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Enhanced Expression of B7.2 (CD86) in Patients with Atopic Dermatitis: A Potential Role in the Modulation of IgE Synthesis¹

Orathai Jirapongsananuruk^*, Michaël F. Hofer^*, Anne E. Trumble^*, David A. Norris and Donald Y. M. Leung^2,*,

^* Department of Pediatrics, The National Jewish Medical and Research Center, Denver, CO 80206; and Department of Dermatology and Department of Pediatrics, University of Colorado Health Science Center, Denver, CO 80262

	Abstract

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Recent studies have suggested that the accessory molecules B7.1 (CD80) and B7.2 (CD86) differ in their capacity to generate Th1 vs Th2 responses. Atopic dermatitis (AD) is a chronic allergic skin disease associated with increased IgE synthesis. To determine the potential role of B7.2 molecules in AD, the present study was conducted to compare the expression of B7.1 vs B7.2 on B cells from patients with AD vs normal subjects or patients with psoriasis. The expression of B7.2 on B cells of AD patients (53.67 ± 3.10%) was significantly higher than normals (38.02 ± 4.95%; p = 0.02) and psoriasis patients (40.19 ± 2.70%; p = 0.006). In contrast, there was no significant difference in B7.1 expression among the three subject groups. Interestingly, total serum IgE from AD patients and normal subjects correlated significantly with B7.2 expression on B cells (r = 0.68; p = 0.004), suggesting a role for B7.2⁺ B cells in IgE synthesis. Indeed, purified B7.2⁺ B cells produced significantly more IgE than B7.2^- B cells in vitro (p = 0.04). Anti-human B7.2, but not B7.1, mAb significantly (p < 0.05) decreased IgE production by PBMC stimulated with IL-4 and anti-CD40 mAb. Furthermore, B7.2⁺ B cells had a significantly higher level of IL-4R and CD23 expression than B7.1⁺ B cells. These data demonstrate the predominant expression of B7.2 in AD, but not psoriasis, and a novel role for this molecule in IgE synthesis.

	Introduction

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Tand B cells play a critical role in the pathogenesis of allergic diseases (1). Activation of resting lymphocytes require at least two signals (2, 3). In addition to the engagement of the TCR with the MHC plus peptide complex expressed on B cells, the interaction between CD40 molecules on B cells and CD40 ligand (CD40L)³ on T cells has been identified as an important costimulatory signal required for switch recombination to IgE synthesis in the presence of IL-4 (4, 5, 6). The engagement of B7 on APCs with CD28/CTLA-4 on T cells is another well-characterized costimulatory pathway for T and B cell activation (7).

At least two members of the B7 family of costimulatory ligands, B7.1 (CD80) and B7.2 (CD86), have now been identified. B7.1 is normally expressed at low levels on "professional" APCs, such as dendritic cells and macrophages, and up-regulated on these APCs as well as on B cells following activation by soluble factors (e.g., cytokines) or ligation of cell surface molecules (e.g., MHC class II and CD40). B7.2 is constitutively expressed on dendritic cells and is rapidly induced on B cells following activation by cross-linking of the Ig receptor or the addition of various cytokines (8). Several studies have demonstrated distinct kinetics and interaction sites between the B7.1/B7.2 and the CD28/CTLA-4 receptor-counter ligand system (9, 10, 11, 12). Such differential binding may have unique signaling properties that affect T cell activation and subsequent Th1/Th2 development. Studies in mice have suggested that the generation of Th2 cells depends mainly on the interaction of CD28 with B7.2 (13). In vitro studies of human T cells reported that B7.2 transfectants preferentially activate Th2-type cytokines whereas B7.1 transfectants skewed responses toward the production of Th1-type cytokines (14). Other reports, however, have not confirmed that B7.2 are involved in Th2 responses. These studies found that both B7.1 and B7.2 were able to costimulate IL-4 and IFN- production from murine T cells (15, 16).

