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The Journal of Immunology, 2002, 168: 2020-2027.
Copyright © 2002 by The American Association of Immunologists

Development of Atopic Dermatitis-Like Skin Lesions in STAT6-Deficient NC/Nga Mice1

Ryouji Yagi*,{dagger}, Haruko Nagai*, Yutaka Iigo{ddagger}, Toshihiro Akimoto{ddagger}, Takao Arai{dagger} and Masato Kubo2,*

* Division of Immunobiology, Research Institute for Biological Sciences, and {dagger} Department of Applied Biological Science, Science University of Tokyo, Noda City, Chiba, Japan; and {ddagger} New Product Research Laboratories III, Daiichi Pharmaceutical Co., Kitakasai, Edogawa, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6-/-) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6-/- NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-{gamma}-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-{gamma} are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-{gamma} production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Atopic dermatitis (AD)3 is a pruritic inflammatory skin disease usually associated with a family history of atopy. T cells and eosinophils are thought to play a major role in the pathogenesis of this disease. The skin lesion in AD reveals a mononuclear cell infiltrate consisting of activated CD4+ T cells expressing IL-4, IL-5, and IL-13 mRNA and protein (1). Most patients have increased serum levels of IgE Ab against many kinds of allergens (2). Evidence suggests that the development and pathogenesis of AD are associated with immunological abnormalities, such as type I allergic reaction (3, 4, 5, 6). However, for many years controversy has surrounded the contribution of allergic or IgE-mediated hypersensitivity reactions to the pathogenesis of AD (7), because in some chronic AD skin lesions the development of AD appears to be independent of plasma IgE levels, but correlates with a significant increase in the number of cells expressing the Th1 cytokine, IFN-{gamma} (8, 9, 10).

NC/Nga (NC) mice, an inbred stain established from Japanese fancy mice by Kondo (Nagoya University, Nagoya, Japan), spontaneously develop an eczematous AD-like skin lesion when kept under conventional care, but not under specific pathogen-free (SPF) conditions (11). The elevation of plasma levels of total IgE has been reported to correlate with the appearance of the AD-like lesion in NC mice, with massive infiltration of CD4+ T cells producing IL-4 and IL-5, and the degranulation of mast cells and eosinophils (12). These pathophysiological observations in AD of NC mice highly resemble those in human AD, and this strain of mouse has thus been considered a useful model to analyze pathologic mechanisms of human AD (13). In NC mice, constitutive tyrosine phosphorylation of Janus kinase 3, a tyrosine kinase responsible for IL-4R-mediated signaling, is thought to be involved in the enhanced sensitivity of B cells against IL-4, leading to the elevation of total IgE levels (14). Moreover, Th2-specific chemokines, TARC and monocyte-derived chemotactic cytokine (MDC), and their receptor, CCR4, have been reported to be highly expressed in the lesions of the NC mouse (15). These findings strongly suggest the possible involvement of Th2 cells in the development of AD-like skin lesions in the NC mouse.

STAT6 is a critical transcriptional factor that regulates IL-4-mediated immune responses. STAT6 is phosphorylated and activated through an IL-4R-mediated signal; it translocates as a phosphorylated homodimer and subsequently regulates IL-4-mediated transcriptional events, including Th2 differentiation, expression of cell surface markers, and Ig class switching to IgE. IL-4-mediated STAT6 activation is an efficient cascade for the generation of Th2 cells during primary T cell activation (16, 17). The disruption of the STAT6 gene in mice has revealed its requirement for the development of Th2 cells and Th2-specific immune responses, such as IgE hyperproduction and atopic bronchial asthma (18, 19, 20, 21).

