Inhibition of Pim1 Kinase Activation Attenuates
Allergen-Induced Airway Hyperresponsiveness
Yoo Seob Shin1*, Katsuyuki Takeda1*, Yoshiki Shiraishi1, Yi Jia1, Meiqin Wang1, Leila Jackson2,
A. Dale Wright2, Laura Carter2, John Robinson2, Erik Hicken2, and Erwin W. Gelfand1
1Division of Cell Biology, Department of Pediatrics, National Jewish Health, Denver, Colorado; and2Array Biopharma, Boulder, Colorado
Pim kinases are a family of serine/threonine kinases whose activity
can be induced by cytokines involved in allergy and asthma. These
examined, to the best of our knowledge, in the development of
allergic disease. This study sought to determine the role of Pim1
kinase in the development of allergic airway responses. Mice were
sensitized, challenged, and rechallenged with allergen in a second-
ary model. To assess the role of Pim1 kinase, a small molecule
inhibitor was administered orally after sensitization and during the
challenge phase. Airway responsiveness to inhaled methacholine,
airway and lung inflammation, cell composition, and cytokine
concentrations were assessed. Lung Pim1 kinase concentrations
were increased after ovalbumin sensitization and challenge. In the
primary allergen challenge model, treatment with the Pim1 kinase
the development of airway hyperresponsiveness, eosinophilic air-
way inflammation, and goblet cell metaplasia, and increased Th2
manner. These effects were also demonstrated after a secondary
treatment. After treatment with the inhibitor, a significant reduction
was evident in the number of CD41and CD81T cells and concen-
ness, airway inflammation, and cytokine production in allergen-
sensitized and allergen-challenged mice. These data identify the
important role of Pim1 kinase in the full development of allergen-
induced airway responses.
Keywords: airway hyperresponsiveness; inflammation; Pim1 kinase;
Asthma is a complex inflammatory disorder, characterized by
persistent airway inflammation and airway hyperresponsiveness
allergen exposure, infectious pathogens, or chemical agents (1,
2). Several clinical and experimental investigations showed that
T cells, and especially Th2-type cells, play a pivotal role in the
development of AHR and eosinophilic inflammation through
the secretion of a variety of cytokines, including IL-4, IL-5, and
IL-13 (3, 4). These cytokines bind to the extracellular Janus
kinase (JAK) receptors and subsequently induce the phosphor-
ylation and activation of signal transducers and activators of
transcription (STATs), which translocate into the nucleus, where
they bind to DNA and affect basic cell functions, including cell
growth, differentiation, and death (5).
Pim kinases represent a family of three serine/threonine
kinases that control cell survival, proliferation, differentiation,
and apoptosis (6–8). Unlike other serine/threonine kinases, these
are regulated via JAK/STAT activation–driven transcription of
the Pim gene, rather than by membrane recruitment and phos-
phorylation (8). The overexpression of Pim kinase has been dem-
onstrated in various human lymphomas, leukemias, and prostatic
cancers (9). The role of Pim-induced oncogenic transformation
was extensively studied in hematopoietic tumors (10–13). Despite
numerous studies on the role of Pim kinase in the development of
tumor cells, studies exploring the role of these kinases in
immune cells have been limited. Pim1 kinase was expressed in
human eosinophils, and played a major role in the IL-5–induced
survival of eosinophils (14, 15). Furthermore, Pim1 expression was
increased in eosinophils from bronchoalveolar lavage (BAL) fluid,
compared with blood from patients with asthma after an allergen
provocation (16). In a recent study, Pim1 kinase was shown to
promote cell survival in T cells (17).
CD41T cells play a central role in the development of al-
lergic inflammation (18). CD41T cells, especially Th2 cells pro-
ducing IL-4, IL-5, and IL-13, were identified in the BAL fluid
and airway tissue in patients with asthma (4). The transfer of
Th2 cells, followed by airway allergen challenge in mice, was
sufficient to induce airway eosinophilia and AHR (19, 20). Re-
cent studies demonstrated increased numbers of CD81T cells
in the lung tissue of patients with asthma (21). These studies
suggest that not only CD41T cells but also CD81T cells may
be essential in the development of AHR and allergic inflamma-
tion (22–25). Subsets of CD81T cells that produce IL-4, IL-5,
and IL-13, but not IFN-g, labeled as Tc2 cells, are known to
increase AHR and airway inflammation (26–28).
In this study, we determined the role of Pim1 kinase in the
in vivo, using a Pim1 kinase inhibitor. The inhibitor was exam-
ined in two distinct models of allergic airway inflammation.
