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Clin Exp Immunol. Oct 2005; 142(1): 21–30.
PMCID: PMC1809492

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

Abstract

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.

Keywords: atopic dermatitis, B cell activation, proliferation, IgE

Introduction

Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease characterized by infiltration of inflammatory cells. Data from previous studies suggested that B cell may be involved in the disease process. It has been shown that total serum IgE levels in human AD patients were markedly increased [14]. The predominance of Th2 cytokine including IL-4, IL-5 and IL-13 expressing cells at the acute phase is a histology hallmark of AD. While chronic skin lesion has significant fewer IL-4 and IL-13 expressing cells, it has greater numbers of IL-5, IL-12 and IFN-γ expressing cells, than in acute AD [57]. IL-4, a critical Th2-type cytokine essential for IgE class switching [8], has been found to have a genetic link in some human patients with AD [9]. Most of the studies demonstrated that there was a significant correlation between disease severity and level of total serum IgE [1,3,4]. But another study indicated that the extent of the dermatitis was not closely associated with the serum IgE concentration [2]. The relationship between IgE elevation and AD development has not yet been established, as a step-by-step study in human patients with AD, i.e. studying the serum IgE before disease onset, at acute disease stage and at chronic disease stage, cannot be done for both practical and ethical reasons. For the same ethical constrain, in vivo studies on the mechanism underlying IgE elevation in human AD patients cannot be performed. For example, B cell proliferation in human patients with AD at various stages of disease development has not been examined. Likewise, B cell populations carrying activation molecules, costimulatory molecules, or IA/IE in human patients with AD at various stages of disease has not yet been determined. We have generated an epidermally expressed IL-4 transgenic (IL-4-Tg) mouse line which develops a skin inflammatory disease, closely resembling human AD on clinical, histological, bacteriological, and serological criteria was generated in our laboratory [10] and the disease occurred in this IL-4-Tg mouse line fulfills the clinical diagnostic criteria for AD in human patients [11]. Furthermore, using cDNA microarray and reverse transcription real-time PCR methodology, we have documented an early up-regulation of predominant Th2 cytokines followed by a late surge of predominant Th1 cytokines in the skin lesion of the IL-4 Tg mice [12]. More recently, our characterization of the inflammatory cells in the different stages of the skin disease revealed that as the disease progresses, there is an increase of proliferation of T cells, an increase of T cell activation markers in the secondary lymphoid organs, and an increase infiltration of T cells to the skin [13]. We now turn our attention to the involvement of B cells. Taking advantage of the IL-4 Tg mouse model of AD, we studied the B cells and B cell products at different stages of disease development, so that we may gain further insight into the roles of B cells in the AD development. Our study results demonstrated that:

  • As the disease progresses from before onset to early disease, and to late disease, there was 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 increase of Th2-driven serum IgG1 and IgE in early disease was followed by significant increase of Th1-driven IgG2a in late disease;
  • The significant increases of activation molecule (IA/IE), proliferation (to LPS), and surface IgE on B cells of the IL-4-Tg mice preceded the up-regulation of serum IgE and disease onset;
  • IL-4 cytokine milieu in the skin-draining lymph nodes increased as the disease progressed. These data implies that, B cells, activated in the secondary lymphoid organs by up-regulation of IL-4, may play a role on the AD disease development by up-regulating serum IgE concentration, which serves as a marker of disease onset.

Materials and methods

Mice

The establishment and genotyping of IL-4 Tg mouse line was published previously [10,12,13]. The IL-4-Tg mice and non-Tg littermates were housed in special pathogen-free cages and fed with normal mouse chow and water without any manipulation. We have previously determined that all IL-4-Tg mice and none of non-Tg mice developed the well-characterized chronic inflammatory skin disease under these conditions [12,13].

Disease phenotype classification

As established previously, skin lesions from IL-4 Tg mice developed for the duration of one week or shorter are defined as early lesions (Tg-EL). Skin lesions developed for three weeks or longer are defined as late lesions (Tg-LL). Tg mice before disease onset is termed Tg-BO [12,13].