To address the functional relevance of IgE and Th2-mediated immune responses in NC mice to the development of AD-like skin lesions, we created STAT6-deficient (STAT6-/-) NC mice that were unable to mount the Th2 cytokine production required for the serum IgE response. Unexpectedly, these mice still elicited pathogenesis of AD-like skin disease, characterized by infiltration of eosinophils and mast cells, suggesting the Th2 cell-independent nature of the pathogenesis of AD in NC mice. We further discuss the possibility that IFN-{gamma} and IL-18, predominantly expressed in affected skin regions, may be more important than Th2 cytokines for the development of AD in NC mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

SPF NC/Nga mice were purchased from Clea (Tokyo, Japan). STAT6-deficient mice with a C57BL/6 background were a gift from Dr. S. Akira (Osaka University, Osaka, Japan) (18). The STAT6-deficient mice have been backcrossed to NC/Nga mice for more than six generations in the SPF condition. STAT6+/- NC mice were selected by genomic PCR using the primer specific for the downstream region of the targeted STAT6 gene and the targeted neo-resistant gene. STAT6+/- NC mice were mated, and STAT6+/+ and STAT6-/- NC mice were selected from their offspring.

Cytokines and Abs

The reagents for ELISA and intracellular cytokine staining, anti-IL-2 (JES6-1A12 and JES6-5H4 biotin), anti-IFN-{gamma} (R4-6A2 and XMG1.2 biotin), anti-IL-4 (BVD4-1D11 and BVD6-24G2 biotin), anti-IFN-{gamma} (XMG1.2) FITC, and anti-IL-4 (11B11) PE, were purchased from BD PharMingen (San Diego, CA). Anti-CD28 mAb (PV-1) was a gift from Dr. R. Abe (Science University of Tokyo, Tokyo, Japan).

Measurement of serum Ig concentration

Blood was drawn from the sinus cavernosus, and serum was separated by centrifugation at 1500 x g for 10 min. Serum levels of IgM, IgG1, IgG2a, IgG2b, and IgE were determined by ELISA using Ab pairs specific for different mouse Ig isotypes (BD PharMingen).

Measurement of cytokine production in primary TCR activation and ELISA

Cells were incubated with anti-CD8 mAb (3.155) at 4°C and were placed on the plate-coated anti-mouse Ig (Cappel, Aurora, OH) to eliminate B and CD8+ T cells. This enrichment resulted in >80% CD4+ T cells. The CD4+ T cell populations (1 x 106 cells/ml) were prepared from lymph nodes and stimulated with plate-bound anti-TCR plus anti-CD28 mAb for 48 h. The culture supernatants were harvested, and the cytokine concentration was analyzed by ELISA as described previously (22).

Measurement of cytokine concentration secreted from effector CD4+ T cells

The enriched splenic CD4+ T cells (1 x 106 cells/ml) were stimulated with plate-bound anti-TCR (H57-597) mAb (30 µg/ml) plus the soluble form of anti-CD28 mAb (PV-1, 5 µg/ml). After 7 days, viable cells were collected and restimulated with plate-bound anti-TCR for 24 h, the culture supernatants were harvested, and the cytokine concentration was analyzed by ELISA.

Dermal histology

The dermal skin were resected, fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned, stained with either H&E or Giemsa solution, and examined by light microscopy for histologic changes.

