Attenuation of Immunological Symptoms of Allergic Asthma in
Mice Lacking the Tyrosine Kinase ITK1
Cynthia Mueller and Avery August2
Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, TH2 cells, and cytokines, increased
mucous production in the lung, and elevated serum IgE. This TH2-type response suggests a prominent role for TH2 cells and their
cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown
to play a role in the differentiation and/or function of TH2-type cells, suggesting that ITK may represent a good target for the
control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immuno-
logical symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil
infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice
lacking ITK. T cells from ITK?/?mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13,
in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our
results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the TH2 cytokines IL-5 and IL-13 and may
be an attractive target for antiasthmatic drugs. The Journal of Immunology, 2003, 170: 5056–5063.
city African Americans and Hispanics, especially children and
young adults (up to three times the rate) (1). This results not only
in morbidity caused by the disease, but also in lost opportunities in
education and the workplace because of children and adults miss-
ing days. Patients with allergic asthma manifest airway inflamma-
tion and show increases in eosinophils, TH2 cells, and cytokines,
increased mucous production in the lung, and elevated serum IgE
(2). These events combined impact the epithelial and smooth mus-
cle cells of the lung leading to airway hyperresponsiveness.
The presence of so many hallmarks of a TH2-type response has
pointed to a prominent role for TH2 cells and, in particular, TH2-
type cytokines IL-4, -5, and -13 in the pathology of this disease.
Introduction of Ag-specific TH2 cells alone or IL-4 and IL-13
alone can induce the majority of these events and lead to airway
hyperresponsiveness in mice, and blocking these cytokines pre-
vents the development of specific symptoms in mice (3–5). Fur-
thermore, blocking IL-4 can relieve some of the symptoms of
asthma in humans (6). The transcription factor GATA-3 regulates
IL-5 production and dominant-negative forms of this protein can
significantly inhibit experimental allergic asthma in mice (7). Find-
ing further targets that are pharmaceutically tractable and that reg-
ulate the development and/or function of TH2 cells would assist in
he prevalence and severity of asthma has increased world-
wide in the last 20 years. In the United States, morbidity
and mortality are disproportionately high among inner
treating this disease. The development of TH2 cells is dependent on
the cytokine milieu in the microenvironment where T cells en-
counter Ag such that in the presence of IFN-? or IL-12, naive T
cells differentiate into TH1 cells and subsequently secrete IFN-?.
Similarly, in the presence of IL-4, naive T cells differentiate into
TH2 cells and subsequently secrete IL-4, -5, and -13 (for review,
see Ref. 8). Although it has been proposed that specific types of
dendritic cells can produce specific cytokines that lead to either
TH1 or TH2 differentiation of T cells, the source of the cytokines
responsible for T cell differentiation in vivo is not clear since T
cells can also produce these cytokines (8). T cell cytokine produc-
tion is dependent on early signaling events initiated by the TCR
and costimulatory signals such as CD28. The combination of TCR
and costimulatory signals result in activation of T cells and ulti-
mately their differentiation into TH1 and/or TH2 cells and subse-
quent cytokine production (9). Costimulatory signals delivered by
CD28 have been reported to be critical in the development of the
two T cell subsets and their corresponding cytokine production
(10, 11). Although there is a significant amount of data suggesting
that CD28 may preferentially affect the differentiation to TH2 cells
in vitro (12, 13), as well as in vivo (11, 14), this remains an un-
clarified point because there are reports of CD28-independent TH2
responses in vitro (15) and in vivo (16). Whether CD28 signals
affect the ability of T cells to develop into TH2 cells or their sub-
sequent secretion of cytokine is not clear; however, CD28 signals
have been demonstrated to be required for the development of
allergic asthma in mice (17–21). The CD28 related costimulatory
molecule inducible costimulator molecule has also been demon-
strated to be involved in regulating the pathology of either a Shis-
tosoma mansoni model of allergic airway disease (22) or an OVA
model of asthma in mice (23). In addition, other targets proposed
for regulating the development of allergic asthma include the ad-
hesion molecule LFA-1 and the chemokine receptors CCR3, 4, and
8 (24–27). These molecules all lie downstream of T cell activation
or are involved in T cell activation and trafficking of cells in the
case of the chemokine receptors.
Triggering the TCR complex results in the activation of a num-
ber of attractive pharmaceutical targets, the tyrosine kinases of the
Src, Syk, and Tec family of tyrosine kinases (for review, see Ref.