First, the development of allergen-induced airway responses was
examined in mice that were systemically sensitized to ovalbumin
(OVA), followed by 3 consecutive days of OVA challenge via
the airways (primary allergen challenge). Second, effects on
established airway disease were examined in mice that were
sensitized and challenged with OVA, and when their allergic
airway inflammation had resolved, they were exposed to a single
provocative airway challenge with allergen (secondary allergen
challenge). This condition may more closely resemble allergic
asthma, where subjects are already sensitized and repeatedly ex-
posed to allergen. The results demonstrated that the administration
of Pim1 kinase inhibitor prevented the development of AHR,
airway inflammation, and BAL cytokine production in mice
(Received in original form June 6, 2011 and in final form November 2, 2011)
* These two authors contributed equally to this work.
This study was supported by National Institutes of Health grants HL-36577 and
AI-77609, and by Array Biopharma.
The contents of this work are solely the responsibility of the authors, and do not
necessarily represent the official views of the National Heart, Lung, and Blood
Institute or the National Institutes of Health.
Correspondence and requests for reprints should be addressed to Erwin W. Gelfand,
M.D., Division of Cell Biology, Department of Pediatrics, National Jewish Health,
1400 Jackson Street, Denver, CO 80206. E-mail: email@example.com
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2011-0190OC on November 10, 2011
Internet address: www.atsjournals.org
Vol 46, Iss. 4, pp 488–497, Apr 2012
sensitized and challenged to allergen. The administration of
Pim1 kinase inhibitor also attenuated the consequences of sec-
ondary challenge in previously sensitized and challenged mice.
These suppressive effects were manifested in both CD41and
CD81T cells, and may help identify a novel role for Pim1
kinase in the development of allergen-induced AHR and airway
MATERIALS AND METHODS
Female BALB/c mice, 8–12 weeks of age and free of pathogens, were
purchased from Harlan Laboratory (Indianapolis, IN). The animals
were maintained on an OVA-free diet. Experiments were conducted
under a protocol approved by the Institutional Animal Care and Use
Committee of National Jewish Health.
Sensitization and Challenge with Allergen
Our experimental protocol for the sensitization and primary and sec-
ondary challenges to allergen was described previously (29). Briefly,
in the primary allergen challenge protocol, mice were sensitized by an
intraperitoneal injection of 20 mg OVA (Fisher Scientific, Pittsburgh,
PA), emulsified in 2.0 mg of alum (AlumImuject; Pierce, Rockford, IL)
on Days 1 and 14, followed by aerosolized OVA challenge (1% in
saline for 20 minutes) on Days 28, 29, and 30. Control mice were
sensitized with PBS followed by OVA challenge in the same way. In
the secondary allergen challenge protocol, mice were sensitized with 10
mg OVA with alum on Days 1 and 7, followed by 0.2% OVA challenge
on Days 14–16 (primary allergen challenge). Fourteen days after the
final primary allergen challenge, mice were challenged again with 1%
OVA for 20 minutes (secondary allergen challenge). A group of mice
were sensitized with PBS, followed by primary and secondary chal-
lenges with OVA. In all groups, assays were performed 48 hours after
the final allergen challenge.
Pim Kinase Inhibitor Treatment
To determine the role of Pim1 kinase in the development of allergen-
induced AHR and airway inflammation, we used the Pim1 kinase inhib-
itor, AR00460770 (Array Pharma, Boulder, CO). To characterize
AR00460770 in vitro, the cellular half-maximal inhibitory concentra-
tion (IC50) and kinase selectivity assays were determined. The cellular
IC50of AR00460770 was analyzed by the Ser112 phosphorylation of
transiently transfected Bcl-2–associated death promoter (BAD) in
HEK-293 cell lines, engineered to express human Pim1 and Pim2
(Millipore, Billerica, MA) and rat Pim3 (Array BioPharma, Boulder,
CO) in conjunction with a DNA vector construct directing the ex-
pression of the Pim kinase substrate glutathione S-transferase (GST)-
BAD (pEBG-mBAD). Cells were treated with serial dilutions of
AR00460770 for 1.5 hours, and then labeled with an antibody specific
for phospho-BAD (Ser112) and an antibody against GST (Cell Signal-
ing Technology, Danvers, MA) as a normalization control. Immu-
noreactivity was detected using infrared (IR) fluorophore–conjugated
secondary antibodies, and quantified on an imager (Aerius; Li-Cor,
Lincoln, NE). The kinase selectivity of AR00460770 was evaluated using
the Kinase Profiler service (Millipore) (30–32). The properties and spec-
ificity of the inhibitor are described in Tables 1 and 2.
Western Blot Analysis
Lung tissue was homogenized, and lysates were cleared of debris and
resuspended in an equal volume of 2 3 Laemmli buffer. Lysates were
loaded onto a 4–10% gradient reducing gel, subjected to electrophore-
sis, and transferred to nitrocellulose membranes. The membranes were
blotted with goat anti-Pim1 (Santa Cruz Biotechnology, Santa Cruz,
CA) and rabbit anti-GAPDH (R&D Systems, Minneapolis, MN), anti-
goat IgG (Invitrogen, Carlsbad, CA), and anti-rabbit IgG (Rockland,
Gilbertson, PA). Images were captured and quantitatively analyzed
using an Odyssey infrared imager (Li-Cor).