Atopic dermatitis (AD) is a prototypic allergic skin disease associated with elevated IgE synthesis and increased B cell expression of CD23 (17, 18, 19). The IgE molecule is thought to play an important role in allergen-driven responses and allergen presentation to CD23-positive B cells in AD (20). Skin infiltrating and peripheral blood T cells from patients with AD express high levels of IL-4 and IL-5, but not IFN-, consistent with the Th2-type cytokine pattern of synthesis (21, 22, 23, 24, 25, 26). The relevance of B7.2 in Th2-predominant human allergic diseases such as AD has not been previously examined. In the current report, we therefore studied the expression of B7.1 vs B7.2 in AD and normal subjects as well as patients with psoriasis, a skin disease known to be associated with Th1-mediated responses (27, 28). We demonstrate an abnormally high expression of B7.2 on B cells from patients with AD, and a potential role for B7.2 in the induction of IgE synthesis.

	Materials and Methods

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Patients and control subjects

We obtained blood from eight patients (three males and five females, aged 27 to 45 yr) with moderate to severe AD (skin involvement more than 20%) and serum IgE ranging from 269 to 9797 IU/ml. None of the patients had previously used systemic corticosteroids. Topical corticosteroids were withheld for greater than 48 h before blood collection. Eight normal healthy adults (four males and four females, aged 27–36 yr) who were skin prick test negative for a panel of common allergens and had serum IgE levels <200 IU/ml were used as the healthy control group. Eight psoriasis patients (four males and four females, aged 28–41 yr) who had not used systemic corticosteroids or UV therapy for greater than 6 mo served as a skin disease control. These psoriasis patients had no history of allergy and serum IgE <200 IU/ml. Informed consent was obtained from all subjects before entry into the study.

Reagents

Human rIL-4 was obtained from Schering Research Institute (Bloomfield, NJ). A mAb against human CD40 (clone 626.1, IgG1) was a kind gift of Dr. Shu Man Fu (University of Virginia, Charlottesville, VA). Mouse IgG was purchased from Southern Biotechnology Associates, (Birmingham, AL). Purified mouse anti-human B7.2 mAb and anti-human B7.1 mAb were purchased from PharMingen (San Diego, CA). Mouse anti-human IL-4R mAb was obtained from Genzyme (Cambridge, MA). Goat anti-mouse FITC was purchased from Caltag Laboratories (Burlingame, CA). Mouse serum was obtained from Sigma (St. Louis, MO). The following mAb were obtained from Becton Dickinson Immunocytometry Systems (San Jose, CA): anti-CD 19 FITC and peridinin chlorophyll-a protein (PerCP), anti-CD23, and anti-B7.1 phycoerythrin (PE), G1CL (mouse IgG1 control; FITC, PE, PerCP) and G2CL (mouse IgG2 control; FITC, PE, PerCP). The anti-B7.2 PE-conjugated mAb was obtained from Ancell (Bayport, MN). The anti-CD23 FITC conjugated mAb was obtained from PharMingen.

Cell preparation and cell cultures

PBMC were isolated from heparinized venous blood from study subjects by density gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden), and washed three times in HBSS (Life Technologies, Grand Island, NY).

T cells were purified from PBMC by magnetic bead separation as previously described (29). Briefly, PBMC were incubated with mAb anti-CD3 (Becton Dickinson) at 4°C for 30 min, then washed two times in HBSS and incubated with magnetic beads coated with anti-mouse IgG (Dynabeads M-450, Dynal, Oslo, Norway) at 4°C for 60 min. Ab-coated T cells bound to the beads were separated from non-T cells using a magnet (Advanced Magnetics, Cambridge, MA). Both T cells and non-T cells were incubated in culture medium (RPMI plus 10% FCS) overnight at 37°C to detach T cells from the magnetic beads (subsequently removed by a magnet), and to separate monocytes/macrophages from B cells by plastic adherence. T cell purity was 96.5% by this separation method. Purified CD19⁺B7.2⁺, and CD19⁺B7.2^- B cells were isolated from B cell preparations by fluorescence-activated cell sorting on an EPICS 752 cell sorter (Coulter, Hialeah, FL) using the anti-CD19 FITC and B7.2-PE mAbs, as previously described (30). Purification by cell sorting resulted in 95.8% of CD19⁺B7.2⁺, and 91.2% of CD19⁺B7.2^- cells.