Immunology Research Laboratories and Department of Veterinary Science, Pennsyl-
vania State University, University Park, PA 16802.
Received for publication January 21, 2003. Accepted for publication March 10, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant RO1-AI51626, a
Johnson & Johnson Focused Giving Program Grant and the Pennsylvania State Uni-
versity Innovative Biotechnology Fund (to A.A.).
2Address correspondence and reprint requests to Dr. Avery August, Department of Vet-
erinary Science, Immunology Research Laboratories, Pennsylvania State University, 115
Henning Building, University Park, PA 16802. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: ITK, inducible T cell kinase; WT, wild type; IN,
The Journal of Immunology
Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00
28). These kinases are critical in the activation of immune cells and
have limited expression patterns, with specific family members
having lymphoid-specific expression (28). In T cells, members of
the first two families of kinases, Lck and Zap-70, are critical for T
cell activation as well as development (29). However, Txk/Rlk, a
Tec family kinase, seems to be dispensable for T cell activation
and development (30). By contrast, inducible T cell kinase (ITK),3
the other Tec kinase expressed in T cells, seems to have a more
prominent role in T cell activation and differentiation (30–37).
Triggering the TCR also results in increases in intracellular cal-
cium, which can activate the protein phosphatase calcineurin and
mediate other downstream effects (38). The influx of calcium in T
cells is controlled by ITK and mice lacking ITK have reduced
calcium increases upon stimulation (37, 39). Indeed, the cal-
cineurin inhibitors cyclosporin A and FK506 have been suggested
as treatments for asthma patients (40). These mice also have re-
duced naive T cell function in IL-2 production and proliferation
when stimulated via the TCR (32, 36, 39). In addition, T cells from
mice lacking ITK either secrete no IL-4 or significantly less IL-4
than normal T cells, an event that seems to be dependent on the
ITK-mediated calcium increase (35, 37).
These data suggest that by regulating the development and/or
function of TH2 cells, ITK may modify the development of allergic
asthma. We have tested this hypothesis and now report that mice
lacking ITK have drastically reduced lung inflammation and mu-
cous production following induction of allergic asthma. This was
probably due to a number of deficiencies in the ITK null mice:
reduced Ag-specific recruitment of T cells to the lung; overall
reduction in cytokine production, but preferential reduction in Ag-
specific secretion of IL-5 and IL-13 by ITK null T cells; reduced
T cell proliferative responses to challenge with the allergen OVA.
However, these mice have high levels of serum and OVA-specific
IgE. Our results suggest that the tyrosine kinase ITK may be an
attractive target for antiasthmatic drugs.
Materials and Methods
Wild-type (WT) (The Jackson Laboratory, Bar Harbor, ME) and ITK null
mice (kind gift from Dr. D. Littman, New York University School of Med-
icine, New York, NY; Ref. 32) on C57BL/6 backgrounds (6–8 wk old)
were used for these experiments. The ITK null mice were backcrossed to
the C57BL/6 at least 10 generations. All mice were kept in microisolater
cages in the animal facilities at Pennsylvania State University and were
provided with food and water ad libitum. All experiments were approved
by the Office of Research Protection’s Institutional Animal Care and Use
Committee at Pennsylvania State.
Allergic asthma induction
Groups of mice (WT or ITK?/?mice) were primed with OVA (Sigma-
Aldrich, St. Louis, MO) or carrier as follows: 50 ?g/ml OVA complexed
with aluminum hydroxide (10 ?g OVA/1 mg alum; Pierce, Rockford, IL)
were injected i.p. on days 0 and 5. Control mice were injected with alu-
minum hydroxide alone. Mice were then daily exposed intranasally (IN)
with OVA (2 mg/ml, 40 ?g total) on days 12 through 15 and sacrificed 24 h
later for analysis.