Measurement of cytokine and chemokine expression in skin

RT-PCR was performed using total RNA isolated from the dermal skin lesion by TRIzol reagent (Life Technologies, Gaithersburg, MD). The sequences of primers used are: eotaxin 1, 5'-atgcagagctccacagcgct-3' and 5'-ttatggttttggagtttggag-3'; eotaxin 2, 5'-ctgtgcctgacctccagaac-3' and 5'-ctaaacctcggtgctattgc-3'; CCR3, 5'-tgctactatcaccagtatcattacc-3'and 5'-gcttgttctttccattttctcacc-3'; CCR4, 5'-atgaatgccacagaggtcac-3'and 5'-ttacaaagcgtcacggaagt-3'; CCR5, 5'-taccagatctcagaaagaaggttttcatta-3'and 5'-gcgtttgacaatgtgttttcggaagaacac-3'; MDC, 5'-atggctaccctgcgtgtccc-3' and 5'-ctaggacagtttatggagta-3'; TARC, 5'-caggaagttggtgagctggtata-3' and 5'-ttgtgttcgcctgtagtgcata-3'; caspase I, 5'-tggtcttgtgacttggagga-3' and 5'-tggcttcttattggcacgat-3'; IL-4, 5'-atgggtctcaacccccagctagt-3' and 5'-gctctttaggctttccaggaagtc-3'; IL-18, 5'-caaagtgccagtgaaccccagaccaga-3' and 5'-acaaaccctccccacctaactttgatg-3'; IL-4, 5'-gagccatatccacggatgcgacaa-3' and 5'-catggtggctcagtactacgagta-3'; IL-5, 5'-ttgacaagcaatgagacgat-3' and 5'-ggctacattaccagtttgag-3'; IFN-{gamma}, 5'-ctcaagtggcatagatgt-3'and 5'-gagataatctggctctgcaggatt-3'; and IL-12 p35, 5'-tctctggacctgccaggtgt-3' and 5'-cctgttgatggtcacgacgcg-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine the relevance of Th2-mediated responses to the development of eczematous AD-like skin lesions, STAT6-deficient mice were backcrossed to the NC/Nga background for six generations under SPF conditions. STAT6+/+ (control) and STAT6-/- NC mice were selected at the age of 6–8 wk and moved from SPF to conventional conditions. After 4 wk or more the control NC mice commenced scratching, and lesions developed in affected regions, including the back, neck, face, and ears. The lesion consisted of hemorrhage and excoriation, and the extent of the lesions varied among individuals (Fig. 1GoA). Under conventional conditions in our animal facility, 10 of 19 (52.6%) mice developed AD-like skin lesions (Fig. 1GoB). Surprisingly, STAT6-/- NC littermates also clearly elicited AD-like skin lesions, and the frequency was even higher than that in control mice (76.9%). When we scored the extent of the lesions on the ears, face, head, and back, there was no difference in the mean values in the score of clinical skin severity between controls and STAT6-/- NC mice (Fig. 1GoB).



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  		FIGURE 1. Frequency and severity of the AD-like skin lesion in STAT+/+ (control) and STAT-/- NC mice. Aa, Control normal NC mice. Ab, The same litter of affected control NC mice. The severe lesions extended into the back, neck, and ear, and the ear was completely scratched out. B, The frequency of skin lesion was 10 of 19 for control littermate NC mice and 10 of 13 for STAT6-/- NC mice. The clinical severity of the AD-like skin lesion was assessed as the AD score with respect to four individual regions (ear, face, head, and back), and the total score was plotted. A score of zero represents normal skin, while a score of 4 represents the most severe skin lesion. {circ}, Control NC mice; •, STAT6-/- NC mice. Mean values of the AD score are indicated by the horizontal bars.

 
We first analyzed immunological abnormalities in STAT6-/- NC mice and compared them with those in control littermates. At the age of 8 mo, IgE levels in the control mice developing AD-like skin lesions were ~10-fold higher than those in unaffected control littermates (Fig. 2GoA). IgM levels in affected control mice were slightly lower than those in unaffected controls, while the serum levels of IgG remained unchanged even after the development of skin lesions. In STAT6-/- NC mice, IgE and IgG1 levels were drastically reduced; IgE was decreased to undetectable levels, while the IgG1 levels were almost 20-fold lower than those in control mice. The development of skin lesions in STAT6-/- NC mice did not enhance the serum IgE or other Ig isotype class levels (Fig. 2GoA). These results indicated that the development of the AD-like skin lesions in STAT6-/- NC mice is independent of the elevation of serum IgE.



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  		FIGURE 2. Serum Ig concentration in AD-like skin lesion developing control and STAT-/- NC mice. Sera were obtained from control and STAT6-/- NC mice with (•) or without ({circ}) AD-like skin lesion at the age of 8 mo, and serum levels of IgM, IgG1, IgG2a, IgG2b, and IgE were determined by ELISA. Each symbol indicates individual mice, and mean values are indicated by the horizontal bars.