Score of the disease severity

Score of the disease severity was determined by the number of locations of skin involvement and the presence of inflammation and scale. The presence of inflammation, scale or skin lesions in each of the following areas, will be assigned one number: left ear, right ear, face, eye, trunk, left leg, right leg, tail. The severity score of the disease will be the sum of total numbers of a given mouse. A total of 15 mice were observed for each group.

Genomic DNA preparation and quantitative determination of IL-4 transgene copy number

Genomic DNA from IL-4 Tg mice was extracted according to the protocol published in a reference book [14]. To quantitatively determine the initial copy numbers of IL-4 in genomic DNA in each Tg-EL and Tg-LL mouse (n = 15 per group), real time PCR was performed. Primers for IL-4 and GAPDH and their plasmid standards were published previously [15]. The protocol for real time PCR was described previously [12]. The PCR was performed in an Mx3000p Real-Time PCR System (Stratagene, La Jolla, CA, USA). In order to normalize for DNA input amounts, the highest copy number of GAPDH of all samples was divided by copy numbers of an individual sample to give a calculating factor, which was then used for normalization.

ELISA measurements of total serum IgE, IgG1 and IgG2a

The concentrations of mouse total serum IgG1 and IgG2a were measured using commercially available ELISA kits from Alpha Diagnostic Int. (San Antonio, TX, USA). For mouse total serum IgE concentrations, ELISA was performed as below. Briefly, anti-mouse IgE monoclonal capture antibody (B.D. Bioscience, San Diego, CA, USA) was first coated on a Microtiter Styrene Immunoloassay plate (DYN-EX Tech, Chantilly, VA, USA) at a concentration of 0·5 mg/ml in PBS at 4°C for overnight. After the plate was blocked with PBS containing 10% FBS at room temperature (RT) for 1 h, serial diluted mouse IgE (B.D. Bioscience) for standard curve and diluted sera (1 : 25) obtained from four groups of mice (non-Tg mice, Tg-BO, Tg-EL and Tg-LL) were applied to the wells and incubated for 1 h at RT. Subsequently, a biotinylated antimouse IgE detection antibody at a concentration of 2 µg/ml (B.D. Bioscience) was applied to the wells for 1 h at RT. After washing, diluted streptavidin-HRP conjugate (Zymed, San Francisco, CA, USA) was added for 30 min incubation. Finally ABST substrate (Zymed) was added and the plate was read at 415 nm in a µQuant plate reader (Bio-TEK, Inc., Winooki, VT, USA). To test the specificity of IgE ELISA, different concentrations of mouse IgG1 (50, 100, 300, 600 ng/ml, Alpha Diagnostic Int.) were used as the antigen. IgG1 concentrations of 50, 100, 300 ng/ml were not detectable by the IgE ELISA; while the 600 ng/ml concentration of IgG1, an equivalent of 0·25 ng/ml of IgE, was detected. This concentration (0·25 ng/ml) was far less than the lowest level of IgE in mouse sera detected (88·3 ng/ml, Fig. 4) indicating that anti-IgE antibody used in IgE ELISA did not pick up sufficient IgG to alter the true value of IgE.

Fig. 4
Total serum concentrations of IgE, IgG1and IgG2a are elevated as the disease develops. Serum samples from non-Tg, Tg-BO, Tg-EL and Tg-LL mice were examined for total IgE levels (n = 15; a), and IgG1/IgG2a (n = 10; b & c) by ELISA as described ...

