4 is controlled under the promoter of the keratin 14 gene (16-19). These mice are normal when they are newborn. However, almost four months after birth, they develop AD-like lesions such as xerosis (dry skin), conjunctivitis, and pruritic skin lesions (16). Many skin lesions appear in ears, necks, and eyes. Forty-three percent of the mice develop

The Roles of Th2-Type Cytokines in the Pathogenesis of Atopic Dermatitis 43

(Figure 1). STAT6 is a transcriptional factor critical for IL-4 or IL-13 signals. The association of the *IL13*, *IL4RA*, and *STAT6* genes with IgE is confirmed by recent Genome Wide

Gene Variant Association Function References

*IL4* -590C/T AD Change in IL-4 production 24-26

Lower affinity with the IL-

production 34, 37-40

44

44

45

28-36

41, 42

13R α2 chain Enhanced stability Transduction of stronger signal via IL-13Rα1

Change in IL-13

SHP-1

2964G/A Serum IgE 43

Decreased binding to

Increased STAT6 activation

AD Serum IgE specific antiallergen IgE

AD Serum IgE specific antiallergen IgE

Hyper IgE syndrome AD

Serum IgE Eosinophil number

Serum IgE Eosinophil number

Serum IgE Eosinophil number

Another group confirmed the association of this SNP with AD (26).

Table 2. Summary of genetic associations in Th2-type cytokines and their related molecules

There exists a SNP in the promoter of the *IL4* gene (-590C/T). This SNP was originally reported to be associated with bronchial asthma (24) and is thought to affect the binding activities to NFAT, resulting in modulating IL-4 production. Transmission disequilibrium testing shows a significantly preferential transmission of the T allele in AD patients (25).

*IL13RA1* 1398A/G Serum IgE 27

Association Studies (22, 23).

Arg110Gln (Arg130Gln, G4257A)


(Gln551Arg)

in18SNP1C/T

GT repeat in exon1

*IL4RA* Gln576Arg

*IL5* -703C/T

*IL13* 

*STAT6* 

**3.1 IL-4 gene** 

skin lesions within 12 months. Inflammatory skin lesions show the presence of *Staphylococcus aureus*, a common infectious complication and exacerbating factor in human AD patients. Histological analyses of the lesions in these mice show spongiosis, acanthosis, dermal and epidermal infiltration of mononuclear cells and eosinophils, and degranulating mast cells in the dermis. Expression of Th2-type cytokines (IL-5, IL-13), Th1-type cytokines (IL-12p40, IFN-γ), and inflammatory cytokines (TNF-α, IL-1β) is upregulated in the skin lesions of these mice. Enhancement of these cytokines except IL-12p40 is even observed in the skin, before the disease onset or in the intact skin (17). Correspondingly, serum levels of IgE and IgG1 are elevated even before the onset, which reflects high expression of IL-4 and IL-13. Afterward, up-regulated serum levels of IgG2a are observed, reflecting high expression of IFN-γ (19). T cells in these mice possess higher proliferating capacity *in vitro* spontaneously or induced by stimulants such as T cell receptor triggering or Staphylococcus enterotoxins A and B compared to T cells in wildtype mice (18). It is of note that the numbers of dendritic cells, macrophages, and NK cells increase in the skin tissues and lymphoid organs of these mice, which suggests that overexpression of IL-4 in skin tissues modulates antigen-presenting activity *in vivo*.

#### **2.2.2 IL-13 transgenic mice**

Zhu's group established IL-13 transgenic mice in which withdrawal of doxycycline induces expression of IL-13 under the control of the promoter region of the keratin 5 gene (20). Six to eight weeks after the withdrawal of doxycycline, AD-like skin lesions (i.e. pruritis, loss of hair, erythema, crusting, excoriation, bacterial pyoderma, and erosions, thereafter dry lichenified skin lesion) appear in these mice. All of the mice develop these features within four months. In the skin lesions, F4/80+ cells including macrophages and dendritic cells, eosinophils, CD4+ cells, and mast cells are accumulated. Expression of various chemokines (CCL2/MCP-1, CCL5/RANTES, CCL11/Eotaxin, CCL17/TARC, CCL22/MDC, CCL27/CTACK), TSLP (a critical cytokine correlated with AD at the interface of epidermis and immune cells), and VEGF (a potent cytokine for angiogenesis), is up-regulated in these mice. Serum levels of IgE and IgG1 are elevated. Furthermore, systemic Th2 skewing is enhanced because the lymphocytes in the lymph nodes of these mice produce more IL-4 and IL-13 upon stimulation of anti-CD3/CD28 antibody (Ab) than do those in the wild-type mice.
