From the Division of Molecular and Clinical Immunology, Faculty of Medicine and Health Sciences,
University of Nottingham, Nottingham NG7 2UH, United Kingdom
Recent reports have indicated that the cysteine protease activity of Der p 1 may play a significant role in its ability to elicit IgE antibody responses, mainly through cleavage of membrane CD23 on B cells and interleukin (IL)-4 synthesis and secretion from mast cells and basophils.
Here we demonstrate for the first time that Der p 1 also cleaves the
subunit of the IL-2 receptor (IL-2R or CD25) from the surface of human peripheral blood T cells and, as a result,
these cells show markedly diminished proliferation and interferon
secretion in response to
potent stimulation by anti-CD3 antibody. Given that the IL-2R is pivotal for the propagation of Th1 cells, its cleavage by Der p 1 may consequently bias the immune response towards Th2
cells, thereby creating an allergic microenvironment.
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Introduction |
We (1, 2) and others (3) have recently demonstrated
that Der p 1, a major allergen of the house dust mite
Dermatophagoides pteronyssinus, proteolytically cleaves CD23
(the low affinity receptor for IgE, also known as Fc
RII)
from the surface of cultured human B cells. Given that
CD23 is involved in the negative feedback regulation of in
vivo IgE synthesis (4), its cleavage by Der p 1 could potentially disrupt the IgE regulatory mechanism, thereby leading to excessive IgE synthesis.
Der p 1 was also shown in vitro to induce mast cell and
basophil degranulation, and stimulate IL-4 synthesis and secretion in the absence of IgE (7). These actions of Der p 1, which were due to its proteolytic activity, are particularly
significant, since mast cells and basophils can support IgE
synthesis by secreting IL-4 and IL-13 and provide contact-mediated help through CD40L (8, 9). Therefore, the protease activity of Der p 1 not only subverts an important
mechanism that controls IgE synthesis, namely the negative
feedback regulation by membrane CD23, it apparently also
provides a cytokine environment conducive for IgE synthesis.
These observations raise the question of whether the enzymatic activity of Der p 1 also perturbs human T cells with
respect to favoring the emergence of a Th2 background,
which would create an allergic microenvironment. In this
paper, we demonstrate that Der p 1 cleaves CD25, the 55-kD
subunit of the IL-2 receptor (the high-affinity form of
which consists of
,
, and
subunits), from the surface of
peripheral blood T cells and, as a result, these cells show
markedly diminished proliferation and IFN-
secretion in response to potent stimulation by anti-CD3 antibody. Given that the IL-2 receptor is pivotal for the propagation of Th1
cells (10), its cleavage by Der p 1 may consequently bias the
immune response towards Th2 cells, which instead depend
on the IL-4 receptor for their propagation.
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Materials and Methods |
Antibody Reagents.
The following PE- or FITC-labeled mouse
monoclonal antibodies to human cell surface markers were purchased: anti-CD25 (ACT-1), anti-CD4 (MT310), anti-CD45RO
(UCHL1), anti-CD2 (MT910), anti-CD3 (UCHT-1), anti-CD8
(DK25; Dako, Buckinghamshire, UK) and anti-CD69 (TP1.55.3;
Immunotech, Luton, UK). Anti-CD3 antibody was purified on
protein G from the supernatant of a hybridoma cell line (OKT3; ECACC, Porton Down, UK).
Der p 1 Preparation.
Der p 1 was purified from lyophilized
house dust mite culture supernatant (SmithKline Beecham Pharmaceuticals, Worthing, UK) by affinity chromatography using
monoclonal anti-Der p 1 antibody (4C1; Indoor Biotechnologies, Clwyd, UK) as previously described (11). The affinity-purified Der p 1 was then passed through a soybean trypsin inhibitor
column to remove traces of serine protease activity. The purity of
the preparation was assessed by silver stain SDS-PAGE (12% gel)
analysis and its identity was confirmed by NH2-terminal sequencing on an automatic amino acid sequencer (Applied Biosystems,
Foster City, CA); the sequence obtained (TNACSINGNA)
matched the previously published sequence of Der p 1 (12).
CD25 Cleavage Assays.