Characterization of lung pathology
Following prime and challenge, mice were sacrificed and lungs were re-
moved. Lungs were fixed in formaldehyde (3%) overnight before embed-
ding and 5-?m sections were cut for staining. Sections were stained with
H&E to examine infiltrating cells and periodic acid-Schiff (PAS) for anal-
ysis of mucous production. In some experiments, one lung was used for
histology and the other was dissociated using collagenase (150 U/ml), and
the resultant cell populations were analyzed using an Advia 1200 Hema-
tology System (Bayer, Norwood, MA). To analyze the T cell population in
the lung, mononuclear cells were isolated from collagenase-dissociated
lungs on a 30%/60% Percoll gradient as described elsewhere (41). Cells
were then reacted with directly conjugated Abs against CD3 (CyChrome;
BD Pharmingen, San Diego, CA) and analyzed by flow cytometry. In some
experiments, these stained cells were permeabilized and stained with anti-
IL-4 (PE) and anti-IFN-? (FITC; all from BD Pharmingen) and analyzed
by flow cytometry, gating on the lymphocyte population as defined by their
forward and side scatter characteristics.
Analysis of T cell response to OVA
Following prime and challenge, mice were sacrificed and splenocytes pu-
rified. Proliferation of T cells to OVA was analyzed by incubating the
isolated splenocytes with OVA at the indicated concentrations. Cultures
were then pulsed with [3H]thymidine for 18 h 2 days after initiating the
culture; the culture was harvested and incorporated radioactivity was de-
termined by scintillation counting. Analysis of cytokine secretion was per-
formed by stimulating 2 ? 105purified CD4?T cells with the indicated
concentrations of OVA and T cell-depleted splenocytes for 96 h. Super-
natants were then harvested and cytokine-specific (IFN-?, IL-4, -5, -10,
and -13) ELISAs were performed according to the manufacturer’s instruc-
tions (BD Pharmingen for the IFN-?, IL-4, -5, and -10, and R&D Systems
(Minneapolis, MN) for IL-13).
Analysis of IgE levels
Before and following prime and challenge, mice were sacrificed and serum
was obtained. Dilutions of sera were analyzed for total IgE and OVA-
specific IgE by ELISA. In the former case, anti-murine IgE (2 ?g/ml,
1/250) was used as capture Abs and HRP-conjugated anti-murine IgE
(1/250) as detection reagents (Southern Biotechnology Associates, Bir-
mingham, AL). To detect OVA-specific IgE, OVA was coated onto the
ELISA wells (20 mg/ml) and dilutions of sera tested with HRP-conjugated
anti-murine IgE were used as detection reagents.
Values were compared using Student’s t test.
ITK null mice exhibit reduced lung inflammation following
allergic asthma induction
The tyrosine kinase ITK has been reported to be involved in the
activation of T cells leading to TH2 cell differentiation in vitro and
in TH2 type responses to pathogens in vivo (35, 37). Since allergic
asthma has many hallmarks of a TH2-type response in humans and
in animal models, we tested whether ITK may be involved in the
allergic inflammatory response in the lung to the model allergen
OVA in a murine model of allergic asthma. In this model, mice are
primed with OVA, then challenged IN with OVA (42). This treat-
ment results in increased inflammatory cell infiltration into the
lung, thickening of the epithelial cells lining the bronchioles of the
lung, mucous secretion, and increases in IgE in the serum of these
mice. We primed mice (WT or ITK?/?on the same backgrounds)
by i.p. injection of OVA/alum on days 0 and 5, then on days 12–15
they were challenged by exposure to OVA IN. On day 16, mice
were sacrificed and analyzed for immunological symptoms of al-
lergic asthma. Control mice were either not primed, but were chal-
lenged, or were primed and not challenged, with similar results
(data not shown).
Isolated lungs were fixed and stained with H&E to determine
leukocyte infiltration (Fig. 1). Our analysis demonstrated that al-
though WT mice develop leukocyte infiltrates in the lung, mice
lacking ITK had much reduced infiltration (compare Fig. 1, a and
b and c and d). WT mice also exhibited thickening of the epithelial
cell lining of the bronchioles, which was not observed in ITK?/?
mice. Analysis of cells obtained following collagenase dissociation
of lungs from similar experiments demonstrated a marked increase
in eosinophil influx in lungs from WT mice primed and challenged
with OVA, but not from unprimed mice or from mice lacking ITK
under any of the tested conditions (Fig. 1e, p ? 0.068). These data
suggest that mice lacking ITK have defective inflammatory re-
sponses to priming and challenge with OVA in this model.
Mucous production in response to allergic asthma has been
shown to be controlled by TH2 cells and/or their cytokines (5). To
5057The Journal of Immunology
determine whether ITK?/?mice were also defective in mucous
production in response to OVA challenge, we stained lung sections
with PAS to detect mucous. Figure 2 confirms our findings in Fig.