Assessment of Airway Function
Airwayresponsivenesswas assessed as previouslydescribed,bymeasur-
ing changes in pulmonary resistance (RL) in response to increasing
doses of inhaled methacholine (MCh; Sigma-Aldrich, St. Louis, MO)
in anesthetized and ventilated mice (33). The values of peak airway
responses to inhaled MCh were recorded.
Bronchoalveolar Lavage and Lung Histology
Lungs were lavaged with 1 ml of Hanks’ balanced salt solution through
the trachea immediately after the assessment of AHR. Numbers of
total leukocytes were counted with a hemocytometer, and cell differ-
entiation was performed on cytospin slides prepared with Wright-
Giemsa stain. The numbers of inflammatory and mucus-containing cells
were quantitated as previously described (34).
Measurement of Cytokines
Cytokine concentrations in BAL fluid and cell culture supernatants
were measured by ELISA, as previously described (34).
TABLE 1. CELLULAR HALF-MAXIMAL INHIBITORY
CONCENTRATIONS FOR PIM INHIBITION WERE DETERMINED
FOR AR00460770 (IC50) BY ASSESSING SER112 PHOSPHORYLATION
OF TRANSIENTLY TRANSFECTED BAD IN HEK CELL LINES
ENGINEERED TO EXPRESS PIM1, PIM2, OR PIM3
Pim1 Pim2 Pim3
AR0046077093 9,200 340
Definition of abbreviations: SER, serine.
Cellular half-maximal inhibitory concentration values are reported in nanomole.
TABLE 2. CHARACTERIZATION OF PIM INHIBITOR AR00460770
Kinase AbbreviationEnzyme IC50(nm)
Proto-oncogene serine/threonine-protein kinasePim1
Proline–alanine–rich sterile (STE)20–related kinase
Tyrosine kinase, non–receptor 2
Ca21/calmodulin-dependent protein kinase II–g
FMS-like tyrosine kinase receptor–3
Platelet-derived growth factor receptors
MAP/microtubule affinity–regulating kinase 1
Ca21/calmodulin–dependent protein kinase II–b
59 AMP-activated protein kinase
Ribosomal S6 kinase
Definition of abbreviations: IC50, half-maximal inhibitory concentration; MAP, mitogen-activated protein.
AR00460770 was tested at 10 mM against 230 kinases in enzymatic assays (Millipore Kinase Profiler). It was deter-
mined to be selective for the three Pim isoforms.
Shin, Takeda, Shiraishi, et al.: Pim1 Kinase Activation in Development of AHR489
Isolation of Lung Mononuclear Cells and Flow Cytometry
Lung mononuclear cells (MNCs) were isolated as described previously
using collagenase digestion, and their cellular composition was identi-
fied as described elsewhere (35).
CD41and CD81T Cell Purification and Cell
The purification of CD41and CD81T cells was conducted as previ-
ously described (24). The purity of CD41and CD81T-cell populations
exceeded 95%, as assessed by flow cytometry.
In cell proliferation assays, an anti-mouse CD3e monoclonal anti-
body (mAb) (5 mg/ml; R&D Systems) was immobilized on 96-well
flat-bottom plates overnight at 48C. Purified CD41and CD81T cells
were incubated the with inhibitor or PBS as vehicle (2 3 105cells/well),
and anti-CD28 mAb (5 mg/ml; R&D Systems) was added to the anti-
CD3–precoated plates and incubated at 378C for 24 hours. After 24
hours, 1 mCi tritium-labeled thymidine per well (Perkin-Elmer, Boston,
MA) was added to 96-well plates for 6 hours and harvested with distilled
water, followed by counting in a microplate scintillation and lumines-
cence counter (Packard, Meriden, CT). The cell viability of CD41and
CD81T cells was assessed 24 hours after incubation with 10 mM of
inhibitor by a vital stain with trypan blue, and determined using an
automated cell counter (Countess; Invitrogen).
Results were expressed as means 6 SEM. The t test was used to de-
termine differences between the two groups. For comparisons between
multiple groups, the Tukey-Kramer test was used. Nonparametric anal-
yses, using the Mann-Whitney U test or Kruskal-Wallis test, were also
applied to confirm that statistical differences remained significant, even
if the underlying distribution was uncertain. Differences were regarded
as statistically significant when P , 0.05.
In Vitro Characterization of AR00460770
The cellular IC50and kinase selectivity of AR00460770 were
determined and, as shown in Tables 1 and 2, exhibited strong
inhibition specific to Pim1 kinase.