PBLs were purified from 20 ml of
heparinized whole blood on a discontinuous Histopaque density
gradient (Sigma Chemical Company, Poole, UK). Cells (5 × 105) were suspended in serum-free AIM V medium (Gibco Life
Technologies Ltd., Paisley, UK) in flat-bottomed, 24-well plates
and were stimulated with PHA (5 µg/ml final concentration) for
3 d at 37°C in a humidified atmosphere of 5% CO2. CD25 cleavage was performed by incubating 105 cells with Der p 1 (preactivated with 5 mM cysteine) in a total volume of 200 µl AIM V
medium for 1 h at 37°C. The cells were centrifuged and the supernatant was tested for soluble CD25 concentration by ELISA
(R & D Systems, Abingdon, UK). The cells were then resuspended in PBS containing 0.5% BSA and 0.1% azide, stained
with PE-labeled anti-CD25 antibody for 30 min at room temperature in the dark, and fixed with 5% formaldehyde. The expression of other T cell surface markers (i.e., CD2, CD3, CD4, CD8,
CD45RO, and CD69) was monitored in the same way using the
appropriate PE- or FITC-labeled antibodies. Cells were analyzed
on a FACScan® (Becton Dickinson, Mountain View, CA) with a
linear fluorescence setting of 660 volts. The fluorescence (FL1 or
FL2) profile versus forward scatter was used to monitor the cells,
and the amplification scale was altered according to the level of
fluorescence. For each sample, 5,000 events were collected and
then analyzed using the flowMATE program (DAKO, High Wycombe, UK).
Anti-CD3-Induced T Cell Proliferation Assay.
PBLs were prepared from heparinized whole blood of nonallergic volunteers as
described above. Anti-CD3 antibody (0.2 µg/ml final concentration) was added in AIM V medium to round-bottomed, 96-well
plates. After 1 h, 105 lymphocytes were added to each well and
incubated overnight at 37°C in a humidified atmosphere of 5%
CO2. On the next day (unless otherwise stated), Der p 1 (with or
without preactivation with 5 mM cysteine) was added to the cell
cultures and incubation continued for another 3 d. 16 h before
termination of the culture, [6-3H]thymidine (specific activity 24.0 Ci/mmol; Amersham Life Science, Buckingham, UK) was added
to each well at a final concentration of 4 µCi/ml. Cells were then
transferred to Unifilter-96 plate GF/C and radioactivity was
counted in scintillation fluid (Microscint O) using a top counter
(both from Canberra Packard Limited, Pangbourne, UK). With
some blood samples, parallel cultures were carried out for cytokine
(IL-2, IL-4, and IFN-
) measurements using Quantikine ELISA
kits (R & D Systems).
To exclude cellular cytotoxicity of Der p 1, the number of
apoptotic and necrotic cells were determined using the Annexin V/FITC kit (Boehringer Ingelheim Bioproducts Partnership,
Heidelberg, Germany).
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Results and Discussion |
We have affinity purified Der p 1 from dust mite extract
and confirmed its identity by NH2-terminal sequencing.
The Der p 1 preparation was tested for its ability to proteolytically cleave functionally important molecules, including CD25, expressed on cultured human T cells. The
data show that Der p 1 cleaves CD25, but not CD2, CD3,
CD4, CD8, CD45RO, or CD69 (Fig. 1). The cleavage of
CD25 by Der p 1 was associated with the release of soluble CD25 into the culture supernatant (Fig. 2 a). This indicates
that Der p 1 causes limited digestion of CD25, since the
CD25 fragment released was detectable with antibody reagents used for measuring spontaneously shed CD25. The
cleavage of CD25 was inhibited by antipain, a low molecular
weight protease inhibitor of microbial origin, thus confirming that the cleavage of CD25 was due to the proteolytic activity of Der p 1 (Fig. 2 b).

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Fig. 1.
The proteolytic effect of Der p 1 on human T cell surface
markers. Paired results represent the expression of markers in the absence
(open bars) and presence (solid bars) of Der p 1 (5 µg/ml). Data presented
are the means of duplicate experiments; SE was <5%.
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Fig. 2.
(a) Der p 1-induced
cleavage of membrane CD25
(filled circles) and concomitant release of soluble CD25 (open circles). (b) CD25 cleavage is
blocked by earlier treatment of
Der p 1 (5 µg/ml) with antipain
(4 µM). Data presented in a and
b are the means of duplicate experiments; SE was <5%.