Flow cytometric analyses

Skin draining lymph nodes (LNs) and spleens were collected from non-Tg, Tg-BO, Tg-EL and Tg-LL mice. A total of 5 × 105 cells from LN and/or the spleen were first incubated with antimouse CD16/32 (B.D. Bioscience) in cold PBS/2% FBS/0·1% NaN3 for 10 min on ice to block Fc receptors. Cells were then labelled with FITC anti-CD19 and PE anti-CD11a/CD69/IgE/IgG1/IgG2a (B.D. Bioscience) /CD44/CD80/CD86/or FITC anti-IA/IE MHC class II (eBioscience, San Diego, CA, USA) and PE anti-CD19 (B.D. Bioscience) for 20 min on ice. Flow cytometry was performed and 10 000 cells were analysed per group on the Calibur FACS system (B.D. Bioscience). Live cells were gated based on 7-aminoactinomycin D (B.D. Bioscience) uptake. To determine absolute numbers of B cells in each spleen and LN from non-Tg, Tg-BO, Tg-EL and Tg-LL mice, single cell suspensions were counted using a haemocytometer. The absolute numbers of CD19+ CD11a+ cells were then obtained by multiplying the total cell numbers of each spleen and LN by the percentage of CD19+ CD11a+ cells determined by flow cytometry.

In all experiments, samples with FITC or PE-conjugated isotype controls were also analysed and no positive staining was found. All antibodies were used at a final concentration of 5 µg/ml.

B cell proliferation assay

LNs were harvested from non-Tg, IL-4-Tg Tg-BO, Tg-EL and Tg-LL mice. B cells were enriched by positive selection using anti-CD19 microbeads according to the manufacturer's instruction (Miltenyi Biotec, Auburn, CA, USA). The purity of B cells determined by flow cytometric analysis was greater than 96%. A total of 2 × 105 purified B cells in triplicate wells were incubated with 50 µg/ml LPS (Sigma, St. Louis, MO, USA) or 50 µg/ml antimouse IgM antibody (Jackson ImmunoResearch, West Grove, PA, USA) in DMEM supplemented with 10% FBS (Sigma) for 72 h at 37 °C in 5% CO2 incubator, and then the proliferation rate was performed using a Cell Counting Kit-8 (CCK-8) from Dojido (Kumamoto, Japan) as described previously [13].

Quantitative analysis of IL-4 in LNs

LNs were collected from non-Tg, Tg-BO, Tg-EL and Tg-LL mice (n = 5 in each group). Total RNAs were extracted by TRIzol (Invitrogen, Carlsbad, CA, USA), treated with DNAse I (Invitrogen), and then reverse transcribed to cDNA using Retroscript Kit (Ambion, Austin, TX, USA). IL-4 cDNA copy numbers were determined by quantitative real time PCR calibrated by standard curve generated by IL-4 plasmid and normalized by GAPDH as described in our previous publication [12].

Statistical analysis

The significance of the variation among different groups was determined by One-Way anova. Analysis and the difference between two groups were determined by Tukey-Kramer Multiple Comparison Test. For correlations between IL-4 transgene copy number with IgE level or disease severity, Spearman's rank correlation test was used. All the analyses were performed by GraphPad Instat Software (San Diego, CA, USA). P-value < 0·05 was considered significantly different.

Results

B cell percentage and total number increase in the LNs but decrease in the spleen of diseased mice

The frequency of CD19+ B cells in LNs steadily increased with the progression of the disease, from 34% in Tg-BO, to 37% and 44% in Tg-EL and Tg-LL, much greater than the 14% in non-Tg mice (Fig. 1a). On the contrary, the percentage of CD19+ B cells among CD11a+ leucocytes in the spleen decreased with the progression of the disease, from 20% in Tg-BO, to 14% in Tg-EL, and to only 4% in T-LL, compared to 20% in non-Tg mice (Fig. 1a). However, the average frequencies of CD19+ cells in the LNs and spleen of Tg mice were 27·4%, 25·6% and 24·1% in Tg-BO, Tg-EL and Tg-LL mice, respectively, which were higher than that of in non-Tg mice (16·9%, Fig. 1a). The average absolute numbers of CD19+ cells in each spleen and LN showed a similar pattern as the percentage of CD19+ cells (Fig. 1b). The combined total numbers of B cells in the spleen and LN were 1·1 × 107, 1·2 × 107 and 0·9 × 107 in non-Tg, Tg-BO and Tg-EL mice, respectively. In Tg-LL mice, the total number of B cells was 2·3 × 107, doubling that of other groups of mice (Fig. 1b).