For cell culture studies, PBMC (at 1 x 10⁶ cells/ml) or purified B cells (1 x 10⁵ cells/ml) were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies) supplemented with 10% heat-inactivated FCS (Life Technologies), 0.5% BSA, 50 µg/ml human transferrin, 5 µg/ml bovine insulin, and 1 µg/ml each of oleic, linolenic, and palmitic acid (Sigma) as previously described (31, 32) in round-bottom 96-well plates (Costar, Cambridge, MA) at 37°C in a humidified 5% CO₂ atmosphere. Purified T cells at 1 x 10⁶ cells/ml were added to microculture wells containing purified B cells, and cultured in the presence of rIL-4 (400 U/ml) with or without anti-CD40 mAb (1 µg/ml) to induce IgE or IgG synthesis. In selected experiments, the effects of anti-B7.1 or anti-B7.2 mAb on IgE synthesis was studied. In these studies, the mouse IgG control, mouse anti-human B7.2 mAb, and the mouse anti-human B7.1 mAb were used at a concentration of 10 µg/ml. Culture supernatants were collected after 14 days and stored at -20°C until assayed.

ELISA for Ig determination

The IgE assay was conducted as previously described (33). Ninety-six-well microtiter plates (Dynatech, Chantilly, VA) were coated with 0.1 ml of a 1:1 mixture of purified monoclonal anti-human IgE (4.15 and 7.12, a kind gift from Dr. A. Saxon, University of California, Los Angeles, CA) diluted in 0.1 M NaHCO₃ at pH 9.6, at a concentration of 20 µg/ml after overnight incubation at 4°C. The wells were blocked with 0.1% gelatin in 0.1 M NaHCO₃ at room temperature for 1 h. Serial dilutions of culture supernatants were incubated in duplicate for 2 h at room temperature and overnight at 4°C, with parallel human IgE standard controls (Pharmacia). The plates were then washed, a 1:100 dilution of affinity-purified biotinylated goat anti-human IgE (Vector, Burlingame, CA) was added, and plates were incubated for 90 min at 37°C. After a subsequent wash, wells were incubated with a 1:1500 dilution of streptavidin-alkaline phosphatase (Tago, Burlingame, CA) for 90 min at 37°C. The wells were then developed with 2 mM p-nitrophenyl phosphate substrate (Sigma) and the OD was read at 405 nm on an Emax microplate reader (Molecular Devices, Menlo Park, CA). The concentrations of IgE in the supernatants were read from an IgE standard curve. The lower limit of sensitivity of this assay was 0.50 ng/ml.

The protocol for IgG assay (33) was identical to that for IgE except that the initial capture Ab was an affinity-purified polyclonal goat anti-human IgG Ab (Tago) diluted in 0.1 M NaHCO₃ at a concentration of 10 µg/ml. The second Ab was biotinylated goat-anti-human IgG (Vector). The IgG standards were obtained from Sigma. The lower limit of sensitivity of this assay was 1 ng/ml.

Cell staining and flow cytometric analysis

Five parameter analysis was performed using a FACScalibur flow cytometer (Becton Dickinson) using FITC, PE, and PerCP as the three fluorescent parameters. Immunofluorescence staining for this multiparameter analysis and methods of cytometer set up and data acquisition were performed as described previously (30). List mode multiparameter data files (each file with forward scatter, side scatter, and three fluorescent parameters) were analyzed by using the Cell Quest MacIntosh program (Becton Dickinson). Analysis was performed using a light scatter gate including only viable lymphocytes and a gate based on expression of CD19⁺ B cells. Negative control reagents were used to verify the staining specificity of experimental Abs.

Statistical analysis

Data are expressed as individual values or the mean for each subject group. Statistical comparisons were made using an unpaired Student t test to compare different groups of study subjects, and a paired Student t test to compare the results from the same subjects. Nonparametric data differences between groups of paired t test and serum IgE level for Pearson correlation were log transformed before being tested. Differences between groups were considered significant at a p value < 0.05.