Lung Pim1 Kinase Concentrations Are Increased after
Sensitization and Challenge with Allergen
To determine the importance of Pim1 kinase after allergen chal-
lenge, we evaluated protein expression levels of the kinase in
lung tissue after the OVA challenge of sensitized mice. Pim1 ex-
pression levels in OVA-sensitized mice were markedly increased
after OVA challenge compared with levels in nonsensitized,
challenged-only mice. This up-regulation was detected in OVA-
sensitized mice 6 hours after their second OVA challenge, and
remained high up to 24 hours after the third OVA challenge
Pim1 Kinase Inhibitor Treatment Prevents Development of
AHR and Airway Inflammation after Primary
To determine the effects of Pim1 kinase inhibitor treatment on
allergen-induced airway inflammation and AHR, mice were
treated with the inhibitor or vehicle during the OVA challenge
phase in the primary allergen challenge model. As shown in
Figures 2A and 2B, vehicle-treated mice developed greater
airway responses to MCh and eosinophil numbers in BAL
fluid after sensitization and challenge with OVA, compared
with sham-sensitized, OVA-challenged mice. Mice treated
with the Pim1 inhibitor at doses of 10, 30, or 100 mg/kg devel-
oped significantly lower airway responsiveness to inhaled MCh
and lower BAL eosinophil numbers, compared with the
vehicle-treated group. Sham-sensitized but OVA-challenged
mice were treated with 100 mg/kg of the inhibitor to assess
its potential effects on smooth muscle contraction. Treatment
with the inhibitor in this way did not alter the development
of increasing RL with increasing concentrations of inhaled
As shown in Figure 2C, the inhibitor treatment of sensitized
and challenged mice reduced the concentrations of IL-4, IL-5,
IL-13, and IFN-g in BAL fluid in a dose-dependent manner.
Figure 1. Expression levels of Pim1 kinase in lungs after
sensitization and challenge with ovalbumin (OVA). Pim1
kinase concentrations were determined by Western blot
analyses in lungs of mice that were sensitized and chal-
lenged with OVA, or that received sham sensitization and
OVA challenge. Expression levels were examined at three
time points: 6 hours after the second OVA challenge, 6
hours after the third OVA challenge, and 24 hours after
third OVA challenge. Experiments were repeated at least
three times. Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was used as a loading control (A), and the aver-
age optical densitometry was expressed by standardizing
to GAPDH (B). *P , 0.05 compared with the sham-sensi-
490 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 46 2012
Significant changes were evident primarily at the highest admin-
istered dose of the inhibitor (100 mg/kg).
Histopathological analyses of lung-tissue sections revealed
that the numbers of inflammatory cells, including eosinophils
in peribronchial and perivascular areas, were increased in mice
after OVA sensitization and challenge, compared with sham-
sensitized and challenged mice (Figure 2D). Similarly, the numbers
of periodic acid–Schiff–positive (PAS1) mucus-containing goblet
cells were increased in the sensitized and challenged mice (Figure
2E). The administration of inhibitor significantly decreased the
numbers of inflammatory cells and PAS1mucus-containing goblet
cells in lung tissue in a dose-dependent manner (Figure 2E).
Inhibition of Pim1 Kinase Attenuates the Development of
AHR and Airway Inflammation in the Secondary Allergen
The airway responses in the primary allergen challenge model
reflect the first immune responses in the lungs, where adaptive
immunity is initiated in response to airborne allergen exposure.
For the most part, patients with asthma have already developed
allergic airway inflammation and airway dysfunction before the
initiation of treatment. Immune responses to allergen and tissue
remodeling of the airways are generally already established. The
secondary allergen challenge model is an approach to examine
the response to allergen provocation where allergen-induced air-
way inflammation is previously established. To determine the
effects of Pim1 kinase inhibition in the secondary allergen chal-
lenge model, we measured AHR, cell composition, and cytokine
concentrations in BAL fluid, 48 hours after a single provocative
allergen challenge. As in the primary allergen challenge model,
vehicle-treated mice developed significantly higher airway re-
sponsiveness to MCh and eosinophils in BAL fluid after OVA
sensitization and secondary allergen challenge. Similar to the
results observed in the primary allergen challenge model, treat-
ment with the Pim1 kinase inhibitor (at 10, 30, and 100 mg/kg)
significantly decreased levels of airway responsiveness and the
compared with vehicle-treated groups (Figures 3A and 3B).
Moreover, a significant reduction in numbers of lymphocytes
and neutrophils was evident at the higher doses of inhibitor.
Assays of BAL cytokine concentrations demonstrated that
IL-4, IL-5, IL-13, and IFN-g were decreased in Pim1 kinase
inhibitor (100 mg/kg)–treated mice that had been sensitized
and challenged with OVA (Figure 3C). Histopathological anal-
yses revealed that Pim1 kinase inhibition decreased numbers of
inflammatory cell in the lungs and goblet cell metaplasia along
the airways (Figure 3D).