Fig. 1
B cell percentage and total number increase in the LNs but decrease in the spleen of the diseased mice while they are activated as the disease progresses. The LNs and spleens were collected from non-Tg, Tg-BO, Tg-EL and Tg-LL mice. Single cell suspension ...

B cells are highly activated as the disease progresses

The percentage of activation markers of CD44 and CD69 on CD19+ B cells increased steadily in both the spleen and the LNs with the progression of the disease (Fig. 1c,d). The percentage of CD44+ B cells in the spleen increased with the progression of the disease, from 15·4% in Tg-BO, to 30·6% in Tg-EL, and to 42·1% in T-LL, compared to 13·2% in non-Tg mice (Fig. 1c). The same trend was observed in the LNs with lower values compared with those in the spleen (Fig. 1c). Similar results were obtained in the percentage of CD69+ B cells (Fig. 1d). Co-stimulatory molecular CD80 on B cells also showed marked increased in these two lymphoid tissues. In the spleen, the percentage of CD80+ B cells elevated from 11·7% in Tg-BO, to 19·3% in Tg-EL, and to 52·6% in T-LL, while non-Tg mice had 12·2% in (Fig. 1e). CD86 on B cells in the spleen also had similar increase as the disease progressed (Fig. 1f). Both CD80 and CD86 on B cells demonstrated similar patterns of increment in the LNs (Fig. 1e,f).

MHC class II molecule (IA/IE) is intensively expressed by B cells as the disease progresses

The frequencies of MHC class II molecule IA/IE in non-Tg and Tg mice at different stages of the disease were about the same as 98% on B cells (data not shown). However, the mean florescence intensity (MFI) of IA/IE steadily increased from Tg-BO, Tg-EL, to Tg-LL stages in LN B cells with the corresponding intensities of 1257·3 ± 181·0, 1773·3 ± 119·0, 2186·5 ± 119·0, respectively, as the disease progressed. MFIs of IA/IE in Tg-EL and Tg-LL mice were significantly higher than that of non-Tg (1016·7 ± 85) (P < 0·01 and < 0·001) (Fig. 1g). Similar results were observed in the spleen B cells (Fig. 1g). Importantly, the MFI of IA/IE in Tg-BO B cells was significantly higher than that of non-Tg mice, indicating the presence of activated B cells before the onset of disease.

B cells isolated from Tg mice with skin disease proliferated significantly higher than that of non-Tg littermates

Using anti-IgM to cross link the surface Igs, generally, we found the proliferation of B cells gradually increased as the disease progressed. B cells from Tg-EL and Tg-LL mice had higher proliferation rates than those of non-Tg and Tg-BO mice (Fig. 2). By using LPS as a polycolonal stimulant, LN B cells demonstrated the same trend of proliferation response as showed in Fig. 2. In addition B cells from Tg-LL mice had spontaneous proliferation without any stimulation, suggesting B cells in Tg-LL stage were highly activated in vivo. Importantly, the B cells in Tg-BO mice also have a higher proliferative response to LPS than those of non-Tg mice, indicating the activation of B cells precedes the disease onset.

Fig. 2
B cells from diseased Tg mice have strong proliferation against stimuli of LPS and anti-IgM. Isolated B cells from LNs of non-Tg, Tg-BO, Tg-EL and Tg-LL mice in triplicate were incubated in the presence or absence of 50 µg/ml LPS or 50 µg/ml ...

