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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The Th2 cytokines, interleukin (IL)-4 and IL-5, have an important role in atopic disease. CD30 is a transmembrane molecule that may be expressed on a proportion of activated T-lymphocytes and has been reported to be a marker for Th2 phenotype. Our objective was to compare the in vitro cytokine responses and
CD30 expression of peripheral blood mononuclear cells (PBMCs) to stimulation with house dust mite antigen (Dermatophagoides pteronyssinus) in atopic asthmatics, atopic nonasthmatics, and normal subjects,
and to see if atopic asthmatic cytokine production correlated with symptomatic disease activity and
whether cytokine production was allergen-specific. Eighteen atopic asthmatics (all were allocated a symptomatic disease score), 6 atopic nonasthmatics, and 7 healthy nonatopic individuals were studied. Resting
serum IL-4 levels were measured, then PBMCs were separated using Lymphoprep density centrifugation
and cultured in modified RPMI 1640 medium. PBMCs were stimulated with IL-2 alone or with D. pteronyssinus (1,000 subcutaneous units/ml) with IL-2 and harvested after 5 and 10 d. Using monoclonal antibodies and flow cytometry we obtained the percentage of CD4+ T cells expressing CD30 and the intensity
of CD30 staining. Culture supernatants were analyzed for IL-4 and interferon 
(IFN-) using an enzyme-linked immunosorbent assay. In 9 atopic asthmatics PBMCs were also stimulated nonspecifically using
phytohemagglutinin (PHA). IL-4 was detectable in the serum of atopic subjects but not in normal subjects.
Stimulation of PBMCs with D. pteronyssinus produced significant amounts of IL-4 in atopic asthmatics
and atopic nonasthmatics, but minimal quantities in normal subjects. Much lower levels of IFN- were
produced by atopic asthmatics in response to D. pteronyssinus compared to atopic nonasthmatics. IFN-
levels had an inverse correlation with asthmatic symptom score. CD4+ T-cell expression of CD30 also correlated inversely with IFN- production and IFN-:IL-4 ratio. PHA produced minimal levels of IL-4 compared to specific allergen stimulation. It is concluded that different groups of atopic patients exhibit different patterns of allergen-induced cytokine production. In vitro allergen-induced cytokine production in
atopic asthmatics correlated with symptomatic disease activity, and is allergen-specific.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Despite the availability of efficacious treatment and much knowledge of the immunopathology, asthma morbidity and mortality are increasing (1, 2). Since the original work by Mosmann and Coffman (3, 4) it has become broadly accepted that the Th2-type cytokines interleukin (IL)-4 and IL-5 have an important role in atopic disease and more specifically in atopic asthma (5). IL-4 is involved in the switch of B cells to IgE production (17) and IL-5 has been shown to cause eosinophil infiltration into the airway wall (18). Both IL-4 and IL-5 have been implicated in the airway hypersensitivity associated with asthma (18, 19). It has been shown that T cells in bronchoalveolar lavage from atopic asthmatics express mRNA for IL-4 and IL-5 (20). Other work has documented that detectable levels of IL-5 may be found in the serum of patients with acute severe asthma (21) and that peripheral blood CD4+ T cells from patients with asthma express cytokine mRNA in a Th2-type pattern and show elevated secretion of cytokines prolonging eosinophil survival (22). Bronchial biopsies have also been shown to have lymphocytes containing mRNA encoding Th2 cytokines (23). This tendency of T-cell clones from atopic patients to present a Th2 cytokine phenotype has also been documented in vitro.
We have already shown that atopic asthmatics express CD30, a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily originally described as a marker on Hodgkin's lymphoma cells (24), on CD4+ T cells following allergen stimulation and that this response is absent in normal individuals and is allergen-specific (Leonard and colleagues, submitted). CD30 has been claimed to be a marker of Th2 cytokine phenotype (25, 26). In this article, we address the issue of whether increased CD30 expression in atopic asthma reflects increased Th2 cytokine production, as it has recently been disputed that CD30 is in fact a marker of the Th2 phenotype (27).