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To assess the biological consequences of Der p 1-induced
CD25 cleavage, we conducted an IL-2R-dependent T cell
proliferation assay. This was carried out by stimulating human T cells with anti-CD3, which is known to induce T
cell proliferation through IL-2 production and IL-2R expression (13). Der p 1-treated cultures showed up to 61%
decrease in T cell proliferation, an effect that was due to
the enzymatic activity of Der p 1 (Table 1). This action of
Der p 1 was most effective within 18-48 h of culture initiation (Fig. 3), and appeared to coincide with the time
course of CD25 expression (14). To further test the hypothesis that Der p 1-induced suppression of T cell proliferation is due to CD25 cleavage, we examined the kinetics
of IL-2, IL-4, and IFN-
production and soluble CD25 release during this window of Der p 1 action. We found that
the early peak of IL-2 production, namely within 6-24 h of
culture initiation, was not significantly affected by Der p 1 (Fig. 4 a), thereby indicating that the Der p 1-induced suppression of T cell proliferation was not due to decreased
IL-2 production or its cleavage by Der p 1. On the other
hand, Der p 1-treated cultures showed marked release of
soluble CD25 as demonstrated by a shift in the soluble
CD25 release curve (Fig. 4 b). Cleavage of CD25 by Der p 1 clearly renders the T cells unresponsive to the proliferative
effect of IL-2, as manifested by a decrease of at least fourfold in IFN-
production (Fig. 4 c). Taken together, our
data suggest that the inhibition of T cell proliferation was
indeed due to the cleavage of CD25, since it has been
shown that T cell proliferation, apart from depending on
the concentration of IL-2, depends on the level of IL-2R
expression and the contact time between IL-2 and IL-2R
(15). At the time points studied, there was very little IL-4
detectable in the T cell cultures (data not shown).
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Table 1
Inhibition of IL-2-mediated Human T Cell Proliferation by Der p 1 Requires Enzymatically Active Protease (i.e., Preactivated with
Cysteine)
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Fig. 3.
Der p 1 suppresses human T cell proliferation mostly within
18-48 h of culture initiation. Results represent cultures with no Der p 1 (open bar) or those to which Der p 1 (10 µg/ml) was added at different
time points (solid bars). The thymidine label was added 80 h after culture
initiation. Data presented are the means of quadruplicate experiments; error bars represent SEM.
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Fig. 4.
Effect of Der p 1 (10 µg/ml; added 6 h after culture initiation) on the time course of Th1 cytokine expression and soluble CD25 release.
Data represent the kinetics of IL-2 expression (a) soluble CD25 release (b), and IFN- expression (c) in the absence (open circles) and presence (filled circles)
of Der p 1. Data presented are the means of duplicate experiments; SE was <5%.
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Given that the CD25 molecule is critical to the anti-CD3 model of T cell proliferation (13), our data show that
Der p 1-induced CD25 cleavage has a significant downregulatory effect on this aspect of T cell function. These findings raise the question of the role of the enzymatic activity
of Der p 1 in perturbing the homeostatic control of the human Th1-like/Th2-like subset distribution. The Th1 and
Th2 cell populations each enhance the development of
cells of the same subset while suppressing the propagation of those of the other subset. Within this cytokine driven
process of polarization of T cell development, IL-2, along
with IFN-
, and IL-4 are considered to be autocrine growth
factors for the Th1 and Th2 cells, respectively (10). Therefore, Der p 1-induced cleavage of CD25 is likely to lead to
impaired development of cells of the Th1 subset and consequent augmentation of those of the Th2 subset. For instance, it has been shown that mite allergen-specific T cell
clones exhibit Th2 cytokine profile (16) and require IL-4
for their optimal growth (17), which means that in an ongoing allergic response to Der p 1, such Th2 cells will not
be sensitive to the enzymatic activity of Der p 1 compared
to IL-2-dependent Th1 cells. Clearly, it would have been
of interest to also monitor the effect of Der p 1 on the IL-4
receptor (CD124), but, due to low levels of expression of
this receptor on peripheral blood T cells, we were unable
to carry out this experiment.
A number of studies indicate that Th2 responses are involved in the pathogenesis of allergic diseases (18), mainly
through the secretion of IL-4/IL-13 and IL-5, which are
responsible for stimulating IgE synthesis and eosinophil
function, respectively. It is also highly relevant in this connection that IL-2 and IL-2R
knockout mice show enhanced synthesis of immunoglobulins including IgE (19), a
finding which is consistent with uncontrolled Th2 cell propagation. Recent reports indicate that the protease activity of Der p 1 plays a significant role in its ability to elicit IgE responses, mainly through cleavage of membrane CD23 on
B cells (1) and IL-4 synthesis and secretion from mast
cells and basophils (7). The data described here suggest yet
another mechanism by which Der p 1 uses its protease activity to bias the immune response in favor of IgE, this time
by preferentially targeting a molecule (i.e., the IL-2R
subunit) which plays a pivotal role in the development and
propagation of Th1 cells, thus favoring the emergence and
dominance of Th2 responses (10).
Address correspondence to Dr. Farouk Shakib, Division of Molecular and Clinical Immunology, Faculty of
Medicine and Health Sciences, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone:
44-0115-9709-123; Fax: 44-0115-9709-125; E-mail: farouk.shakib{at}nottingham.ac.uk
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