 
IgE class switching in B cells is regulated by IL-4 secreted from Th2 T cells, and the requirement of STAT6-mediated signaling for the development of these cells has been clearly demonstrated by the absence of Th2 immune responses in STAT6-deficient mice (18, 19, 20). However, an alternative Th2 developmental pathway independent of IL-4-mediated signaling has been demonstrated in parasitic infection and antigenic stimulation systems (23, 24, 25). Thus, we next analyzed Th1 and Th2 cytokine production in activated T cells from spleens of STAT6-/- NC and control mice. The committed effector CD4+ T cells were induced by a combination of anti-TCR and anti-CD28 Abs. After 7 days of culture, T cells were harvested and restimulated with anti-TCR Ab for 24 h, and the concentrations of IL-2, IFN-{gamma}, IL-4, IL-5, and IL-10 in the supernatants were analyzed by ELISA. The splenic T cells from the control mice developing skin lesions showed slight increases in IL-4 and IL-5 production, 2- to 3-fold increases in IFN-{gamma} and IL-10, and no changes in IL-2 compared with unaffected controls. In STAT6-/- NC mice, Th2 cytokine levels, including IL-4, IL-5, and IL-10, were almost undetectable even after the mice developed skin lesions (Fig. 3GoA). However, the increased production of IFN-{gamma} was still observed in STAT6-/- NC mice following the development of skin lesions (Fig. 3GoA). Furthermore, the cytokine production profile in T cells from skin-draining lymph nodes proximal to the skin lesions was very similar to that in the committed CD4+ T cells from spleen (data not shown).



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  		FIGURE 3. Cytokine production and Th cell development in AD-like skin lesions in control and STAT-/- NC mice. A, Cytokine production of the activated effector T cells from control and STAT-/- NC spleens. The CD4+ T cells were separated from spleen cells of control and STAT-/- NC mice with (•) or without ({circ}) AD-like skin lesions, and Th cell differentiation was introduced by stimulation with anti-TCR and CD28 mAbs for 7 days. The differentiated T cells were restimulated with anti-TCR mAb, and the concentrations of cytokines in the supernatant were measured by ELISA. Each symbol indicates individual mice, and mean values are indicated by the horizontal bars. B, Profile of Th cell development from spleens of control and STAT-/- NC mice. The preactivated CD4+ T cells were prepared as described in A and restimulated with anti-TCR mAb in the presence of monensin. After permeabilization, cells were stained with anti-IFN-{gamma}-FITC and anti-IL-4-PE.

 
We also studied Th1 and Th2 differentiation in CD4+ T cells from STAT6-/- NC mice by single cell analysis. CD4+ T cells were preactivated with anti-TCR and anti-CD28 mAb to initiate Th cell differentiation, and IL-4- or IFN-{gamma}-producing Th cells were examined by intracellular cytokine staining after 7 days of culture. Predominant skewing toward IL-4-secreting Th2 cells was found in normal NC mice, and this profile became more prominent with the development of AD-like disease (Fig. 3GoB). In contrast, STAT6-/- NC mice showed no Th2 development; instead, most T cells skewed toward IFN-{gamma}-secreting Th1 cells (Fig. 3GoB). The proportion of Th1 cells was significantly increased in mice developing the disease. Therefore, although the genetic background of NC mice has a propensity to facilitate the Th2 response, Th2-mediated effector functions, including Th2 cytokine production, and subsequent IgE responses may not be responsible for the development of AD-like skin disease in NC mice.