Surface IgE, IgG1 and IgG2a are increased on B cells in the diseased mice

We measured the surface IgE, IgG1 and IgG2a on CD19+ B cells isolated from non-Tg and Tg mice without any stimulation. There is a progressive increase of cell surface IgE-bearing B cells from 26·9% (Tg-BO), to 32·2% (Tg-EL), to 72·0% (Tg-LL), which were all significantly higher than that of non-Tg mice (1·9%, P < 0·001, Fig. 3). The increase of surface IgG1-bearing B cells had the same trends as IgE (Fig. 3). Similar increase was observed in IgG2a-bearing B cells, but the significant increase only occurred in the Tg-EL and Tg-LL mice, and not in the Tg-BO mice (Fig. 3). Furthermore, the maximum frequencies of B cell expressing surface IgG1 and IgG2a were 9·1% and 3·8% in Tg-LL mice, 3·6% and 1·8% in Tg-EL mice, which were much lower than those B cells expressing surface IgE, suggesting a predominant B cell activation towards genes switching for epsilon heavy chain. The significant increase of cell surface IgE and IgG1 in Tg-BO mice again indicates the B cells activation has occurred prior to the disease onset.

Fig. 3
Surface IgE, IgG1 and IgG2a on B cells are significantly increased in the diseased mice. LNs were collected from non-Tg, Tg-BO, Tg-EL and Tg-LL mice. Single cell suspension from each group was labelled with FITC anti-CD19 and PE anti-IgE/IgG1/IgG2a without ...

Serum IgE, IgG1 and IgG2a are elevated in the diseased mice

As the disease evolved, the levels of serum IgE significantly increased from 88·3 ± 54·5 ng/ml at Tg-BO, to 1109·8 ± 862·0 ng/ml at Tg-EL (P < 0·001 compared to Tg-BO and non-Tg), then to 1584·6 ± 707·4 ng/ml at Tg-LL (P < 0·001 compared to Tg-BO and non-Tg) (Fig. 4a). There was no significant difference between Tg-EL and Tg-LL (P > 0·05) (Fig. 4a). Serum IgG1 (7·8 mg/ml) levels in Tg-LL mice were also elevated significantly compared to those of Tg-EL (3·4 mg/ml), Tg-BO (0·2 mg/ml) and non-Tg mice (0·34 mg/ml) (P < 0·001, in all three, Fig. 4b). The IgG1 level in Tg-EL mice was also significantly higher than that of Tg-BO and non-Tg mice (P < 0·001). By contrast, IgG2a (6·1 mg/ml) in Tg-LL, but not in Tg-EL mice, was significantly increased comparing to non-Tg mice or Tg-BO mice (Fig. 4c).

The increase of serum IgE correlates with percentage increase in IL-4-expressing keratinocytes as the disease progresses

Statistic analysis combining the current finding (Fig. 4) and our previous data [12] indicated that there was a strong correlation between the percentage of IL-4-producing keratinocytes and total serum IgE (coefficient r = 0·9007, Fig. 5a), thus as the disease progressed from Tg-BO, to Tg-EL and Tg-LL, the more keratinocytes expressed IL-4, the higher the level of serum total IgE was observed. Correlation between IL-4 transgene copy numbers and the levels of total serum IgE at Tg-EL and Tg-LL stages (coefficient r = 0·162 and −0·043, respectively, Fig. 5b, ,c),c), however, was not demonstrated. Furthermore, correlation between IL-4 transgene copy numbers and severity scores at Tg-EL and Tg-LL stages (coefficient r = −0·094 and −0·036, respectively, Fig. 5d,e) was not found.

Fig. 5
Total serum IgE correlates with percentage increase in IL-4-expressing keratinocytes as the disease progresses. Serum IgE levels determined by ELISA were correlated with either the IL-4-expressing keratinocytes (a) or with the IL-4 transgene genomic DNA ...

Progressive increase of IL-4 mRNA levels in LN

Using quantitative real time PCR analysis we found that IL-4 mRNA expression in Tg-BO mice increased compared to that of non-Tg mice and continued to increase as the disease progressed to Tg-EL stage although there were no statistic significance (P < 0·05). The expression reached the highest point at Tg-LL stage, and it was significantly higher than those of other groups (P < 0·05).