	Results

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Elevated B7.2 and CD23 expression on B cells of AD patients

Freshly isolated PBMC from patients with AD and psoriasis, and from normal subjects were analyzed by flow cytometry for the expression of B7.2, B7.1, and CD23 on their B cells. As shown in Figure 1, B7.2 expression was significantly higher on B cells from AD patients (mean ± SEM = 53.67 ± 3.10%) as compared with normal subjects (38.02 ± 4.95%; p = 0.02) or psoriasis patients (40.19 ± 2.7%; p = 0.006). Similarly, CD23 expression was significantly higher on B cells from AD patients (80.33 ± 2.67%) as compared with normal subjects (65.48 ± 4.17%; p = 0.01) and psoriasis patients (66.55 ± 4.5%; p = 0.02). In contrast, B7.1 expression on B cells from AD patients (15.44 ± 2.09%), although lower than the other two study groups, did not show any significant differences compared with normal subjects (20.69 ± 3.99%; p = 0.26) and psoriasis patients (20.3 ± 5.27%; p = 0.41).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Increased B7.2 and CD23 expression on B cells from AD patients. Freshly isolated PBMC from AD patients (black circles), normal subjects (white circles), and psoriasis patients (squares) were analyzed by flow cytometry for expression of either B7.1, B7.2, or CD23 on CD19+ B cells. The data indicate that B cells from patients with AD have significantly higher levels of B7.2 and CD23, but not B7.1, expression than normal subjects or patients with psoriasis. Results from individual and mean values are shown.

Serum IgE levels were analyzed from AD patients and normal subjects, and compared with B7.2 expression on B cells from freshly isolated PBMC of these subjects. As shown in Figure 2, B7.2 expression on B cells correlated significantly with log serum IgE (r = 0.68, p = 0.004). In contrast, B7.1 expression on B cells did not demonstrate this correlation (r = 0.08, p = 0.76). These data suggested a possible role for the B7.2 molecule in the induction of IgE synthesis.

View larger version (11K): [in this window] [in a new window]   FIGURE 2. Serum IgE correlates significantly with B7.2, but not B7.1, expression on B cells. Serum IgE values of AD patients and normal subjects were determined by ELISA. Expression of B7.2 (upper panel) and B7.1 on B cells was determined by flow cytometry as in Figure 1. The correlation between B7.2 (lower panel) and B7.1 expression on B cells and log IgE was done using a Pearson correlation test.

In these experiments, purified B7.2⁺ vs B7.2^- B cells from normal subjects, isolated by cell sorting, were cultured with rIL-4 in the presence and absence of anti-CD40 or T cells as described in Table I. Culture supernatants were analyzed for IgE and IgG synthesis after 14 days of culture. Following stimulation with IL-4 plus anti-human CD40 mAb, IgE production was significantly higher in cultures containing T cells plus B7.2⁺ B cells (line 7) than cultures with T cells plus B7.2^- B cells (line 8) in three of three experiments (p = 0.04). In the absence of T cells, the overall IgE production was lower but did show a higher level of IgE synthesis by B7.2⁺ (line 5) as compared with B7.2^- B cells (line 6) used in experiments 1 and 2 (Table I). In contrast, no significant differences were observed in the level of IgG synthesis by B7.2⁺ vs B7.2^- cells under all culture conditions examined.

To directly test the hypothesis that the B7.2 molecule played a role in the induction of IgE synthesis, we examined whether mAb to B7.2, as compared with B7.1, inhibited IgE synthesis following stimulation of PBMC from eight normal subjects with rIL-4 and anti-human CD40 mAb. As shown in Figure 3 (left), IgE production by PBMC cultured with anti-B7.2 mAb (8.44 ± 2.94 ng/ml) synthesized significantly lower levels of IgE than PBMC cultured with the mouse IgG isotype control (34.35 ± 17.04 ng/ml; p = 0.01). In contrast, IgE production from cultures with anti-human B7.1 mAb (46.86 ± 18.64 ng/ml) was not significantly different from the mouse IgG isotype control. Furthermore, IgE production in cultures with anti-B7.2 mAb demonstrated significantly lower levels of IgE synthesis than cultures with anti-B7.1 mAb (p = 0.004). IgG production (see Fig. 3, right) was not significantly affected by the addition of these different Abs (p > 0.05).

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Anti-human B7.2, but not anti-human B7.1, mAb inhibits IgE synthesis. PBMC from eight normal subjects were cultured for 14 days in IL-4 (400 U/ml) and anti-CD40 mAb (1 µg/ml) in the presence of either mouse IgG control (10 µg/ml), mouse anti-human B7.2 (10 µg/ml), or mouse anti-human B7.1 (10 µg/ml). IgE (left) and IgG (right) were determined in culture supernatants as described in Materials and Methods. Results from eight individual subjects were expressed as mean ± SEM.

