B-cell isotype control in atopy and asthma assessed with cDNA array technology

Martin H. Brutsche1, Ingrid Carlen Brutsche1, Peter Wood3, Nesrin Mogulkoc2, Adnan Custovic2, Jim Egan2, and Ashley Woodcock2

1 Pulmonology, University Hospital of Basel, CH-4031 Basel, Switzerland; 2 North West Lung Research Centre, South Manchester University Hospital Wythenshawe, Manchester M23 9LT; and 3 Department of Biological Sciences, University of Manchester, Manchester M13 9WL, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

B-cell isotype switching and the production of IgE is regulated by a variety of gene products through different mechanisms. A better understanding of these processes has the potential to identify markers of disease and new therapeutic targets. The aim of the study was to investigate human B-cell isotype control and IgE production in atopy and asthma with cDNA array technology. Eighteen atopic asthmatic, eight atopic nonasthmatic, and fourteen healthy control subjects were included. Peripheral blood mononuclear cells were separated by gradient centrifugation, mRNA was purified, and the reverse-transcribed probes were hybridized to cDNA membranes. Group differences were assessed with the Mann-Whitney U-test. Twenty-three of seventy-eight tested IgE-related genes had significantly altered expression in atopy and asthma compared with that in the healthy subjects. The differentially expressed genes include surface molecules involved in T- and B-cell interaction and activation, cytokines, intracellular signaling products, and transcription factors. In conclusion, both atopic nonasthmatic and atopic asthmatic individuals had activated proinflammatory pathways, a minimal requirement for B-cell isotype switching, and a clear net pro-IgE cytokine climate.

complementary deoxyribonucleic acid; bronchial asthma; immunoglobulin E; B-cell isotype switch; gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ATOPY IS CHARACTERIZED by increased synthesis of IgE specific for common allergens (14). Cross-linking of IgE with an allergen results in rapid release of a variety of mediators, including histamine, leukotrienes, prostaglandins, and proteases, which account for many of the inflammatory changes underlying the symptoms of immediate hypersensitivity. In addition, cell activation involving IgE leads to a prolonged inflammatory response lasting several hours after allergen exposure (10, 22), although it is well established that an influx and activation of inflammatory cells such as neutrophils, eosinophils, and T cells are central to the chronicity of the response. Continuous low-grade daily allergen exposure (5, 6) thus results in an ongoing inflammatory stimulus leading to chronic inflammation such as seen in asthmatic airways and the nasal mucosa in allergic rhinitis. A better understanding of the induction and regulation of IgE synthesis in B cells is crucial for the elucidation of the pathogenesis of IgE-dependent disorders such as asthma. In addition, it enables refinement of therapies targeting IgE.

Production of IgE and other immunoglobulins results from reciprocal activation of T and B cells (28). As a key initial step in the chain of events necessary for the induction of immunoglobulin production, resting B cells bind allergens through their membrane-bound antigen-specific immunoglobulin. After internalization of the antigen receptor complex, antigens are processed and presented to T cells as peptide fragments in association with the class II major histocompatibility complex. Induction of IgE synthesis by human B cells requires two signals (25, 26): signal 1 results from cognate interaction between membrane-bound receptors and ligands expressed by activated helper T and B lymphocytes, whereas signal 2 involves the T cell-derived cytokines interleukin (IL)-4 and/or IL-13. Engagement of the B-cell antigen CD40 by the CD40 ligand (CD40L), expressed on T cells, leads to isotype switching during immunoglobulin synthesis. The CD40- CD40L interaction is well established (for a review, see Ref. 25a) as a key signal for the induction of isotype switching, whereas the elucidation of the role of other cell-cell interactions, for example, through adhesion molecules, needs further study. An important counteracting cytokine for IgE synthesis is interferon (IFN)-gamma , which is produced mainly by T lymphocytes (19). Several surface molecules and cytokines and various hormones have been shown to modulate IgE synthesis in vitro, suggesting that a complex network of molecular events is involved in the production of IgE. However, the relevance of these factors for IgE production in vivo requires further elucidation.