1, indicating that although WT mice had increased mucous pro-
duction by the goblet cells lining the bronchioles (Fig. 2, a and b),
the bronchioles from ITK?/?had much reduced to almost absent
mucous production (Fig. 2, c and d). These results suggest that ITK
may regulate the TH2-type response and/or the resultant cytokine
production that leads to mucous production in this model of aller-
Reduced T cell infiltration in the lung of ITK null mice
following allergic asthma induction
Examining lung-derived cells for the presence of T cells indicated
that although there was an increase in the percentage of T cells (of
the total lymphocyte population) in the lungs of WT mice upon
OVA challenge, the lungs from the ITK?/?mice exhibited no
increase in the percentage of T cells compared to the control ITK
null mice (Fig. 3, a and b). Analysis of these lung-derived T cells
for intracellular cytokine (IFN-? and IL-4, classical TH1 and TH2
cytokines, respectively) indicated that while approximately 1.7%
of the T cells from WT lungs had intracellular IL-4, none of the T
cells obtained from ITK null lungs had intracellular IL-4. No
IFN-? was detected in the T cells from either the WT or ITK null
lungs (Fig. 3c). These data suggest that reduced Ag-specific T cell
recruitment to the lung may in part underlie the reduced responses
we observe in the ITK null mice.
Increased total and Ag-specific IgE in ITK null mice
Serum IgE levels correlate with asthmatic symptoms (43). To de-
termine whether the ITK?/?mice were generating an IgE response
against the allergen, we tested their serum for total IgE and OVA-
specific IgE. Under these immunization conditions, WT mice had
increased serum IgE whereas WT control mice had low levels of
IgE comparable to untreated naive WT mice (Fig. 4a). Surpris-
ingly, mice lacking ITK had higher levels of serum IgE than con-
trol WT mice in the unimmunized state (Fig. 4a, p ? 0.004). This
increased serum IgE level was surprising given the proposal that
ITK may be involved in regulating TH2 responses. B cell class
switch to IgE can be controlled by the levels of IL-4, IFN-?, as
well as IL-10 and perhaps ITK null mice have altered serum levels
of these IFN-? and IL-10, thus reducing the potential negative
induction of allergic asthma. WT (a and b) or ITK?/?(c and d) mice were
primed twice, 5 days apart with OVA/alum (c and d) or alum alone (a and
c) then exposed IN to OVA (all mice) on days 12–15 as described in
Materials and Methods. Twenty-four hours after the final OVA intranasal
exposure, lungs from mice were fixed, paraffin-embedded, sectioned (5
?m), and stained with H&E. a, WT mouse primed with alum and exposed
to OVA IN. b, WT mouse primed with OVA/alum and exposed to OVA
IN. c, ITK?/?mouse primed with alum and exposed to OVA IN. d,
ITK?/?mouse primed with OVA/alum and exposed to OVA IN. Note the
thickening of the epithelial cell layer lining the bronchioles and inflam-
matory infiltration in the WT mouse primed and exposed (b) and the lack
of such thickening and infiltration in the lung from the mouse lacking ITK
(d). Original magnifications, ?10. e, Reduced eosinophil infiltration in the
lungs of mice lacking ITK following induction of allergic asthma. Lungs
from similarly treated mice were isolated and dissociated as described in
Materials and Methods. The resultant cell populations were analyzed using
an Advia 120 Hematology Analyzer for neutrophils (neut), monocytes
(mono), basophils (baso), and eosinophils (eosin). Lymphocytes were also
detected but no significant changes were detected in this population (data
not shown). Profiles from the lungs of control mice profiles were similar to
those seen in the ITK?/?mice and were omitted for clarity of presentation.
Note the significant difference in the percentage of eosinophils in the lungs
from the primed and challenged ITK?/?mice compared to WT mice (p ?
0.068). Similar results were seen when control mice were primed with
OVA/alum but were only exposed to PBS IN. Representative of more than
seven separate experiments with at least three mice per group.