Next, we examined the possibility that the mechanisms regulating eczema formation in STAT6-/- NC mice may differ from those in STAT6+/+ mice. In a comparison of histological sections of dermal skin from the head and back of unaffected NC mice and NC mice that had developed lesions, it was found that the lesions showed a significant thickening of the dermis and epidermis and a cellular infiltrate in the dermis that contained eosinophils (Fig. 4GoB, H&E staining) and mast cells (Fig. 4GoC, Giemsa staining). The skin lesions elicited in STAT6-/- NC mice had histological features very similar to those of control NC mice (Fig. 4Go, D and E). Therefore, we concluded that there were no significant differences in the pathogenesis of skin lesions between control and STAT6-/- NC mice. These findings clearly indicate that the infiltration of eosinophils and mast cells tightly correlates with the development of disease, but increased IgE levels and Th2 cells were not necessary for these infiltration events.



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  		FIGURE 4. Histologic analysis of dermal skin lesion from control and STAT6-/- mice. The normal dermal skin was resected from the head of unaffected control mice (A). The skin lesions were resected from head of the affected control (B and C) and STAT6-/- mice (D and E). The sections were stained by H&E (HE; B and D) and Giemsa solution (C and E). Strong staining with HE indicates eosinophils (open arrows). Strong staining with Gimza indicates mast cells (closed arrows). Magnification, x66.

 
The above observations in STAT6-/- NC mice raised the question of whether the development of skin lesions is regulated by immunological responses via Th cells. It would be possible that nonimmunological stimuli under conventional conditions produce skin itching with the infiltration of eosinophils and mast cells. To investigate this possibility, we studied the pathogenesis of lymph nodes. The cervical, axillary, and brachial lymph nodes in the affected NC mice were markedly enlarged compared with those in unaffected littermates (Fig. 5GoAa). The enlargements were specifically restricted to lymph nodes proximal to the skin lesions, while distal inguinal and popliteal lymph nodes in the affected NC mice remained normal (Fig. 5GoAa). The enlarged cervical lymph node contained 10 times more cells compared with that of normal littermate NC mice (data not shown), suggesting that large numbers of T and B cells specifically migrate into the lymph nodes proximal to the skin lesion elicited in the ear and head. Indeed, histological and FACS analyses revealed that the cervical lymph node in the affected mice were replete with T and B cells (Fig. 5Go, Ab and B). The proportion of B220+ B cells in the cervical lymph node was markedly higher than that in the distal inguinal lymph node, and more than half of the B cells showed low expression of surface IgM, indicating that many plasma cells migrate into the proximal lymph node (Fig. 5GoB). This was consistent with the histological features of the cervical lymph nodes in affected mice (Fig. 5GoAb). Moreover, T cells in the cervical lymph nodes showed an enhancement of CD62 ligandlow, CD44high-activated phenotype (Fig. 5GoB and data not shown). These results indicated that site-specific inflammation may contribute to the development of skin lesions.



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  		FIGURE 5. Enlargement of cervical lymph node (LN) and cytokine production in the cervical LN T cells from the AD-developing NC mice. A, Appearance of LN. B, Histologic analysis of cervical LN in the AD-developing NC mice. Ly, lymphocyte (T cells); PC, plasma cells. C, Cytokine production at primary stimulation. The CD4+ T cells were separated from lymph node cells of control and STAT-/- NC mice with (•) or without ({circ}) AD-like skin lesions and stimulated with anti-TCR mAb. The concentrations of cytokines in the supernatants were measured by ELISA. Each symbol indicates an individual mouse, and mean values are shown by the horizontal bars.

 
Next, we studied the relevance of T cell-mediated immune responses at the site of the developing disease by assessing cytokine production. Purified CD4+ T cells from cervical lymph nodes were prepared and stimulated with anti-TCR and CD28 mAbs for 48 h. The levels of Th2 cytokines, IL-4 and IL-5, produced by lymph node T cells from the control affected mice were increased in some mice compared with those in unaffected controls, whereas IL-4 and IL-5 levels did not correlate with the development of AD in STAT6-/- NC mice (Fig. 5GoC). In contrast, IFN-{gamma} production was 3- to 4-fold higher in most AD-developing mice, and an increase in IFN-{gamma} production was consistently found in STAT6-/- NC mice (Fig. 5GoC). This indicates that accumulation of the IFN-{gamma}-producing T cells is consistently observed at lymph nodes proximal to the dermal skin lesion.