Understanding the compartmentalization of atopic disease is at the heart of research into atopy; in other words,
if atopic patients invariably respond to allergen with the
Th2 cytokine phenotype, then why don't all atopic patients
develop asthma and why do some atopic patients have
their disease confined to the skin or upper respiratory
tract? To address this issue, and in view of recent work attempting to implicate concentrations of the house dust
mite allergen Dermatophagoides pteronyssinus to the presence and severity of asthma (31), we have studied house dust mite allergen-induced peripheral blood mononuclear
cell (PBMC) cytokine production in atopic asthmatics,
atopic nonasthmatics, nonatopic asthmatics, and normal individuals. It is accepted that IL-4 and interferon- (IFN-)
have reciprocal regulatory effects on Th2 development in
vitro (15, 32). IL-12 has also been shown to inhibit the
development of Th2 phenotype (35). It has been shown
that an imbalance between IFN- and IL-4 production exists in atopic subjects (9, 36). Along these lines it has been
argued that the most relevant parameter in determining
allergic hypersensitivity reactions is not whether T cells reactive to a given allergen are present in any given individual, or whether they respond, but what is the cytokine pattern with which they respond (9), i.e., the balance between
Th1 cytokine (IFN-) and Th2 cytokine (IL-4). In atopic
patients, the response to allergen is skewed toward IL-4
and away from IFN-. What has not been defined is how
allergen-induced cytokine production in vitro relates to the
real-world clinical situation. In serum-free medium it has been shown that virtually 100% of atopic and normal individuals show T cell proliferation in response to inhalant
allergens (37). This article compares the allergen-induced
cytokine responses, IL-4 and IFN- production, in different subject groups and tries to determine whether patterns
of cytokine production correlated with symptomatic disease activity.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Study Population
Blood samples were taken from 18 atopic asthmatics (for
demographic data, see Table 1), 6 atopic nonasthmatics (6 males, age range 27-36 yr, all of whom had atopic rhinitis),
and 7 normal individuals (2 males, 5 females, age range
24-45 yr). All subjects had a clinical history, including history of medications such as 
2 agonists (BAs), inhaled steroids (ISs), and theophyllines (THs) (see Table 1). Physical
examination and pulmonary function tests were also performed, as was skin-prick testing for a range of common allergens, house dust mite (D. pteronyssinus), Lolium perenne
(LP), grass pollen antigen, rye grass, cat, dog, Aspergillus (ASP), and tree antigens (Table 1). For the purposes of this article, atopic asthma was defined as skin test positive to D. pteronyssinus with or without reactions to other allergens,
with a history of wheeze, and abnormal pulmonary function
test (PFT) and/or documented airway hyperresponsiveness
to histamine challenge. Atopic nonasthma was defined as
skin-prick test positive to D. pteronyssinus with or without reactions to other allergens, with normal pulmonary function,
no history of wheeze, and no evidence of airway hyperresponsiveness to histamine challenge. Normal subjects had
negative skin-prick tests to all aforementioned allergens, no
history of wheeze, normal PFTs, and nonreactivity to histamine airway challenge. All subjects were nonsmokers. A positive skin test was defined as a wheal 5 mm or greater than
the control wheal 15 min after application of the relevant allergen. The size of any wheal was measured in millimeters
and translated into a score (1+, 5 mm; 2+, 6-10 mm; 3+, 11-
15 mm; 4+, > 15 mm).
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Symptomatic Disease Activity Score
In the asthmatic patients, forced expiratory volume in 1 s (FEV1) varied from 31% to 110% predicted. The FEV1 on the first clinic visit did not correlate well with asthmatic patient symptoms and requirement for rescue bronchodilator use, a finding not unique to our group of patients, as PFTs have been shown to sometimes correlate poorly with patient morbidity and quality of life (38), and merely provide a "snapshot" of airway caliber at the time of measurement. Therefore all subjects with asthma were allocated a symptomatic disease activity score based on frequency of wheeze, use of rescue bronchodilator medication in the preceding 3 mo, and clinical findings on auscultation (see Table 2). Various investigators have developed scores incorporating criteria such as these (42, 43). We deliberately did not use traditional asthma severity scores (44), as these tend to be adversely affected if the patient is taking inhaled steroids regularly, despite the fact that they may be well controlled and asymptomatic.
Serum Cytokine Analysis
For serum analysis 10 ml of clotted blood was taken from
the antecubital fossa. The blood was spun at 3,000 rpm and
the serum separated, stored at 
20°C, and analyzed later
for IL-4 and IFN- using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN). Cell culture supernatants were analyzed with the
same kits for IL-4 and IFN-. The detection sensitivity of
the IFN- and IL-4 ELISAs was 3 pg/ml. This minimum detectable dose of IFN- and IL-4 for ELISA kits was determined by adding two standard deviations to the mean optical density value of 20 zero-standard replicates and
calculating the corresponding concentration from the standard curve. An IFN-:IL-4 ratio was also obtained for each
individual by expressing IFN- as picograms per milliliter
and dividing by the IL-4 level (in picograms per milliliter).