Reduced lung inflammation in mice lacking ITK following
induction of allergic asthma. WT (a and b) or ITK?/?(c and d) mice were
primed and challenged as described in Fig. 1 legend. Twenty-four hours
after the final OVA IN exposure, lungs from mice were fixed, embedded in
paraffin, sectioned (5 ?m), and stained with PAS to detect mucous. a, WT
mouse primed with alum and exposed to OVA IN. b, WT mouse primed
with OVA/alum and exposed to OVA IN. c, ITK?/?mouse primed with
alum and exposed to OVA IN. d, ITK?/?mouse primed with OVA/alum
and exposed to OVA IN. Note that the dark areas lining the goblet cells in
the lung of the WT mouse primed and exposed are stained with the PAS
stain and mucous is being produced by these cells (b). Also note the very
low production of mucous in the lung from ITK?/?mouse and the lack of
hyperplasia of these cells (d). Original magnifications, ?20.
Reduced mucous production in mice lacking ITK following
5058ROLE OF ITK IN ALLERGIC ASTHMA
influence of these cytokines on class switch to IgE (44, 45). To
determine whether ITK?/?mice have altered serum levels of IL-4,
IFN-?, or IL-10, we tested serum from WT and ITK?/?mice for
levels of IFN-?, IL-10, and IL-4. Our analysis showed that ITK
null and WT mice both had low levels of serum IL-4 and IL-10 and
equivalent levels of IFN-? (IFN-?: WT, 681.6 ? 136.3 pg/ml;
ITK?/?, 611.5 ? 14 pg/ml; IL-4: WT, 8.5 ? 2.4 pg/ml; ITK?/?,
none detected; IL-10: WT and ITK?/?, none detected). However,
it is not clear whether these levels would lead to the increased
circulating IgE levels in the ITK null mice.
Analyzing serum levels of IgE in ITK?/?mice primed and chal-
lenged with OVA revealed no change in total IgE, but similar to
the WT mice, they did respond with increased OVA-specific IgE
(Fig. 4b, p ? 0.001 for ITK?/?mice and p ? 0.0007 for WT
mice). There was no significant difference in the levels of OVA-
specific IgE in WT vs ITK?/?mice. Since OVA is a T-dependent
Ag, these data indicate that although ITK?/?T cells may have
altered TH2 development and/or function, they may still be able to
generate sufficient TH2-type cytokines to allow for the develop-
ment of an IgE-mediated response. This OVA-specific IgE re-
sponse, however, did not accompany an increase in the inflamma-
tory response in the lung, suggesting perhaps a difference in
systemic vs mucosal immune responses in these mice or the lack
of recruitment of eosinophils to the lungs that can respond, leading
to the pathology seen in WT mice. However, it has been noted that
increased IgE does not always accompany the lung pathology seen
in allergic asthma (46).
Reduced T cell proliferation and altered cytokine production by
ITK null T cells in response to allergen stimulation
Because it appeared that ITK?/?mice had a TH2-type humoral
immune response to OVA, which is a T cell-dependent Ag (47),
we next determined whether ITK?/?T cells could respond to
OVA following priming and challenge. Splenic cells from mice
following induction of allergic asthma. a, WT (top panels) or ITK?/?
(bottom panels) mice were primed and challenged as described in Fig. 1
legend. Lungs were then isolated and dissociated as described in Fig. 1
legend and stained with Abs to CD3 and data were analyzed by gating on
the lymphocyte population. Shaded areas indicate staining with anti-CD3
while the open lines are control Abs. b, Lung cells from WT (top panels)
or ITK?/?(bottom panels) mice primed and challenged as described in
Fig. 1 legend were stained with Abs to CD3, permeabilized, and then
stained with Abs to IFN-? and IL-4. Data shown represent the analysis of
the CD3?population for intracellular IFN-? (y-axis) and IL-4 (x-axis)
Reduced T cell infiltration in the lungs of mice lacking ITK
with OVA in mice lacking ITK. WT (left) or ITK?/?(right) mice were
primed and challenged as described in Fig. 1 legend. Twenty-four hours
after the final OVA intranasal exposure, serum from these mice as well as
naive mice were analyzed for IgE levels by ELISA. Sera from the follow-
ing mice were analyzed: primed with OVA/alum and exposed to OVA (I);
primed with alum and exposed to OVA (striped bars); and naive mice (?).
a, Total IgE levels (1/10 dilution). Note that naive and alum-only primed
WT mice have low levels of total serum IgE levels (the horizontal line
represents the limit of detection of the assay). By contrast, naive and
unprimed ITK?/?mice have significantly elevated total serum IgE levels
compared with similarly treated WT mice (p ? 0.004). ITK?/?mice also
exhibited significantly higher total serum IgE compared to WT mice fol-
lowing prime and challenge (p ? 0.002). b, OVA-specific IgE levels (1/50
dilution). Note that naive and alum-only primed WT and ITK?/?mice
have low levels of OVA-specific serum IgE levels (the horizontal line
represents the limit of detection of the assay). By contrast, both primed WT
and ITK?/?mice have significantly elevated OVA-specific serum IgE lev-
els compared to unprimed mice (p ? 0.0007 for WT mice and p ? 0.001
for ITK?/?). There was no significant difference in the levels of OVA-
specific IgE seen in WT vs ITK?/?mice.