In the present study, we sought to develop a novel approach to study the regulation of human B-cell isotype control resulting in IgE synthesis. In this first study of its kind, we restricted our investigation to peripheral blood of atopic individuals, although it is likely that additional important information will be obtained from studying the lymphoid tissue where IgE is produced. In addition to seeking evidence for a broad range of genes being differentially expressed in atopic disease, we have sought to identify differences between asthmatic individuals with the disease ranging from mild to severe in an attempt to provide insight into the determinants of disease severity. Gene expression was studied with cDNA array technology to standardize array data for comparison purposes, focusing the analysis and modeling to genes known to be involved in B-cell isotype control and the production of IgE.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Fourteen nonatopic control subjects (12 women; mean age 42 yr, range 27-65 yr), eight atopic nonasthmatic (AN) subjects (1 woman; mean age 36 yr, range 23-49 yr), and fifteen atopic asthmatic (AA) subjects (12 women; mean age 41 yr, range 18-66 yr) gave informed consent and participated in the study. Atopy was defined as a wheal 3 mm or larger in diameter than the negative control wheal on skin prick testing to a range of four common aeroallergens in the United Kingdom (Dermatophagoides pteronyssinus, cat, dog, and mixed grasses). Atopic asthma was defined as symptomatic bronchial hyperreactivity or reversibility in a sensitized individual. All patients were diagnosed and treated for asthma before being enrolled in the study, and no attempt was made to change their medication. The asthmatic subjects were scored according to the Aas (1) asthma severity score. The Aas score is a five-step scale clinical score that takes into account events occurring during the previous year. Patients with atopic asthma had a range of disease severity (I, n = 2; II, n = 1; III, n = 4; IV, n = 9; V, n = 2). They were taking inhaled corticosteroids (beclomethasone equivalent dose: none, n = 2; 400 µg/day, n = 2; 800 µg/day, n = 1; 1,000 µg/day, n = 3; 1,500 µg/day, n = 3; 2,000 µg/day, n = 6). None had received oral or parenteral corticosteroids for at least 2 mo.

Isolation of peripheral blood mononuclear cells and cDNA hybridization. Peripheral blood was drawn, and peripheral blood mononuclear cells (PBMCs) were separated immediately by gradient centrifugation followed by washing in AIM-V serum-free culture medium. Purified mRNA (TRIzol Reagent, Life Technologies, Paisley, UK; Oligotex, QIAGEN, Crawley, UK) was reverse transcribed with an oligo(dT) primer mix and labeled with [32P]dATP (Amersham Life Sciences). After an overnight hybridization onto membranes with an immobilized probe cDNA for 609 gene products in duplicate (Atlas, Clontech, Palo Alto, CA; the complete list of genes with accession numbers is published at http://www.clontech.com), quantification was performed by autoradiography and phosphorimaging.

Standardization of quantitative hybridization signals. To compare the results of different hybridization experiments, the data had to be standardized for nonspecific variation. This was done by expressing the results relative to the geometric mean of the 100 genes with the greatest expression (GM100; the assumption was made that PBMCs express similar amounts of mRNA with comparable overall mean hybridization intensities over the range of 609 gene products; Fig. 1), which proved to be superior to other methods of standardization.