Preparation of Peripheral Blood Mononuclear Cells for Cell Culture
Twenty milliliters of heparinized blood was taken, shaken well, and diluted in an equal volume of phosphate-buffered saline (PBS). The PBMCs were then separated using Lymphoprep density centrifugation (Nycomed Pharma AS, Oslo, Norway), washed twice in PBS, and then made up to a concentration of 1 × 106 cells/ml using RPMI 1640 culture medium (Sigma Aldrich Co., Dorset, UK) with 10% fetal calf serum, 1.25% penicillin/streptomycin, and 1.25% glutamine. The cell suspension was then plated out onto a 24-well culture plate with 2 ml/well.
Allergen Stimulation
To the culture wells was added either IL-2 alone (5 ng/ml;
R&D Systems) or house dust mite antigen (D. pteronyssinus, 1,000 subcutaneous units/ml; ALK A/S, Horsholm,
Denmark) with IL-2. Wells were harvested after 5 and 10 d, the supernatants being stored for cytokine analysis of
IFN- and IL-4 levels using relevant ELISA kits as mentioned previously. Proportions of CD4+ T cells expressing
CD30+ were determined using fluorescently labeled monoclonal antibodies and flow cytometric analysis, as described in the section CELL SURFACE MARKER ANALYSIS.
The harvest times were established, having carried out
time course experiments that determined that CD30 was
expressed optimally 10 d after antigen stimulation. For
wells cultured past day 5 the medium was changed after
5 d and more IL-2 added at a dose of 5 ng/ml.
Nonspecific Stimulation
Phytohemagglutinin (PHA), 1 µg/ml, was added to the culture wells. Wells were harvested at 48 and 72 h to find the percentage of CD4+ T cells expressing CD30, and the supernatants were stored for cytokine analysis. These harvest times were chosen because time course experiments showed that CD30 expression and cytokine production were optimal at this time poststimulation with PHA, and because after this time CD30 expression fell progressively to minimal amounts by 5 d.
Cell Surface Marker Analysis
The percentage of CD4+ T cells expressing CD30 was determined by fluorescent staining and flow cytometry (FACScan; Becton Dickinson, Mountain View, CA), using a combined lymphocyte and CD3 gate. The mean fluorescence intensity of CD30 (CD30 MFI) staining was also noted. The antibodies used were directly conjugated to fluorochromes, CD3-phycoerythrin (PE), CD4-PE-cyanin 5 (Cy 5), CD8-PE-Cy 5, and CD30-fluorescein isothiocyanate (FITC), all from Dako (Glostrup, Denmark). To rule out nonspecific staining a nonspecific mouse isotype control antibody conjugated to the relevant fluorochrome was used in parallel with each of the mentioned monoclonal antibodies. Antibodies were incubated with samples at 2-4°C for 30 min, washed twice with PBS, and fixed with 2% para- formaldehyde prior to FACScan analysis.
Statistical Analysis
As cytokine levels and percentage of CD4+ T cells expressing CD30 did not follow a Gaussian distribution, nonparametric tests were used and values expressed in the
text are medians unless otherwise stated. Samples were
compared with controls using a Wilcoxon matched-pairs
test (45). Mann-Whitney tests were used for independent
intergroup analysis. Nonlinear regression analysis was
used to assess whether a relationship existed between the percentage of CD4+ cells expressing CD30 (% CD4CD30),
IL-4, IFN- and symptom score (38). These analyses were
carried out using GraphPad Prism software (GraphPad
Software, Inc., San Diego, CA).
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Results |
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Materials & Methods
Results
Discussion
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Serum IL-4 in Atopic and Normal Patients
Interleukin 4 was seen in the serum of atopic patients (mean ± SEM = 32.6 pg/ml ± 28.6; n = 18) but was undetectable (< 3 pg/ml) in the serum of the healthy non-atopic individuals (n = 7).