The observation in Table I that IL-4 stimulated a higher level of IgE synthesis in purified B7.2⁺, but not B7.2^-, B cells suggested a differential expression of the IL-4R in these two cell types. To address this possibility more directly, we analyzed freshly isolated PBMC from AD patients and normal subjects for the expression of IL-4R and CD23 on their B7.1⁺ vs B7.2⁺ B cells. In Figure 4 (upper panel), IL-4R expression on B7.2⁺ B cells in AD patients (53.28 ± 6.46%) was significantly higher than on B7.1⁺ B cells (34.83 ± 6.30%; p = 0.02). Similarly, the expression of IL-4R expression on B7.2⁺ B cells in normal subjects (46.23 ± 7.03%) was also significantly higher than on B7.1⁺ B cells (23.49 ± 4.39%; p = 0.009).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. IL-4R and CD23 expression is significantly higher on B7.2+ B cells than B7.1+ B cells in both AD and normal subjects. Freshly isolated PBMC from AD and normal subjects were analyzed by flow cytometry for expression of IL-4R (upper panel) and CD23 (lower panel) expression on B7.1+ and B7.2+ B cells. Results for individual values and the mean values are shown. Black circles indicate surface molecule expression on B7.2+ B cells while open circles indicate surface molecule expression on B7.1+ B cells.

	Discussion

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Several studies support the concept that B7.1 and B7.2 have distinct functions in the generation of Th1 and Th2 responses. In the murine experimental allergic encephalomyelitis model, treatment with anti-B7.1 mAb leads to Th2 responses while anti-B7.2 mAb has the opposite effect (13). In a study by Freeman et al. (14), B7.2 provided an initial signal to induce naive T cells to become IL-4 producers, and repetitive costimulation with B7.1 resulted in high levels of IL-2. The blocking of splenic APCs with anti-B7.1 and anti-B7.2 mAb also resulted in Th2- and Th1-type response, respectively (34). However, several studies could not confirm differential effects of B7.1 vs B7.2. A study by Lanier et al. (15) indicated that both B7.1 and B7.2 could costimulate anti-CD3 Ab-induced proliferation as well as IL-2 and IFN- production by murine T cells. Natesan et al. (16) demonstrated that both B7.1- and B7.2-transfected Chinese hamster ovary cells were able to costimulate IL-4 production and up-regulate IFN- mRNA.

Considering these conflicting results, it is important to determine whether B7.1 and B7.2 molecules are differentially up-regulated in human allergic disease. In this report, we have studied AD as a prototypic allergic disease known to be associated with a predominant Th2 response and markedly elevated serum IgE levels (1, 21, 22, 23, 24, 25, 26). The current study is the first to demonstrate the predominant expression of B7.2 in AD patients compared with normal subjects or patients with psoriasis, a Th1-mediated skin disease. Furthermore, we found a significant correlation between B7.2, but not B7.1, expression on B cells with the magnitude of serum IgE levels suggesting a potential role for this molecule in the induction of IgE synthesis.

To study the potential role of B7.2 in IgE synthesis, we investigated whether B7.2⁺ B cells produced higher levels of IgE than B7.2^- B cells. As shown in Table I, following stimulation with IL-4 and anti-CD40 mAb, B7.2⁺ B cells produced more IgE than B7.2^- B cells, particularly in the presence of T cells. This is explained in part by our current observation that B7.2⁺ B cells express significantly higher IL-4R and CD23 than B7.1⁺ B cells. Both of these surface molecules are known to have an important role in promoting IgE synthesis (35). Our observation that IL-4 and anti-CD40 mAb induce higher levels of IgE synthesis in the presence of T cells is consistent with previous observations that CD40 plus IL-4 stimulation provides better signaling for IgE production in PBMC than IL-4 alone (36). In the absence of T cells, B7.2⁺ B cells produced less IgE than in the presence of T cells following the stimulation of IL-4 and anti-CD40 mAb, but still showed a higher level of IgE production from B7.2⁺ B cells than B7.2^- B cells. However, only two of our three experiments demonstrated IgE production in the absence of T cells following IL-4 and anti-CD40 mAb. The observation that not all individuals respond to IL-4 and anti-CD40 mAb has also been reported by Zhang et al. (36).