Discussion

As in any allergic or hypersensitive disorder, IgE is a prominent component of AD. Understanding the role IgE plays in AD therefore is an important step towards the understanding the pathophysiology of the disease. Towards that end we investigate the activation state of the B cells, their production of IgE, IgG1 and IgG2a, and relationship of these findings to the clinical disease progression.

One of the major functions of B cells is to produce Igs. In human patients with AD, humoral immune response is involved by high levels of serum IgE [14]. Our data on total serum IgE concentration reveals that the IgE concentrations in IL-4-Tg mice at EL and LL stages are substantially increased, whereas the IgE concentration remains essentially unchanged in IL-4-Tg mice of BO stage (Fig. 4a). The increase of IgE concentration occurs in the Tg mice with skin disease, and not in those Tg mice before disease onset suggests that elevated serum IgE concentration could be considered as a marker of the disease. The data on total serum concentrations for IgG1 and IgG2a reveals that Tg mice in LL stage had significantly higher total Igs in comparison to that of non-Tg littermates (Fig. 4b,c). In Tg mice of EL stage, their IgG1, but not IgG2a, had significantly higher concentrations than that of non-Tg mice. These findings supported the notion that at the early disease stage the total serum Ig production was under stronger influence of Th2 cytokines and that at the late disease stage, the total serum Ig production was under equally strong influence of both Th1 and Th2 cytokines. In fact, this notion was supported by our prior findings of an early up-regulation of Th2 cytokines and a late surge of Th1 cytokines in our studies on cytokine expressions in the skin [12].

Having determined the up-regulation of Ig production in our IL-4-Tg mice, we first asked the question whether there is a relationship between the copy number of IL-4 transgene and the IgE production, since IL-4 is the most critical cytokine for epsilon heavy chain switching of the B cells [8,16]. To our surprise, we found no correlation between the IL-4 transgene copy number and the total serum IgE concentrations, regardless of the disease stage (Fig. 5b,c). Consistent with the findings of noncorrelation between IL-4 transgene copies and IgE concentrations and between IgE concentrations and disease severity, there is also no correlation between IL-4 transgene copies and disease severity (Fig. 5d,e). However, when correlation between total serum IgE concentrations and percentage IL-4+ keratinocytes [12] was performed, we found a strong correlation between them (Fig. 5a). We interpret these data to suggest that the epidermal expression of IL-4, rather than original epidermal IL-4 transgene copies, influence the serum IgE production and disease progression [12].

This raises the question, what is the mechanism underlying the up-regulation of IgE production in this animal model? We investigated it by first examining the surface-bound IgE in B cells among the different groups: non-Tg mice and IL-4-Tg mice at BO, EL, and LL stages. The data from our flow cytometric analyses indicate that as the disease progresses, the percentage CD19+ B cells that express surface-bound IgE increases as well (Fig. 3). The highest percentage IgE-carrying B cells were detected in the IL-4-Tg mice of LL stage (Fig. 3). Similar increases were observed in the percentage B cells expressing surface-bound IgG1, another Th2-type Ig (Fig. 3). Thus, the increase the total serum IgE concentrations in the Tg mice at EL and LL stages is probably due to the increase in the percentage of B cells that produce IgE. By contrast, the increase of B cell surface IgG2a was documented in IL-4-Tg mice at EL and LL stages, but not in Tg mice at BO stage (Fig. 3). Thus the data indicate that B cells were activated earlier for the production of Th2 type Igs than that of Th1 type Ig and these findings were consistent with our previous documentation of an early up-regulation of Th2 cytokines followed by a late surge of Th1 cytokines in the skin [12].