Allergen Stimulation of Peripheral Blood Mononuclear Cells in Different Subject Groups
Minimal amounts of CD30 expression and IL-4 production
were seen after 5 d of culture (data not shown); therefore
results refer to the 10-d harvest time point. PBMC supernatants from atopic asthmatics (n = 18; Figure 1a and Table 3) contained significant amounts of IL-4 (92.8 pg/ml) in
response to D. pteronyssinus stimulation compared to control IL-2-stimulated culture wells (8.5 pg/ml; P = 0.0014).
In these patients, significantly less IFN- was detected in
D. pteronyssinus-stimulated wells (1,136 pg/ml) compared to control IL-2-stimulated wells (1,387 pg/ml; P = 0.0245). The
median percentage of CD4+ T cells expressing CD30 in
atopic asthmatics was 21.9% in D. pteronyssinus-stimulated
wells as opposed to 12.1% in control wells (P = 0.0003).
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For atopic nonasthmatics (n = 6; Figure 1b and Table 3),
D. pteronyssinus-stimulated culture wells contained median
IL-4 levels of 60.5 pg/ml compared to control wells (5.5 pg/
ml; P = 0.03). In these patients, IFN- production was not
significantly different between D. pteronyssinus-stimulated
(1,582 pg/ml) and control wells (1,726 pg/ml; P = 0.625).
The percentage of CD4+ T cells expressing CD30 was
higher in D. pteronyssinus-stimulated wells (14.7%) than in
control wells (6.6%) in atopic nonasthmatics (P = 0.03).
In the healthy nonatopic subjects (n = 7; Figure 1c and
Table 3), D. pteronyssinus produced no significant difference in the production of IL-4 (22.6 pg/ml) and IFN-
(1,778 pg/ml) or CD4CD30 (5.4%) expression compared to
control IL-2-wells (IL-4 < 3 pg/ml; IFN- = 1,700 pg/ml;
% CD4CD30 = 3.66%).
Can Allergen-induced IL-4, IFN-, and CD30 Discriminate
Subjects with Atopic Asthma from Subjects with Atopy but
without Asthma and Normal Subjects?
(1,136 pg/ml; n = 18) than
did atopic nonasthmatics (1,582 pg/ml; n = 6; P = 0.0427),
and also compared to normal subjects (1,778 pg/ml; n = 7;
P = 0.019). Atopic nonasthmatic IFN- levels did not differ from those of normal subjects (P = 1). The IFN-:IL-4
ratio was significantly lower in atopic asthmatics (14.1; n = 18) compared to atopic nonasthmatics (42.3; n = 6; P < 0.016), and compared to normal individuals (120.9; n = 7;
P = 0.032). The ratio also correlated inversely with the
percentage of CD4+ T cells expressing CD30 (r = 0.51)
(Figure 2b).
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Correlation of Allergen-induced IL-4 and IFN-
Production with Symptomatic Disease
Activity in Atopic Asthma
There was no correlation between IL-4 production and
symptomatic disease activity score. There was an inverse
correlation between IFN- production and disease activity
score (r = 0.79) (see Figure 2a). For each patient with
asthma we calculated an IFN-:IL-4 ratio to take account
of the balance between Th1 activity (IFN-) and Th2 activity (IL-4) and found an inverse correlation between this ratio and disease activity score (r = 0.78) (see Figure 2b).
There was also an inverse correlation between the percentage of CD4+ T cells expressing CD30 and both IFN- production (r = 0.60) and IFN-:IL-4 ratio (r = 0.51) (Figures 3a and 3b).
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Comparison of Allergen and Mitogen-induced IL-4 Production in Patients with Atopy
Among the nine patients who received PHA as well as allergen stimulation, PHA-stimulated IL-4 production peaked at 48 h (3.65 pg/ml) and was significantly less than D. pteronyssinus-stimulated IL-4 production (83.2 pg/ml ± 30.3; P < 0.008) (Figure 4).
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Discussion |
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Materials & Methods
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Discussion
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This article not only presents further evidence in support of the role of Th2 cytokines in atopic disease, but also provides evidence that different groups of atopic patients may respond differently to allergen stimulation of PBMCs with respect to cytokine profiles. It has been stressed that the important factor in allergic hypersensitivity reactions is not whether there are allergen-specific T cells present that respond to any given allergen, but rather how they respond with regard to cytokine production (9). This is supported by work showing that virtually 100% of normal and atopic individuals show a proliferative response to inhalant allergens when stimulated in a serum-free medium (37).