A role for CD28/B7 interactions in IgE synthesis has been previously suggested by other investigators. In a study by Life et al. (37) using anti-CD28 in cultures containing tonsillar B cells, preactivated T cell clones, and IL-4, IgE but not IgG production was inhibited in a dose response fashion. Recently, Gause et al. (38) demonstrated that the increased serum IgE response to goat anti-mouse IgD was blocked in CD28 knockout mice. Our current study demonstrates a differential role for B7.1 vs B7.2 in IgE synthesis. Thus, we found that anti-B7.2, but not anti-B7.1, mAb blocked IgE synthesis by PBMC following stimulation with IL-4 and anti-CD40 mAb. Consistent with our current finding, Tsuyuki et al. (39) have recently reported that airway allergen challenge up-regulates the expression of B7.2 on B cells from the lung, and airway administration of anti B7.2 mAb inhibited local IgE production in a mouse model of asthma. Recently, we have also found an increased expression of B7.2 on B cells from atopic asthmatics (40). Taken together, these data suggest that increased B cell expression of B7.2 is not specific to AD but is observed in other human allergic diseases as well. However, our observation that B cells from patients with psoriasis have normal levels of B7.2 expression indicates that our current observations are selective for the atopic response, and not simply a nonspecific response to inflammation or immune activation.

To further examine the potential role of B7.2⁺ B cells in IgE synthesis, we investigated B7.1⁺ and B7.2⁺ B cells for the expression of IL-4R and CD23 as these two molecules are known to be involved in the augmentation of IgE responses. As shown in Figure 4, the expression of IL-4R and CD23 on B cells of both AD patients and normal subjects was significantly higher on B7.2⁺, as compared with B7.1⁺, B cells. It has been previously reported that the mean fluorescence intensity of CD40 expression on B cells of AD patients is higher than in normal subjects (41). Our study demonstrates that patients with AD also have an abnormally high expression of B7.2 on their B cells. Interestingly, anti-CD40 mAb causes increased expression of B7.2, IL-4R, and CD23 on B cells (42, 43, 44, 45). Maximal expression of B7.2 required signaling through both CD40 and IL-4R (46). Ligation of B7 with CD28/CTLA-4 results in CD40L expression and IL-4 secretion (47), which induce CD23 expression on B cells (48). Recently, Macaulay et al. (49) also demonstrated that Ag-specific B cells preferentially induce CD4⁺ T cells to secrete IL-4, and that the interaction between CD40 and CD40L is important for IL-4 production by T cells.

Based on these observations, we propose the following model: after Ag exposure, elevated IgE expression and CD23 on Ag-specific B cells facilitate Ag presentation to T cells (20). T cells are activated by cognate (primary signal) and noncognate interactions (costimulatory signals) resulting in IL-4 secretion and Th2-type cell responses. CD40 engagement, as a costimulatory signal, up-regulates IL-4 production on T cells (49), and IL-4R expression (45) on B cells, subsequently leading to enhanced IgE synthesis. CD40 also up-regulates B7.2 expression (42), and B7.2, as an important costimulatory signal, increases CD40L expression and IL-4 secretion (47). This results in a significant amplification loop that augments IgE production. Our current data of higher expression of B7.2, CD23, and IL-4R on B cells of AD patients than of normal subjects are consistent with this model and point to a novel mechanism for high level IgE synthesis in AD patients. The predominant expression of IL-4R and CD23 on B7.2⁺ B cells, and the capacity of anti-B7.2 Ab to block IgE synthesis suggests B7.2 may be a therapeutic target in allergic diseases.

	Acknowledgments

 
We thank Susan Leung for her assistance in the recruitment of patients for our studies, and Maureen Plourd-Sandoval for preparation of this manuscript.

	Footnotes

 
¹ This work was supported by Public Health Services Research Grants HL36577 and AR41256, and General Clinical Research Center Grant 5 MO1 RR00051 from the Division of Research Resources as well as the University of Colorado Cancer Center, Denver, CO.

² Address correspondence and reprint requests to Dr. Donald Y. M. Leung, Department of Pediatrics, The National Jewish Medical and Research Center, 1400 Jackson Street, Room K926, Denver, CO 80206. E-mail address:

³ Abbreviations used in this paper: CD40L, CD40 ligand; AD, atopic dermatitis; PerCP, peridinin chlorophyll-a protein; PE, phycoerythrin.

Received for publication October 30, 1997. Accepted for publication December 22, 1997.

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