The next logical question we asked was what mechanism is accounting for the increase in the percentage of IgE-producing B cells. Since the total number of B cells increased in Tg-LL mice, but not in Tg-EL mice (Fig. 1), increased B cell activation could account for the elevated level of serum IgE in Tg-EL mice. Data from our flow cytometric analyses strongly indicate that the B cells are becoming more activated as the disease progresses, the percentage of B cells carrying very early activation molecule CD69 increases progressively as the disease progresses in both the skin-draining lymph nodes and spleens (Fig. 1). CD69 is one of the cell surface proteins which is rapidly up-regulated after B cell activation [17,18]. Similar increase was observed in the percentage of B cells expressing memory/activation/adhesion molecule CD44 (Fig. 1). CD44 is a cell adhesion molecule present on the surface of activated T cells and B cells and is the major cell surface receptor for the glycosaminoglycan, hyaluronan, which is an integral component of extracellular matrix [19]. CD44 is now known to be up-regulated in response to antigenic stimuli, and can mediate leucocyte rolling on endothelial cells and tissue matrix, and lymphocyte activation and homing. In addition, it may participate in the effector stage of immune response, and have broader functions in cellular signalling cascades [1924]. Together, increases in CD44+ and CD69+ B cells of the Tg mice as the disease progresses support the notion that there is constant replenishment of activated memory B cell subset in these lymphoid organs.

It is well known that B cells express costimulatory molecules (CD80 and CD86) upon activation [25,26] and that these molecules have a pivotal role in T cell activation [27]. In the present experiment, we found that the percentage B cells expressing CD80 and CD86 increased as the disease developed (Fig. 1). Additional data supporting an activated state of the B cells are provided by the fluorescence intensity of MHC class II molecules (IA/IE) on CD19+ B cells. As the disease progresses, this intensity increases in B cells derived from both skin-draining lymph nodes and spleen (Fig. 1). A previous study shows that the increase in expression of IA may reflect a proximal event in B cell activation and may enhance subsequent cellular interactions involving B cells [28]. Furthermore, when measuring B cell proliferation by anti-IgM antibody to cross-link surface Ig on B cells or by a polyclonal stimulus LPS, we found that B cells from IL-4-Tg mice at EL or LL stage of the disease proliferated significantly higher than that of non-Tg littermates. In addition, B cells from Tg mice at LL stage proliferated spontaneously in the absence of stimulant (Fig. 2). These data provides strong indication that B cells in Tg mice with skin disease are strongly activated. Together, we interpret our findings to indicate that the increase of immuloglubins (IgE, IgG1 and IgG2a)-producing B cells is primarily due to the activation of B cells.

We further asked the question, ‘What are the possible mechanisms responsible for the B cell activation in our model’. Since IL-4 is required for the Ig heavy epsilon chain switching for the production of IgE [8,16], we examined the IL-4 milieu in the secondary lymphoid organ where T and B cells are interacted. Our findings of progressive increase of IL-4 mRNAs in the LN as the disease progresses (Fig. 6) seems to support a notion that B cells are being activated in these secondary lymphoid organs, leading to enhanced production of IgE as revealed by our study. These data further support the notion that the expression of IL-4, rather than the original transgene copies, influences the IgE production as demonstrated by the correlation of IgE production with percentage IL-4-expressing keratinocytes (Fig. 5a).

Fig. 6
Progressively increased expression of IL-4 in LNs as the disease develops. Total RNAs collected from LNs of non-Tg, Tg-BO, Tg-EL and Tg-LL mice were reversely transcribed and the IL-4 cDNA copy numbers were determined by quantitative real time PCR. Experimental ...

In summary, our data show that in our mouse model of AD, up-regulation of IgE and IgG1 production parallels the development of the disease. This elevation of IgE and IgG1 production is primarily due to B cell activation, which was likely due to a strong IL-4 milieu present in the secondary lymphoid organ. Importantly, the increase of IgE and IgG1 production can be considered as a marker of the disease onset. 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 further supports the involvement of B cell in the disease development.

Acknowledgments

This work is supported in part by NIH grants (R01 AR47667, R03 AR47634, and R21 AR48438, L.S.C).

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