Decrease of CD41and CD81T Cells in the Lungs of Sensitized
and Challenged Mice after Treatment with the Pim1
Because both CD41and CD81T cells are potent effector cells in
the development of allergic inflammation, their numbers were
examined after treatment with inhibitor in sensitized and chal-
lenged mice. Lungs from OVA-sensitized and OVA-challenged
mice that received either inhibitor or vehicle were excised, and
lung MNCs were purified. Numbers of CD41and CD81T cells
were determined by flow cytometry. As shown in Figure 4, the
overall number of CD41T cells was significantly lower in the
Figure 2. Effect of Pim1 kinase inhibition on airway responses after
primary allergen challenge. The effects of the Pim1 kinase inhibitor
were determined in the primary allergen challenge model. (A) Changes
in pulmonary resistance (RL) in response to increasing doses of meth-
acholine (MCh). (B) Cell composition in bronchoalveolar lavage (BAL)
fluid. Macro, macrophages; Lympho, lymphocytes; Eos, eosinophils;
Neu, neutrophils. (C) BAL fluid cytokine concentrations. (D) Lung tissue
histology after staining with hematoxylin and eosin (H&E). (E) Lung
tissue histology after staining with periodic acid–Schiff (PAS). Quanti-
tative analyses of inflammatory and PAS1cells in lung tissue were
performed as described in MATERIALS AND METHODS. Mice were sham-
sensitized, followed by OVA challenge (PBS/OVA), or sensitized and
challenged with OVA (OVA/OVA). The Pim1 inhibitor, AR00460770,
was administered at doses of 1, 10, 30, or 100 mg/kg.#P , 0.05,
compared with PBS/OVA vehicle. **P , 0.05, compared with OVA/OVA
AR00460770 at 1 mg/kg.
AR00460770 at 30 mg/kg.
##P , 0.05, compared with PBS/OVA
Shin, Takeda, Shiraishi, et al.: Pim1 Kinase Activation in Development of AHR 491
inhibitor-treated mice (1.48 6 0.26 3 106cells/lung, versus 3.09 6
0.35 3 106cells/lung in vehicle-treated mice). CD81T cells were
also significantly decreased after Pim1 kinase inhibition, from
0.57 6 0.21 3 106cells/lung to 0.29 6 0.06 3 106cells/lung. These
results demonstrate that Pim1 kinase inhibition in vivo reduces
the numbers of CD41and CD81T cells that accumulate in the
lungs of sensitized and challenged mice.
Reduction of CD41and CD81T-Cell Proliferation and
Cytokine Production In Vitro after Pim1 Kinase
To examine the proliferative capacity of T cells after the inhibi-
tion of Pim1 kinase, CD4 and CD8 T cells were isolated and
purified from spleens and incubated with a combination of anti-
CD3 and anti-CD28 for 24 hours. The cell viabilities of CD41
or CD81T cells were determined in the presence of 10 mM of
the inhibitor. After 24 hours, inhibitor treatment did not show
significant effects on cell viabilities compared with vehicle con-
trol samples (from 90.0–90.3% in CD41T cells, and from 80.2–
82.8% in CD81T cells, respectively). In a dose-dependent man-
ner, the Pim1 kinase inhibitor reduced the CD41and CD81
T-cell proliferation triggered by the combination of anti-CD3
and anti-CD28. In stimulated cell cultures, increased concentra-
tions of IL-4, IL-5, IL-13, and IFN-g were detected. Treatment
with the inhibitor decreased the concentrations of all of these
cytokines in a dose-dependent fashion (Figure 5).
In these initial studies using an experimental model of asthma,
we demonstrated that concentrations of Pim1 kinase were ele-
compared with concentrations in the lungs of nonsensitized,
challenged-only mice. Pim kinases are among the few to be
up-regulated during cell activation through increased transcrip-
tion (8). There are three subtypes of Pim kinases. Pim1 and
Pim2 are primarily restricted to hematopoietic cells, and Pim3
is expressed in brain, kidney, and mammary tissue (36). To date,
only a single study demonstrated that the expression of Pim1
was induced in human immune cells, and that was in eosinophils
from either whole blood or BAL fluid after stimulation with
IL-5 (16). Concentrations of Pim1 kinase in BAL eosinophils,
compared with blood eosinophils, were further increased after
segmental allergen challenge in patients with asthma (16). Link-
ing the increases in Pim1 kinase concentrations in sensitized and
challenged mice to function, we demonstrated that sensitized
and challenged mice treated with a Pim1 kinase inhibitor developed
significantly lower levels of AHR, cytokines, eosinophilic airway
inflammation, lung inflammatory cell accumulation, and goblet
cell metaplasia. The effects of the kinase inhibitor in vivo on the
development of AHR, BAL eosinophilia, airway inflammation,
and goblet cell metaplasia were seen at doses (10–30 mg/kg)
that, based on the cellular activity of AR460770 (Table 1), were
specific to Pim1 kinase inhibition. The suppressive effects asso-
ciated with Pim1 kinase inhibition were observed in both the
Figure 2. (Continued)
492AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 46 2012
primary and secondary allergen challenge protocols. The pri-
mary challenge model reflects the initial immune responses of
the airways to airborne allergen exposure, and best defines the
development of lung allergic responses. In contrast, the second-
ary allergen challenge model detects airway secondary immune
responses in lungs on a background of previously established
Figure 3. Effects of Pim1 kinase inhibition on airway responses in the secondary allergen challenge model. The effects of Pim1 kinase inhibition were
determined in the secondary allergen challenge model. (A) Changes in RLin response to increased doses of MCh. (B) Cell composition in BAL fluid.