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Fig. 1.   Example of a cDNA array after hybridization. With this technique, mRNA can be copied into radiolabeled cDNA with reverse transcriptase so that the relative abundance of individual mRNAs is reflected in the cDNA product. Thus the intensity of the hybridization signal for a given gene product is a result of its relative abundance in the target sample. This method has been proven to provide excellent specificity and reproducibility (7, 20, 21). mRNA species comprising 1:10,000-100,000 of the mass of the target poly(A)+ RNA, which corresponds to ~1 transcript/100,000, could readily be detected (7, 9). Single-strand DNAs with a length of 200-600 bases of known genes can be fixed to a membrane and probed with the RNA-derived cDNA probe. The intensity of the hybridization signal for a given gene is a result of its relative abundance in the RNA-derived DNA probe. Each gene product, including positive and negative controls, is represented in duplicate. Insets: effect of the standardization procedure against the 100 most expressed genes (GM100) on the hybridization results of 48 different experiments. Box plots show the distribution of the results in each experiment before (left) and after (right) this procedure, which enables direct comparison of results. Each experiment is represented by 1 box. The shaded boxes cover 25-75% of data, with the median value shown as a thick solid line. Error bars indicate 10 and 90%. open circle , Outliers; *, extreme values. Over the range of 609 different gene products, the assumption has been made that peripheral blood mononuclear cells (PBMCs) express similar amounts of mRNA with comparable overall mean hybridization intensities. It can therefore be expected that the distribution of the signals resulting from the different experiments must be similar. Ideally, a standardization method would be able to produce a unique distribution for all experiments. Shown is the efficacy of the GM100 method for this standardization process; e.g., experiments B000, B060, B108, and B114 had tendencies for higher values before the application of GM100, which was corrected to a large extent after standardization.

Verification of cDNA array data: immunofluorescent staining and flow cytometry. Several identified differentially expressed genes have been validated on a mRNA level by RT-PCR [for example, IL-4, IL-6, IFN-gamma , vascular endothelial growth factor, and transcription factor CP2 (TFCP2)], on a protein level by ELISA [for example, transforming growth factor (TGF)-beta 1 to -beta 3], and on a receptor level by double-stained fluorescence-activated cell sorter (FACS) analysis [for example, integrin family of genes (ITG) B4 and alpha -subunit of IL-2 receptor (IL-2Ralpha )].

For FACS analysis, another 20 control, 22 AN, and 25 AA individuals with the same inclusion and exclusion criteria as above were recruited. Five milliliters of venous whole blood was centrifuged for 5 min at 750 g at 4°C, the plasma was discarded, and the erythrocytes were lysed in two rounds of 4:1 Boyles' medium for 4 min at 37°C. The cells were then washed in phosphate-buffered saline. At the end of this procedure, the blood leukocytes were incubated with a panel of directly conjugated monoclonal antibodies (10 µl each) for 45 min at 4°C. Each of the fluorescein isothiocyanate (FITC)-conjugated CD4, CD8, and CD19 monoclonal antibodies was combined with phycoerythrin-conjugated CD25, resulting in a panel of three probes per individual completed with a negative control. During FACS acquisition with a FACScan flow cytometer and CellQuest software (Becton Dickinson, Oxford, UK), lymphocytes, monocytes, and polynuclear cells were identified by their forward- and side-scatter characteristics and electronically gated. Acquisition was carried out until 10,000 events within the lymphocyte gate were acquired independent of the total cell number.

Statistical analysis. Data were analyzed with SPSS for Windows 9.0 (SPSS, Chicago, IL) statistical package. Multiple Mann-Whitney U-tests and Kruskal-Wallis tests were used to screen for differential gene expression after standardization with the GM100 method as appropriate. Expression ratios (ERs) were calculated by dividing the geometric mean of the expression of a particular gene in AN or AA subjects by the geometric mean of the expression in control subjects. A graphical approach was taken in addition to statistical significance testing to identify group differences between the phenotypes. Figure 2 shows a dot plot comparing log-transformed and standardized gene expression in healthy control and AA subjects. For FACS analysis, absolute values of the staining intensities were found to be nonparametric and were compared with the Mann-Whitney U-test for two independent groups or Kruskal-Wallis test to compare all three groups (control, AN, and AA). ANOVA was used to compare the data of the quadrant analysis between the groups. A conventional significance level of 0.05 was taken.


