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
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ABSTRACT |
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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
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INTRODUCTION |
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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)-, 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.
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METHODS |
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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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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-,
vascular endothelial growth factor, and transcription factor CP2
(TFCP2)], on a protein level by ELISA [for example,
transforming growth factor (TGF)-
1 to -
3], and on a receptor
level by double-stained fluorescence-activated cell sorter (FACS)
analysis [for example, integrin family of genes (ITG) B4 and
-subunit of IL-2 receptor (IL-2R
)].
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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RESULTS |
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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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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-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-
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-2R (CD25).
IL-2R
, 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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DISCUSSION |
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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- 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-
expressed on T cells can promote IgE
synthesis in vitro. Upregulation of TNF-
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-
(16), IFN-
(16), IL-8 (11),
IL-10 (18), IL-12 (12), and TGF-
(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-
, TGF-
, 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-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-, IL-8), intracellular signaling molecules (e.g., SYK, TYK,
PYK), and transcription factors (e.g., STAT6, TFCP2, NF-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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