(C) BAL fluid cytokine concentrations. (D) Lung tissue histology after staining with H&E. (E) Lung tissue histology after staining with PAS. Quan-
titative analyses of inflammatory and goblet cells were performed as described in MATERIALS AND METHODS. Mice were sham-sensitized, followed
by OVA challenge (PBS/OVA), or sensitized and challenged with OVA (OVA/OVA). The Pim1 inhibitor was administered at doses of 1, 10, 30,
or 100 mg/kg. Control groups received vehicle (n ¼ 8). *P , 0.05, compared with OVA/OVA vehicle or OVA/OVA AR00460770 at 1 mg/kg.
#P , 0.05, compared with OVA/OVA AR00460770 10 mg/kg. **P , 0.05, compared with OVA/OVA vehicle or OVA/OVA AR00460770 at 1 mg/kg.
Shin, Takeda, Shiraishi, et al.: Pim1 Kinase Activation in Development of AHR493
allergic airway inflammation. In both models, the reduction of
eosinophils in the BAL appeared at lower doses of inhibitor,
and these doses did not alter BAL cytokine production levels.
Together with the data on human eosinophils (16), our findings
suggest that the inhibition of Pim1 kinase directly affects eosin-
ophil function. The comparable suppressive effects of Pim1 ki-
nase inhibition after both primary and secondary allergen
challenge imply that the activation of this kinase plays a critical
role in both the initiation and recall responses induced by aller-
gen challenges of sensitized mice.
Pim1 kinase controls cellular survival, and the inhibition of
this kinase can cause cell death. The antibody targeting of
Pim kinase synergistically enhanced the cytotoxicity of antican-
cer agents, and the inhibition of Pim1 kinase triggered the death
of leukemic cells (37, 38). To address the possibility that the
suppressive effects of Pim1 kinase inhibition on allergen-
induced airway responses were attributable to its toxic effects
on immune cells, an evaluation of cell viability was performed.
At concentrations up to 10 mM, the inhibitor had no effect
on cell number or viability, suggesting its effects on airway
responses were not attributable to drug-mediated cellular tox-
icity. These findings are similar to those described in Pim-
deficient mice, in which the rate of apoptosis of T cells was
not different than in wild-type mice (36). Furthermore, treat-
ment with Pim1 kinase inhibitor in sham-sensitized but OVA-
challenged mice did not alter airway responsiveness to MCh,
indicating that the inhibitor less likely exhibited toxic effects on
lung resident cells, including airway smooth muscle. Microarray
data indicate that Pim1 is expressed in airway epithelial cells,
indicating the potential for additional suppressive effects of the
inhibitor on airway epithelial cell function, as well as eosinophil
and T-cell function in the development of AHR and allergic
airway inflammation (39). Although the specificity of the inhib-
itor was demonstrable in vitro, the effects of the inhibitor on
other cells cannot be completely ruled out at this time.
The pathogenesis of asthma is complex, and the role of Th2
CD41T cells secreting proallergic cytokines such as IL-4, IL-5, and
Figure 4. The effects of Pim kinase inhibition on numbers of CD41and
CD81T cells. In OVA-sensitized and OVA-challenged mice, the num-
bers of CD41T cells (CD4) and CD81T cells (CD8) in the lungs of mice
treated with the Pim1 kinase inhibitor (OVA/OVA AR00460770) or ve-
hicle (OVA/OVA vehicle) were determined. Mononuclear cells isolated
from lungs were stained with anti-CD3, anti-CD4, and anti-CD8 for
flow cytometry analysis, as described in MATERIALSAND METHODS. The data
shown are representative of three independent experiments. *P , 0.05,
compared with vehicle.