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Fig. 2.   Dot plot comparing the gene expression in healthy control subjects (x-axis) with the expression in atopic asthmatic subjects (y-axis). Both axes give log-transformed gene expression values standardized with GM100 method. Each gene is represented by its mean expression together with its confidence limit (30%) in both axes designed as an oval. Significance levels of Mann-Whitney U-tests are color-coded. The brighter the color, the more significant the difference in gene expression between the 2 groups. The more distant from the line of identity, the greater the difference in gene expression. The smaller the oval, the lower the variability and the greater the chance of significance. This visual approach is useful for rapid screening of candidate genes and integrates the individual spread of data in the 2 axes in addition to the group means.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of genes involved in B-cell isotype control and IgE production: comparison between control, AN, and AA subjects. Twenty-three of seventy-eight genes present on the cDNA arrays used in this study, which were related to the human B-cell isotype control and the production of IgE, had significantly altered expression in atopy and asthma compared with that in the healthy subjects (Table 1, Figs. 2-4). Most of those genes were similarly altered in both atopy and asthma compared with those in control subjects. We did not identify any individual gene products, which were expressed in all normal control subjects and not at all in atopy or asthma or vice versa. Figure 5 shows an attempt to integrate the results obtained in AN and AA subjects in a model of a human B-cell isotype control.

                              
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Table 1.   Relative gene expression in atopic individuals with and without asthma compared with that in healthy control subjects



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Fig. 3.   Scatterplot comparing relative expression (RE) levels of atopic nonasthmatic (AN) and atopic asthmatic (AA) subjects. Not differentially expressed genes are plotted around the center of this graph (x, 0; y, 0). They do not seem to contribute to the tested phenotypes. Genes with the same trend in AN and AA subjects follow a 45° diagonal line (bottom left to top right). Such genes point to similarly dysregulated pathways in both phenotypes. However, genes that fall out of this pattern, like HOXA1, insulin growth factor binding protein 1 (IGFBP1), interleukin (IL)-1 receptor type 2 (IL-2R2), and nuclear factor (NF)-kappa B1, may be of particular interest. They could indicate pathways with some phenotype specificity. However, this way of looking at the data potentially overemphasizes genes with a large expression variability. An alternate way to screen the large amount of information is given in Fig. 4.



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Fig. 4.   Scatterplot of log-transformed P values giving an overview of differential gene expression in atopy and asthma. According to the 4 quadrants separated by significance levels (-3, representing a P value of 0.05), genes with asthma-specific (upper left) and atopy-specific (lower right) differential expression can be distinguished from not differently expressed genes (lower left) and genes that were altered in atopy and asthma (upper left). The more significantly different the expression, the more right or the higher up a gene was plotted. Only expressed genes are given here. They were labeled with HUGO short names whenever possible. This graph allows a rapid overview of the differences in gene expression between different phenotypes, taking the reproducibility of an altered expression into account and not the x-fold up- or downregulation compared with the reference. This approach is thus complementary to the information gathered in Fig. 3. Examples of particularly interesting genes: asthma specific [interferon (IFN)-gamma -stimulated gene factor-3, inhibitor of apoptosis (IAP), integrin family of genes (ITG) A4], atopy or nonasthma specific [TYK2, IFN-gamma R1, tumor necrosis factor (TNF) receptor-associated factor (TRAF), ITGA6], and atopy and asthma dysregulated [heat shock protein (HSP) B1, IFN-alpha R2, IL-12Rbeta 1, IL-1R1, transcription factor CP2 (TFCP2), ITGB1].



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Fig. 5.   Model of human B-cell isotype control with respect to IgE production in atopy and asthma with integrated gene expression results. Arrows, gene expression in AN and AA individuals relative to the expression in healthy control subjects (see Figs. 3 and 4, Table 1). The main results found in atopy and asthma were summarized and pooled together and thus represent a simplification of the real observations. EBV, Epstein-Barr virus; LPS lipopolysaccharide; Epo, erythropoietin; LFA, lymphocyte function-associated antigen; LTA, lymphotoxin A; TGF, transforming growth factor; CD30L and CD40L, CD30 and CD40 ligands, respectively. Genes were named by HUGO short names, and GenBank accession numbers are given in Table 1.