To further examine whether the infiltration of cytokine-producing cells into the skin is associated with the development of disease, we analyzed the expression of cytokines, chemokines, and chemokine receptors in the skin lesions by RT-PCR. The expression of the Th2 cytokines, IL-4 and IL-5, was not detectable even in the AD-like skins from STAT6+/- NC mice. In contrast, IFN-{gamma} expression was consistently found in the skin of AD-developing NC mice regardless of STAT6 (Fig. 6GoA). IL-18 is known to be a potent inducer of IFN-{gamma} production in conjunction with IL-12. Consistent with the increased IFN-{gamma} level, the expression of both IL-18 and IL-12 was clearly up-regulated in the skin lesions (Fig. 6GoA). Caspase 1, a cleaving enzyme that converts inactivated IL-18 to its active form, was prominently expressed in the skin, but the basal expression was not characteristic of NC mice, because skin from BALB/c and C57BL/6 mice exhibited the same level of caspase 1 expression (Fig. 6GoB). Consistent with a previous observation in caspase 1 transgenic mice that spontaneously developed AD-like skin disease with an elevation of serum IL-18 (26), caspase 1 expression became more prominent in the skin from AD developing NC mice (Fig. 6GoA). These results suggest that the cytokine environment that promotes the production of IFN-{gamma}-producing cells was generated at the site of the developing AD-like skin disease, and that this process resulted in the infiltration of IFN-{gamma}-producing T cells into dermal skin lesions from proximal lymph nodes. Therefore, it is reasonable to speculate that the infiltration of IFN-{gamma}-producing T cells may be associated with eczema formation in NC mice.



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  		FIGURE 6. Total RNA was isolated from the dermal skin of normal and affected control NC mice. The expressions of cytokines, chemokines, and chemokine receptors were analyzed by RT-PCR using the set of primers described in Materials and Methods.

 
The expression of inflammatory chemokines MDC in the skin of the head was not detectable even after the development of disease, while TARC and eotaxin 1 were prominently expressed. TARC expression was slightly increased with the development of disease, but eotaxin 1 was not affected. However, a dramatic enhancement of expression of eotaxin 2 and CCR, such as CCR3, -4, and -5, was found to coincide with the development of skin lesions (Fig. 6GoA). Eotaxin 2 has been identified as a CC chemokine with eosinophil-selective chemoattractant activity, and its expression is regulated by allergen challenge and IL-4 (21). However, the present study clearly demonstrated that the increased eotaxin 2 expression at the skin lesion-developing site in NC mice was STAT6 independent (Fig. 6GoA). The increased eotaxin 2 and CCR3 expression was found only when IFN-{gamma} was constitutively expressed at the skin. Therefore, we speculate that the elevation of these chemokines and receptors may result from the IFN-{gamma}-mediated responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The NC mouse has been proposed as a model of human AD, which is described as a Th2-type disease at least in the initiating phase. Indeed, the disease-developing NC mice revealed predominant Th2 responses and elevated serum IgE levels. However, our finding in STAT6-/- NC mice clearly demonstrated that the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin lesions were elicited in STAT6-/- NC mice that had completely impaired Th2 differentiation and IL-4-mediated IgE class switching. Both control and STAT6-/- NC mice revealed equivalent clinical severity in the AD skin lesions characterized by hypertrophy of the dermis and epidermis and massive infiltration of eosinophils and mast cells. Cytokine and chemokine expression analysis demonstrated that IFN-{gamma}-producing T cells were accumulated at the local lymph nodes, and that IFN-{gamma}, IL-18, and IL-12 were highly expressed at the skin site that developed AD-like disease, suggesting that the infiltration of IFN-{gamma}-producing cells may account for eczema formation in NC mice.