Figure 5. Effects of Pim1 kinase inhibition on cell proliferative responses
and cytokine production from CD41and CD81T cells. Purified spleen
CD41and CD81T cells were preincubated with the Pim1 kinase inhibitor,
followed by anti-CD3 and anti-CD28 stimulation. (A) Cell proliferation
assays were performed 24 hours after anti-CD3/anti-CD28 stimulation,
and calculated from the uptake of tritium-labeled thymidine (n ¼ 8).
CPM, counts per minute. (B) Quantitation of cytokine concentrations in
supernates from anti-CD3/anti-CD28–stimulated CD41cells. (C) Quanti-
tation of cytokine concentrations in supernates from anti-CD3/anti-CD28–
stimulated CD81T cells. CPM, counts per minute. *P , 0.05, compared
with vehicle-treated cells.
494 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 462012
IL-13 has been emphasized (1, 3, 19). The role of IFN-g may be
more complex. In several studies, IFN-g was shown to be a po-
tent suppressor of allergen-induced AHR, lung pathology, and
Th2 cytokine production (40–42). In other studies, IFN-g was
shown to be essential in the development of AHR (43), and the
administration of IFN-g was shown to be ineffective in prevent-
ing AHR or airway eosinophilia (40). In patients with asthma,
elevations of IFN-g were shown to correlate with asthma sever-
ity (44) and bronchial hyperresponsiveness (45). Given the dis-
tribution of Pim1 kinase, several points of action likely explain
the attenuation of AHR, BAL cytokine concentrations, and
lung inflammation in primary or secondary allergen challenge
models. Effector CD41and CD81T cells play a role in the
pathogenesis of asthma through cell proliferation to specific
allergens, followed by prolonged survival (25, 45). T cells are
also required for the development or initiation of airway eosin-
ophilia and goblet cell metaplasia through the release of specific
cytokines (46, 47). The inhibition of Pim1 kinase altered the
activities of these effector cells in the airways, because both
the numbers of CD41and CD81T cells in the lungs and
BAL cytokine concentrations were decreased in inhibitor-
treated, sensitized, and challenged mice. Further, according to
in vitro experiments, inhibitor treatment demonstrated suppres-
sive effects on the proliferation of CD41and CD81T cells
induced in response to stimulation with anti-CD3 and anti-
CD28. In parallel, concentrations of cytokines, IL-4, IL-5, IL-
13, and IFN-g, in stimulated cell culture supernates were also
reduced in a dose-dependent manner. Of interest here, Pim-
deficient T cells were shown to display normal proliferative
responses to high concentrations of anti-CD3 and IL-2, but
Pim was required for normal proliferation at lower levels of
the stimuli (36). Together, the results suggest that under exper-
imental conditions in vivo and in vitro, the inhibition of Pim1
kinase limits responses through interference with the expansion
and activities of critical effector cells, CD41and CD81T cells
in the airways, and possibly eosinophils, as reported elsewhere
(18–25, 48, 49).
to regulate common substrates such as BAD or 4EBP1 (50), the
downstream activities of individual Pim kinases may be differ-
ent. Targeting Pim1 kinase may account for effects on allergic
lung responses in other ways. The suppressor of cytokine
signaling-1, c-myc, Pim-associated protein-1, protein tyrosine
phosphatase U2S, and heterochromatin protein 1 are all poten-
tial downstream targets of Pim1 kinase (51–56). Recently, the
nuclear factor of activated T cells (NFAT) was reported to be
a potential downstream substrate of Pim1 kinase (57). The reg-
ulation of NFAT activity was shown to be important for the
normal selection of thymocytes and may play a role in the func-
tional development of T cells. The down-regulation of NFAT
may play a role in the suppression of both CD41and CD81
T-cell proliferation and T-cell cytokine production as a down-
stream substrate of the kinase.
Asthma is a chronic airway inflammatory disease, as demon-
strated by the infiltration and proliferation of inflammatory cells
in the airways (48, 59). As a result, interference with inflamma-
tory cell accumulation in the airways and cell expansion repre-
sent potential strategies in the treatment of inflammatory
diseases such as asthma. Despite the successful introduction of
anti-inflammatory drugs and immunomodulators in the treat-
ment of autoimmune diseases, few have produced similar ben-
efits in asthma (60, 61). This finding may suggest that the
inflammatory pathways in asthma differ from those in other
diseases, and novel strategies are required. To date, compelling
evidence for the involvement of Pim1 kinase in the develop-
ment and progression of several different diseases has made it
a potential pharmaceutical target. Limited numbers of Pim ki-
nase inhibitors have been available for study. One recent study
using a Pim1 kinase inhibitor demonstrated therapeutic effects
in chronic lymphocytic leukemia (37). The data reported here
demonstrate for the first time, to the best of our knowledge, that
targeting Pim1 kinase effectively reduces the development of
the full spectrum of allergen-induced lung inflammatory responses,
at least in part through limiting the expansion and activities of
effector CD41and CD81T cells. As such, the inhibition of
Pim1 kinase represents a novel therapeutic target in the treat-
ment of asthma.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgments: The assistance of Ms. Diana Nabighian in the preparation of
the manuscript is gratefully acknowledged.