In accordance with the two-signal model, where a minimum of two distinct signals is needed to induce IgE synthesis, we found an upregulation of the IL-4 receptor (IL-4R; AA and AN subjects). IL-4 itself had a twofold increased expression in AN and AA subjects. This increase, however, was not significant due to expression variability. IL-13 was not expressed in all groups. The intracellular effector of IL-4, IL-13, or signal transducer and activator of transduction (STAT) 6 had higher levels of expression in both atopic groups, which, however, did not reach significance. The expression of the gene for CD40 together with the CD40 receptor-associated factor-1 [also called the tumor necrosis factor (TNF) receptor-associated factor (TRAF-3)] was higher in both AN and AA subjects compared with that in the control group. CD40L was not expressed in all groups. Formation of a high-affinity IL-2R is regulated primarily through induction of IL-2Ralpha . We found significantly higher IL-2Ralpha levels, which are a marker of T-cell activation, in AA subjects. The downregulation of IL-2Rbeta in AA subjects was difficult to interpret; IL-2 itself was not measurable in all groups.

Among other relevant IgE-promoting cytokines, IL-6 (AN and AA subjects), lymphotoxin A (formerly TNF-beta ; AN subjects only), and TNF-alpha (AN subjects only) were found to be upregulated. IL-5, IL-9, and their receptors were not expressed in all groups. Among the cytokines, which normally preferentially promote IgM and IgG synthesis, IL-10 was upregulated in AN subjects only. Apart from IFN-gamma receptor 1 (IFN-gamma R1), which is downregulated in AN subjects, IFN-gamma itself and related products were not differentially expressed across all groups. TGF-beta 1, -beta 2, and -beta 3 and TGF-beta receptor 3 (TGF-beta R3) were unchanged in all groups, and IL-8 was downregulated in a nonsignificant fashion. We found the type I IFNs IFN-alpha 10 and IFN-beta 1 upregulated in AN subjects and IFN-alpha receptor 2 (IFN-alpha R2) downregulated in AN and AA subjects. IFN-alpha R2 (AN and AA subjects), a potent inhibitory receptor for IFN-alpha and IFN-beta , and the tyrosine protein kinase TYK2 (AN subjects only), involved in type I IFN signaling, were significantly downregulated compared with those in the control subjects. Additionally, we found higher levels of the IFN type I consensus sequence binding protein IFN-stimulated gene factor 3 in AA subjects. Thus there seems to be a differential expression for type I IFNs with, however, an unclear net effect but at least a partial upregulation of type I IFN signaling. IL-12 was below the limit of detection but had an upregulated receptor in AN and AA subjects (Figs. 3 and 4).

Higher levels for CD86 were seen in atopic and asthmatic individuals. ITG-AL, also known as CD11A or lymphocyte function-associated antigen-1alpha chain, was significantly downregulated in both AN and AA subjects. CD30 showed unchanged expression in all groups.

The receptor for prostaglandin E2 (PTGER-2) was significantly upregulated in AA subjects.

Comparison of gene expression between asthmatic individuals and atopic subjects without asthma. In general, expression for genes related to IgE production was very similar in atopic and asthmatic individuals, with mostly gradual differences between those groups. For several genes with significant differences in expression in AN compared with control subjects, AA subjects showed similar but attenuated trends as AN subjects.

Differences between AA and AN individuals were identified in relation to TNF pathways. Subjects with atopy without asthma had higher TNF-alpha levels, with unchanged TNF receptor (TNFR) 1 or TNFR2 levels, whereas asthmatic individuals had unchanged TNF-alpha and TNFR1 expression levels but higher TNFR2, the high-affinity TNFR, expression levels. Furthermore, asthma-specific upregulated genes include IL-2Ralpha , a marker of T-cell activation, PTGER-4, a receptor for PGE involved in tissue remodeling, and the protein tyrosine kinases LCK and SYK, involved in T- and B-cell receptor signaling. For these gene products, AN subjects had similar but attenuated trends. Furthermore, AA subjects had normal nuclear factor (NF)-kappa B1 expression and increased NF-kappa B receptor (NFR-kappa B; ER 1.79), whereas AN subjects had upregulated NF-kappa B1 (ER 3.66) and normal NFR-kappa B expression.

Genes of the heat shock protein (HSP) group were more dysregulated in AA than in AN subjects compared with control subjects. This was true for HSPB1, HSPD1, and HSPA1A.