Under conventional conditions, >50% of control NC mice spontaneously developed eczematous AD-like lesions around the age of 6 mo or later. The lesions appear to occur as a result of extensive scratching, based on the behavior of the mice. In this strain the level of serum IgE gradually increases and shows relatively high basal levels compared with age-matched mice of other strains. All the mice developing disease showed further elevation of serum IgE levels of about 10-fold (Fig. 2Go) and increased Th2 cytokines, such as IL-4, IL-5, and IL-10 (Fig. 3GoA). These observations in control NC mice were consistent with several previous reports that the Th2-mediated immune response is probably responsible for the development of skin lesions in NC mice (11, 12, 14, 27). In many AD patients increased Th2 cytokines and serum IgE levels are regarded as a specific feature reflecting the development of disease (3, 4, 5, 6, 28). These previous findings suggest that the NC strain would be a useful animal model for human AD (11).

However, STAT6-/- NC mice can elicit AD-like skin lesions to the same extent as control NC mice. The lesions completely fulfilled the pathologic criteria of the disease and were characterized by thickening of the outer layer and stratum spinosum of the epidermis and infiltration of eosinophils and mast cells in the dermis (Fig. 4Go). These results clearly demonstrated that the presence of IgE and Th2 cells is not a prerequisite for the development of skin lesions in NC mice. However, the results in this study could not completely exclude the involvement of an alternative IL-4 signaling pathway in the development of AD, because T and B cells from STAT6-/- NC mice can respond to IL-4 stimulation with a proliferative response and tyrosine phosphorylation of IL-4R and Janus kinase 1 (data not shown).

These findings agree with previous reports that the Ag sensitization elicited the AD-like skin lesions in IgE-deficient mice (29). The thickening of the skin layer in the lesions at the OVA-sensitized site of IgE-deficient mice was similar to the skin lesions in STAT6-deficient NC mice. The infiltration of eosinophils and mast cells in STAT6-deficient NC mice was elicited in the absence of IL-4- and IL-5-producing T cells. However, this Ag-sensitized mouse model indicates the functional significance of IL-4 and IL-5 in the infiltration of eosinophils (29). These results indicate that the mechanisms regulating skin disease in NC mice differ from the Ag sensitization elicited by the AD-like skin lesions. The presence of IgE and Th2 cells is not a prerequisite for the development of AD-like lesions of NC mice at least in the initiating phase, although we cannot rule out the possibility that Th2 cytokines and IgE are responsible for the pathogenetic events in the chronicity and exacerbation of this disease. Current studies in humans have reported that the subgroup of AD patients with normal serum IgE levels and without specific IgE-mediated sensitization is characterized as nonallergic dermatitis (8, 9, 10, 30, 31). Therefore, the development of eczematous AD-like lesions in NC mice may contribute to the pathogenesis of nonallergic dermatitis. The elevated serum levels of IgE and the development of Th2 responses in normal affected NC mice may be consequences of the progression of the pathogenesis of AD-like disease.

The regional lymph nodes proximal to the skin developing the lesions exhibited massive enlargement elicited by the accumulation of activated IFN-{gamma}-secreting T cells and plasma cells (Fig. 5Go). Inconsistent with previous observations (1), expression analysis of cytokines showed no expression of IL-4 and IL-5 at lesional skin (Fig. 6Go). The increased IFN-{gamma} production tightly correlates with the eczema formation regardless of the STAT6-mediated signaling. Many potent inducers of IFN-{gamma} production, such as caspase 1, IL-12, and IL-18, were significantly up-regulated at the skin lesion (Fig. 6Go). Caspase 1 converts IL-18 from an inactivated to an activated form by functioning as a cleaving enzyme, and the cleaved IL-18 acts as a potent inducer of IFN-{gamma} production in conjunction with IL-12 (32, 33). It is well documented that IL-12 produced by Langerhans cells, eosinophils, and keratinocytes is a potential mediator for the induction of IFN-{gamma} in T cells (34, 35, 36). Therefore, the cytokine microenvironment at pathologic sites leads to the accumulation of IFN-{gamma}-secreting T cells during the development of skin lesions.