Increased IgE production following priming and challenge
5059The Journal of Immunology
primed and challenged with OVA were incubated with OVA and
T cell proliferation was determined after 3 days of incubation (Fig.
5). WT mice responded to OVA stimulation by proliferating (Fig.
5). Splenic cells from ITK?/?mice by contrast proliferated poorly,
confirming that these mice have reduced T cell responses.
Analysis of cytokine production in response to OVA stimulation
following the allergic asthma induction protocol indicated that ITK
null T cells from draining lymph nodes produced fewer cytokines
of both TH1 and TH2 type (Fig. 6a). Of note, however, is that while
very little IL-4, -5 or, -13 was detected when these cells were
stimulated in vitro, they were able to produce some IFN-? and
IL-10 (Fig. 6a). Qualitatively similar results were observed when
splenic T cells were stimulated (Fig. 6b). Although T cells from
ITK null mice were able to produce more cytokines of both TH1
and TH2 types, quantitatively they were more defective in the pro-
duction of IL-4 and, to a greater extent, IL-5 and IL-13. Their
production of IFN-? and IL-10 was not affected as much (Fig. 6b).
Thus, while mice lacking ITK have reduced T cell responses to
antigenic stimulation, the production of TH2 cytokines, in partic-
ular IL-5 and IL-13, is more affected by the lack of ITK. These
data do suggest, however, that mice lacking ITK are able to secrete
low levels of IL-4 that may be sufficient to generate an Ag-specific
IgE response. However, the reduced levels of T cells in the lung,
coupled with the reduced production of IL-5 and in particular IL-
13, may be the reason for the significant lack of pathological re-
sponse in the lung we observed during allergic asthma induction in
A large body of data suggests that TH2 cells play a critical role in
the development of allergic asthma, in both mouse models as well
as in humans (2–5). Understanding the mechanisms regulating the
development and/or function of T cell subsets will be critical for
designing therapies that specifically control the development of
and/or treatment of this disease. Although the manipulation of the
development of these T cells by cytokine-specific targeting repre-
sents a promising avenue, these approaches generally tend to in-
volve the use of large proteins that may be costly and difficult to
administer. Further analysis of proteins that may control the de-
velopment and/or function of these cells may result in the discov-
ery of better pharmaceutical targets to which small molecule ther-
apeutics can be developed. In this report, we demonstrate that in
the absence of the Tec family tyrosine kinase ITK, mice are largely
resistant to the immunopathological symptoms in a model of al-
lergic asthma. Specifically, ITK null mice had drastically reduced
levels of infiltration of eosinophils and, in particular, of T cells in
the lung. The T cells that were in the lungs of ITK null mice were
not producing detectable levels of IL-4. Similarly, lymph node T
cells from ITK null mice produced no detectable IL-4, -5, and -13,
while producing low levels of IFN-? and IL-10. Similar results
were observed using splenic T cells although these cells produced
more IFN-? and IL-10. Surprisingly, ITK null mice had elevated
levels of total IgE and generated a normal anti-OVA IgE response,
although this was not sufficient to have a significant effect on the
pathological symptoms of the disease. Our data confirm and extend
previous reports of ITK being involved in the development of a
TH2 response in vivo (35, 37).
with OVA in mice lacking ITK. WT or ITK?/?mice were primed and
challenged as described in Fig. 1 legend. Twenty-four hours after the final
OVA intranasal exposure, splenocytes from these mice were incubated
with the indicated concentration of OVA and proliferation was analyzed
48 h later by pulsing with [3H]thymidine for 18 h. The cells were then
harvested and incorporated3H was counted. The results are expressed as
proliferation index, calculated as fold increase in [3H]thymidine incorpo-
ration in OVA-stimulated cells over cells incubated without OVA. All cells
proliferated similarly in the presence of Con A/PMA/ionomycin stimula-
tion. Note that WT cells had significantly higher proliferation when stim-
ulated with OVA compared to ITK?/?cells. Also note that although not
statistically significant, OVA-stimulated ITK?/?cells also reproducibly
proliferated higher than nonstimulated cells from control mice.