It is well established that an imbalance between IFN-
production and IL-4 production exists in atopic disease (9,
11, 13, 36), but this is the first work to show that PBMCs
from atopic asthmatics, in response to allergen stimulation
in vitro, produced significant amounts of IL-4 and diminished amounts of IFN-. Moreover, while atopic nonasthmatic individuals also produced significant amounts of IL-4,
these subjects produced significantly greater quantities of
IFN- compared to the atopic asthmatic subjects. Therefore
what we have demonstrated suggests that the difference between having atopy and asthma is not related to IL-4
production, but may in fact be related to the level of IFN-
response to allergen stimulation. If this in vitro result reflects the in vivo situation it may have major implications
for future therapeutic interventions in asthma.
While other work has shown that PBMCs from mite-sensitive patients with bronchial asthma show increased
production of IL-4 and decreased IFN- following stimulation with Dermatophagoides (46), the present work goes
further in relating not only these parameters but also
CD30 expression to the contemporaneous clinical situation in vivo. We found, within a group of atopic asthmatics, that cytokine production by PBMCs in vitro correlated
with a symptomatic disease activity score. Of some interest
is the observation that it is reduced IFN- production rather
than raised IL-4 production that shows a significant correlation (inverse) with the patient symptom score. This result
raises the intriguing suggestion that it is the lack of an inhibitory Th1 cytokine response to allergen exposure that may
be the important factor in deciding asthma severity.
Furthermore, the inverse correlation between the IFN-:
IL-4 ratio and symptomatic disease score supports the viewpoint that it is the balance between Th1 (IFN-) and Th2
(IL-4) cytokine production that is important, rather than
the absolute levels of Th2 cytokines produced in response
to allergen stimulation, that decides clinical severity (9).
This correlation of IFN- production and IFN-:IL-4 ratio
with a symptomatic disease activity score is extremely important in that it suggests that this in vitro system is clinically relevant and may be used to monitor disease activity
in vitro. In the past, criticisms have been made of in vitro
systems of allergen stimulation of T-cell clones and cell
lines not being relevant to the situation in vivo (9). This article correlating our in vitro findings with patient symptoms has addressed this issue.
Whether CD30 is a marker of Th2 is currently under debate (27). The present finding that greater proportions
of CD4+ T cells from atopic asthmatics express CD30 compared to atopic nonasthmatic and healthy nonatopic individuals and that cultures of PBMCs expressing high percentages of CD30+CD4+ cells produce less IFN-, implies
that CD30 expression is associated more with Th2-type
cells, rather than Th1-type cells. The possibility that CD30
expression is associated more with the differentiation of
Th2 cells is consistent with reports that in cell lines CD30 has been shown to be associated with differing effects
ranging from proliferation to differentiation and apoptosis
(24); and, furthermore, CD30-CD30 ligand interaction has
been shown to positively influence the development of the
Th2 phenotype (26).
In summary, while many workers have shown an imbalance between IFN- and IL-4 production in atopic patients, this is the first work to demonstrate that different
groups of atopic patients respond differently to allergen
with regard to IFN- production and that within atopic
asthmatics that IFN- production may be inversely correlated with symptomatic disease severity.
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Footnotes |
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Address correspondence to: Leonard W. Poulter, Department of Clinical Immunology, Royal Free Hospital School of Medicine, Rowland Hill Street, Hampstead, London, UK.
(Received in original form October 1, 1996 and in revised form February 11, 1997).
Abbreviations: Aspergillus, ASP;2 agonist, BA; cyanin 5, Cy 5; enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC;
inhaled steroid, IS; interferon , IFN-; interleukin, IL; Lolium perenne,
LP; mean fluorescence intensity, MFI; nerve growth factor, NGF; peripheral blood mononuclear cells, PBMCs; phosphate-buffered saline, PBS;
phycoerythrin, PE; pulmonary function test, PFT; phytohemagglutinin,
PHA; theophylline, TH; helper T cell types 1 and 2, Th1 and Th2; tumor
necrosis factor, TNF.
Acknowledgments: Dr. Leonard and Dr. Tormey are supported by a British Council Chevening Research Scholarship. The authors are particularly grateful to Prof. Conleth Feighery, Department of Immunology, St. James's Hospital, Dublin, for his help and advice and use of his laboratory facilities. The authors thank Kamal Ivory, Richard Tilling, Dr. Alex Whelan, and Rena Willoughby for helpful advice with this work. This work was supported by Glaxo/Wellcome UK Ltd.
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References |
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Introduction
Materials & Methods
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Discussion
References
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