On the basis of these observations in STAT6-/- NC mice, the pathogenesis of the skin lesion was postulated to be regulated by IFN-{gamma}-mediated responses. The expression profile of chemokines in the skin lesion showed increased expression of TARC, eotaxin 2, and CC chemokine receptors even in STAT6-/- NC mice. The TARC/MDC-CCR4 system has been shown to be involved in the Th2-mediated disease process (37, 38). However, consistent with the observation in NC mice that IFN-{gamma}, but not IL-4, induces TARC expression in keratinocytes (15), our results indicate that the enhancement of TARC expression at the pathological site correlated with the increased IFN-{gamma} expression. Eotaxin 2 is newly identified potent chemoattractant for eosinophils. Eotaxin 2 and its receptor, CCR3, were predominantly expressed in lesional skin. It was previously reported that IFN-{gamma} plays a role as a potent inducer for eotaxin 2 and CCR3 (39). Therefore, we speculate that the elevation of these chemokines and receptors may result from IFN-{gamma}-mediated responses. Recently, it has been demonstrated that CXCR3, predominantly expressed on human IL-2-stimulated T cells, is a receptor for IFN-{gamma}-inducible protein 10 (IP10) and monokine induced by IFN-{gamma} (Mig) (40). The induced IP10 and Mig activate eosinophils through CXCR3 that is also expressed on eosinophils (41). It would be possible that this IFN-{gamma}-induced IP10/CXCR3 pathway plays a role in the disease site of NC mice, although we have not analyzed IP10, Mig, and CXCR3 expression in this study.

The pathological significance of IFN-{gamma} proposed in this paper is consistent with previous reports using IFN-{gamma}-deficient or transgenic mice indicating that IFN-{gamma}-secreting cells play a significant role in allergic eczema formation (29, 31). Moreover, recent reports demonstrated that transgenic mice expressing caspase 1 in keratinocytes spontaneously developed AD-like skin disease with an elevation of the active form of IL-18 in serum (37). Recent linkage analysis demonstrated that the locus responsible for skin disease in NC mice is on chromosome 9, and the IL-18 gene is near the locus (42). It is therefore reasonable to speculate that the increased caspase 1, IL-18, and IL-12 expression in the skin microenvironment may result in the increased IFN-{gamma} production, leading to the induction of eotaxin 2 expression that acts as a chemoattractant for CCR3+ eosinophils. However, at this point the evidence to support this idea remains correlative. We have attempted to directly examine the role of IFN-{gamma} by the administration of neutralizing Abs, but it was difficult to control the timing of administration, since the appearance of clinical signs varies among individuals. Therefore, a nonimmune reaction may account for the development of the pathogenesis in NC mice, and the IFN-{gamma}-favored skin microenvironment may be a result of the pathogenesis in NC mice. The establishment of Rag2-, caspase 1-, IL-18-, or IFN-{gamma}-deficient NC mice will directly answer these questions.


   Footnotes
 
1 This work was supported by the grant from the Ministry of Education, Science, and Culture (Japan), the Kao Foundation for Arts and Sciences, and the Naito Foundation. Back

2 Address correspondence and reprint requests to Dr. Masato Kubo, Division of Immunobiology, Research Institute for Biological Sciences, Science University of Tokyo, Yamazaki, Noda City, Chiba 278-0022, Japan. E-mail: raysolfc{at}rs.noda.sut.ac.jp Back

3 Abbreviations used in this paper: AD, atopic dermatitis; IP10, inducible protein 10; MDC, monocyte-derived chemotactic cytokine; Mig, monokine induced by IFN-{gamma}; SPF, specific pathogen free; TARC, thymus and activation-regulated chemokine. Back

Received for publication August 31, 2001. Accepted for publication December 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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