Reduced T cell responses following priming and challenge
priming and challenge with OVA. WT or ITK?/?mice were primed and
challenged as described in Fig. 1 legend. Twenty-four hours after the final
OVA intranasal exposure, CD4?T cells were purified from the draining
lymph nodes (a) or spleens (b) from these mice and were incubated with
OVA (1 mg/ml) along with T cell-depleted APCs, and supernatants were
harvested 96 h later for cytokine analysis. a, IFN-?, IL-4, -5, -10, and -13
secretion in response to OVA stimulation by draining lymph node CD4?
T cells. b, IFN-?, IL-4, -5, -10, and -13 secretion in response to OVA
stimulation by splenic CD4?T cells. ND, None detected.
Reduced cytokine production by ITK null T cells following
5060 ROLE OF ITK IN ALLERGIC ASTHMA
T cell activation is dependent on early signals delivered by the
TCR and CD28. We and others have demonstrated that both the
TCR and CD28 activate the Tec family kinase ITK (31–33, 36,
39). ITK activation leads to activation of phospholipase C?1 via an
unknown mechanism, perhaps by tyrosine phosphorylation, lead-
ing to induction of calcium signaling (39). A number of transcrip-
tion factors that regulate cytokine production are regulated by cal-
cium levels, most notable NFAT (48). The lack of ITK (on the
BALB/c background) has been reported to result in significantly
reduced IL-4 production in the absence of a significant effect on
IFN-? secretion, which in vitro can be rescued by increased cal-
cium levels by ionomycin (37). On the genetic background that we
have used in our studies, C57BL/6, the lack of ITK results in a
more generalized cytokine production defect, with decreases in
both IFN-? as well as IL-4 and IL-5 when T cells are polarized in
vitro (35). However, in Ag-specific recall responses to S. mansoni
egg Ag, reduced IL-4 and increased IFN-? was observed in vitro
(35). These published data suggest that although ITK may gener-
ally regulate T cell responses in vitro, in vivo, ITK may preferen-
tially regulate TH2-type responses.
Our finding of much reduced eosinophilic infiltration and mu-
cous production in the lungs of ITK knockout mice may be the
result of a number of factors that seem to be deficient in ITK null
mice. The reduced T cell infiltration in the lung may result in a
reduced overall T cell response to the allergen. However, this ac-
companied an apparently normal B cell/T cell-dependent IgE Ab
response, suggesting that there is a systemic anti-OVA T cell re-
sponse in these mice and either the level of IL-4 made in these
mice is sufficient for efficient class switch or that another source of
IL-4 exists that can regulate this response. The reduced T cell
infiltration may be the result of reduced chemokine and/or chemo-
kine receptor expression in these mice that drive T cell recruitment
to the lung (49) or reduced adhesion of these cells in the lung.
Indeed, ITK has been shown to be involved in TCR-induced ad-
hesion via the ?1integrins (34). Alternatively, the reduced pro-
duction of IL-4 by ITK null T cells may underlie their lack of
significant Ag-specific recruitment to the lung since IL-4 null TH2
cells have been reported to be defective in this process (50). The
production of IL-4 and chemokine and/or chemokine receptor ex-
pression for lung migration may be linked and future experiments
will investigate these possibilities.
The reduced production of IL-5 is another factor that may un-
derlie the reduced eosinophilic infiltration in the ITK null mice.
IL-5 has been proposed as one of the major factors which regulate
eosinophil infiltration in the lung during allergic asthma develop-
ment (51). However, if the recruitment is hampered, then these
cells are less likely to be able to contribute to the pathology of the
disease. A similar situation exists for IL-13, which has been re-
ported to be involved in the production of mucous by goblet cells
lining the lung in allergic asthma (5). The ITK null T cells pro-
duced much less IL-13 than WT cells, and this coupled with the
reduced T cell presence in the lung may underlie the almost com-
plete lack of mucous production in these mice.