Altered gene expression according to asthma severity. Comparing subjects with Aas (1) severity scores of <= 3 vs. >3, we identified higher CD86 levels in the more severe group. Within the TNF pathway, AA subjects in the more severe group had higher TRAF-3 and lower TNF-alpha inositol hexaphosphate levels. More severe AA subjects had greater expression of extracellular signal-regulated kinase 1 (PRKM3; also called ERK1), and mitogen-activated protein kinase-activated protein kinase. The same difference was observed for HSPF1 and the lymphoid-specific transcription factor POU2F2.

Verification experiments with FACS analysis for IL-2Ralpha (CD25). IL-2Ralpha , also called CD25, the marker for T-cell activation, which was found upregulated in AA subjects by cDNA hybridization, was also significantly upregulated on lymphocytes of asthmatic individuals (P < 0.001) as determined with FACS technology. This proved to be the case due to increased expression on CD4+ lymphocytes. The percentage of CD4+ lymphocytes positive for CD25 was increased 3.2-fold in AA subjects. In addition, the analysis of the staining intensity for CD25 showed higher staining intensities on CD4+ lymphocytes in AA subjects (P = 0.002) and also in the AN subjects (P = 0.03), although there was a true upregulation of CD25 on CD4+ lymphocytes in AA subjects in terms of percent positivity and the degree of staining for CD25 on those cells. In CD8+ and CD19+ cells, no significant difference for CD25 could be found. Figure 6 shows the staining results from a representative normal and AA individual. Thus these findings represent a validation of cDNA technology.


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Fig. 6.   Box plots of typical fluorescence-activated cell sorter double-staining experiments for CD4 and IL-2RA (CD25). A: healthy subject. B: AA individual. PE, phycoerythrin. As seen in B, a small proportion of CD4+ lymphocytes are positive for CD25, which is not the case in the healthy individual. This phenomenon was also detected by cDNA array technology.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Until now, studies have focused on a very limited number of selected gene products based on availability and current understanding. With cDNA or oligonucleotide arrays, several hundred to thousands of gene products can be assayed in a single experiment, opening a new, eventually genomewide dimension to gene expression studies (4, 7, 9, 13, 21). Using this technique, we were able to identify substantial numbers of differentially expressed genes in the peripheral blood of AN and AA subjects compared with those in healthy control subjects. With this approach, many reported observations could be verified simultaneously for their relative in vivo importance. Also, several new hypotheses could be generated. For the sake of clarity, we focused on gene products related to B-cell isotype control.

Many of the differences in gene expression were similar in AN and AA individuals and were thus related to atopy rather than to asthma. Along these lines, all atopic individuals had a marked activation of proinflammatory cascades in peripheral blood. Therefore, our findings confirm the hypothesis that atopy is characterized by a significant proinflammatory drive, which is in accordance with the fact that atopy is a major risk factor for the subsequent development of different atopic diseases in children (23) and adults (8). With respect to the different aspects of B-cell isotype control, we were able to document the presence of all minimal requirements for the induction of IgE production (Fig. 5). Also, there seems to be a balance between "pro-IgE" and "anti-IgE" cytokines, of which members of both types were found upregulated in peripheral blood of AN and AA subjects. However, in an attempt to weigh the expression of pro-IgE against anti-IgE cytokines, clearly pro-IgE cytokines dominate in AN and AA subjects.