We found that IFN-? and IL-10 production by ITK null T cells
in the spleen, while also reduced, was less affected. In the lymph
nodes, IFN-? and IL-10 production was drastically reduced in re-
sponse to Ag-specific stimulation. However, what may be impor-
tant in these mice may not necessarily be the absolute amounts of
cytokine being produced, as they can still mount a reasonable im-
mune response (as witnessed by the production of OVA-specific
IgE), but the ratio of these cytokines to each other. Indeed, al-
though IFN-? can be suppressive in the development of symptoms
in this model, it can also cause increased lung inflammation al-
though not mucous production (52). Viewed this way, ITK null T
cells produce a higher ratio of IFN-?:IL-4, IFN-?:IL-5, and IFN-
?:IL-3, which may have a suppressive effect on the TH2 cytokine-
driven inflammation and accompanying eosinophil infiltration and
mucous production. Similarly, the role of IL-10 in the develop-
ment of allergic asthma has been controversial, with knockout
studies suggesting that IL-10 can either enhance or suppress its
development (53, 54). However, more recent studies have pro-
posed a prominent role for IL-10 in suppressing the development
of allergic asthma, although the source may be T regulatory cells
(55, 56). One possibility is that the ratio of this cytokine in relation
to the others being produced may be a critical factor in regulating
the symptoms seen in this disease.
One seemingly paradoxical finding is the increased levels of
serum IgE found in the ITK null mice. Although IL-4 is critical for
B cells to class switch to IgE production (57) and the ITK null
mice produce less IL-4 than WT mice, they produce almost normal
levels of IL-10 (at least when assayed from splenic T cells). In
human cells, IL-10 has been reported to inhibit IL-4-mediated B
cell class switch to IgE; however, IL-10 can also increase IgE
production by IL-4 and IL-4/CD40-stimulated B cells that have
already class switched to IgE (44). Thus, ITK null mice may have
elevated levels of IgE due to an increase IL-10:IL-4 ratio produced
by ITK null T cells, leading perhaps to reduced class switch to IgE
by B cells, but increased production of IgE by those cells that do
undergo class switch. Alternatively, other cell types such as NK or
NK-T cells, mast cells, dendritic cells, or eosinophils may be able
to produce high levels of IL-4 in ITK null mice, leading to in-
creased IgE production.
Fowell et al (37) have recently shown that T cells from BALB/c
mice lacking ITK are defective in the production of IL-4 and in the
typical TH2-type immune response to Leishmania. They also dem-
onstrated that even in the presence of exogenous IL-4, ITK null T
cells could not secrete IL-4 in vitro, suggesting either that ITK
regulates the ability of these T cells to respond to IL-4 and induce
the necessary chromatin changes for IL-4 production or that ITK
itself regulates those changes (37). Schaeffer et al. (35) however
reported slightly different results, that T cells from C57BL/6 mice
lacking ITK as well as those lacking both ITK and Rlk/Txk have
an overall reduction in TH1 or TH2 cytokine production after TH1
or TH2 condition skewing in vitro. However, in a schistosome egg
challenge model, mice lacking ITK continued to exhibit a TH2-
type defect along with reduced Ag-specific TH2 cytokine produc-
tion in vitro, whereas those lacking both ITK and Rlk/Txk had an
apparently normal TH2-type response in vivo and cytokine pro-
duction in vitro (35). Thus, the in vitro and the in vivo responses
differed in the latter animals; however, the in vivo results suggest
perhaps that the absence of both ITK and Rlk/Txk results in a
default TH2 pathway in vivo for the production of these cytokines
(35). The differences observed by these investigators may be the
result of the different genetic backgrounds used for their studies.
However, although the in vitro differentiation may have led to
slightly different results, in vivo, both models point to a defect in
either TH2 cell differentiation or TH2 cytokine production in mice
A role for ITK in regulating other transcription factors has also
been recently demonstrated by Miller and Berg (58), who recently
showed that ITK null T cells are defective in Fas ligand expression
due to defective up-regulation of transcription factors of the early
growth response family (58). These transcription factors are reg-
ulated by NFAT (48). Thus, the previously published work along
with our data suggest that T cells lacking ITK may not only be
defective in TH2 cell differentiation and/or cytokine production,
but may also be defective in the expression of molecules regulating
T cell migration to the lung. Taken together, these results suggest
5061 The Journal of Immunology
that the tyrosine kinase ITK may represent a good target for de-
veloping drugs to treat allergic asthma.
We are grateful to Dr. Dan Littman for the generous gift of the ITK?/?
mice and Drs. Pamela Correll and Margharita Cantorna (Pennsylvania
State University) for insightful discussions.
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5063The Journal of Immunology