A variety of cytokines can modulate IL-4- and IL-13-mediated IgE synthesis. IgE synthesis can be enhanced by the cytokines IL-5 (17) and IL-6 (26) in vitro. Although IL-5 and its receptor were not expressed in our samples, IL-6 was consistently upregulated in both AN and AA subjects. An involvement of TNF-alpha and its receptors TNFR1 and TNFR2 for the induction of IgE synthesis was suggested in a study of Aversa et al. (3). They demonstrated that TNF-alpha expressed on T cells can promote IgE synthesis in vitro. Upregulation of TNF-alpha in AN subjects and the high-affinity TNFR2 in AA subjects was found in the present study. Thus TNF pathways are dysregulated in atopy and asthma and may contribute to increased IgE production. On the other hand, factors that have been shown to downregulate IgE synthesis in vitro include IFN-alpha (16), IFN-gamma (16), IL-8 (11), IL-10 (18), IL-12 (12), and TGF-beta (29). In our study, we found an upregulation of IL-10 in AN subjects, and there are arguments of a net increased type I IFN signaling in both AN and AA subjects. The upregulation of the receptor for IL-12 is more difficult to interpret. However, with an ER of 6.95 in AA subjects, this gene was among the most dysregulated genes. IL-12 levels could not be detected in peripheral blood in all groups. It is unclear whether a lack of pro-Th1 IL-12 signaling led to an upregulation of its receptor. IL-8, IFN-gamma , TGF-beta , and related genes were not differentially expressed or not expressed in our assay.

Asthma-specific alterations in gene expression could be identified, including members of the TNF family of genes and the NFR-kappa B gene, which is known to induce the transcription of the high-affinity IL-2R2 (CD25) (2). And consistently, IL-2R2, a marker of T-cell activation, was upregulated in an asthma-specific way, compatible with an increased level of T- (and B-) cell activation in asthmatic individuals.

We were able to verify these results on a protein level with FACS double staining, underlying the validity of cDNA hybridization technology. CD25 has been found to be upregulated on CD4+ lymphocytes of AA subjects. This documents evidence of increased activation of circulating CD4+ T cells in stable asthma without allergen challenge. Validation of cDNA array data, as shown here, is not only important to confirm a true up- or downregulation of the many "significant" genes but may also help to attribute the changes to specific cell types.

From an analysis of peripheral blood, limited conclusions can be made about the phenomena, which might occur at the site of inflammation. The picture in the circulation might represent a mirror image of the true situation where "disease-modulating" cells have migrated into the tissue. On the other hand, atopy is clearly a systemic phenomenon involving activation of many genes. Recently, Till et al. (24) found an equivalent cytokine production and proliferative response of T cells from bronchoalveolar lavage fluid and peripheral blood in AA subjects after segmental allergen challenge. Minshall et al. (15) reported an increased IL-5 expression by T cells in the bone marrow of ovalbumin-sensitized BALB/c mice 6 h after allergen challenge compared with that in nonsensitized control mice. We therefore hypothesized that a potential primary alteration in gene regulation, either genetically anchored or triggered by environmental factors, would also be present away from the actual site of inflammation, the lung. Also, an approach in peripheral blood is cheap, easy, and noninvasive and therefore widely applicable. Cell sequestration and the contribution of the different cell types to a specific gene expression pattern need to be further investigated in gene expression studies comparing lung tissue and peripheral blood samples.

Taken together, our results document a clear pro-IgE climate in atopic and asthmatic individuals compared with that in healthy control subjects. Most of the differences in gene expression were related to atopy, being similar in atopic and asthmatic individuals. Different therapeutic targets for an intervention in the regulation of IgE, such as surface markers involved in T- or B-cell interaction (e.g., CD86, cytotoxic T-lymphocyte antigen-4, CD28, CD40), cytokines (e.g., TNF-alpha , IL-8), intracellular signaling molecules (e.g., SYK, TYK, PYK), and transcription factors (e.g., STAT6, TFCP2, NF-kappa B), could be identified. Finally, cDNA array technology proved to be useful and may be complementary to DNA-based studies to analyze interactive and multidimensional pathways as shown here for B-cell isotype control and the production of IgE.


    ACKNOWLEDGEMENTS

We thank Prof. Tony Hegarty and Dr. Jacky Ohanian for permission to use the phosphorimager. Many thanks as well to Ratko Djukanovic and Frazer Smillie for advice.


    FOOTNOTES

I. C. Brutsche was supported by the Uarda Frutiger Foundation and the Swiss National Foundation.

Address for reprint requests and other correspondence: M. H. Brutsche, Pulmonology, Univ. Hospital of Basel, Petersgraben 4, CH-4031 Basel, Switzerland (E-mail: mbrutsche{at}uhbs.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 July 2000; accepted in final form 13 November 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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