1. Busse WW, Lemanske RF Jr. , Asthma. N Engl J Med 2001;344:350–362.
2. Umetsu DT, McIntire JJ, Akbari O, Macaubas C, DeKruyff RH.
Asthma: an epidemic of dysregulated immunity. Nat Immunol 2002;3:
3. Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL,
Donaldson DD. Interleukin-13: central mediator of allergic asthma.
4. Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM,
Corrigan C, Durham SR, Kay AB. Predominant Th2-like bron-
choalveolar T-lymphocyte population in atopic asthma. N Engl J Med
5. Aaronson DS, Horvath CM. A road map for those who don’t know
JAK-STAT. Science 2002;296:1653–1655.
6. Bachmann M, Moroy T. The serine/threonine kinase Pim-1. Int J Bio-
chem Cell Biol 2005;37:726–730.
7. Wang Z, Bhattacharya N, Weaver M, Petersen K, Meyer M, Gapter L,
Magnuson NS. Pim-1: a serine/threonine kinase with a role in cell
survival, proliferation, differentiation and tumorigenesis. J Vet Sci
8. Amaravadi R, Thompson CB. The survival kinases Akt and Pim as
potential pharmacological targets. J Clin Invest 2005;115:2618–2624.
9. Nawijn MC, Alendar A, Berns A. For better or for worse: the role of
Pim oncogenes in tumorigenesis. Nat Rev Cancer. 2011;11:23–34.
10. Amson R, Sigaux F, Przedborski S, Flandrin G, Givol D, Telerman A.
The human protooncogene product p33Pim is expressed during fetal
hematopoiesis and in diverse leukemias. Proc. Natl. Acad. Sci. USA
11. Valdman A, Fang X, Pang ST, Ekman P, Egevad L. Pim-1 expression in
prostatic intraepithelial neoplasia and human prostate cancer. Pros-
12. Cibull TL, Jones TD, Li L, Eble JN, Ann Baldridge L, Malott SR, Luo
Y, Cheng L. Overexpression of Pim-1 during progression of prostatic
adenocarcinoma. J Clin Pathol 2006;59:285–288.
13. Nieborowska-Skorska M, Hoser G, Kossev P, Wasik MA, Skorski T.
Complementary functions of the antiapoptotic protein A1 and serine/
threonine kinase Pim-1 in the BCR/ABL-mediated leukemogenesis.
14. Temple R, Allen E, Fordham J, Phipps S, Schneider HC, Lindauer K,
Hayes I, Lockey J, Pollock K, Jupp R. Microarray analysis of eosi-
nophils reveals a number of candidate survival and apoptosis genes.
Am J Respir Cell Mol Biol 2001;25:425–433.
15. Andina N, Didichenko S, Schmidt-Mende J, Dahinden CA, Simon HU.
Proviral integration site for Moloney murine leukemia virus 1, but not
phosphatidylinositol-3 kinase, is essential in the antiapoptotic signal-
ing cascade initiated by IL-5 in eosinophils. J Allergy Clin Immunol
16. Stout BA, Bates ME, Liu LY, Farrington NN, Bertics PJ. IL-5 and
granulocyte–macrophage colony–stimulating factor activate STAT3
and STAT5 and promote Pim-1 and cyclin D3 protein expression in
human eosinophils. J Immunol 2004;173:6409–6417.
17. Fox CJ, Hammerman PS, Thompson CB. The Pim kinases control
rapamycin-resistant T cell survival and activation. J Exp Med 2005;
Shin, Takeda, Shiraishi, et al.: Pim1 Kinase Activation in Development of AHR495
18. Busse WW, Coffman RL, Gelfand EW, Kay AB, Rosenwasser LJ.
Mechanisms of persistent airway inflammation in asthma. A role for
T cells and T-cell products. Am J Respir Crit Care Med 1995;152:388–
19. Cohn L, Homer RJ, Marinov A, Rankin J, Bottomly K. Induction of
airway mucus production by T helper 2 (Th2) cells: a critical role for
interleukin 4 in cell recruitment but not mucus production. J Exp Med
20. Hogan SP, Matthaei KI, Young JM, Koskinen A, Young IG, Foster PS.
A novel T cell–regulated mechanism modulating allergen-induced
airways hyperreactivity in BALB/c mice independently of IL-4
and IL-5. J Immunol 1998;161:1501–1509.
21. Azzawi M, Bradley B, Jeffery PK, Frew AJ, Wardlaw AJ, Knowles G,
Assoufi B, Collins JV, Durham S, Kay AB. Identification of activated
T lymphocytes and eosinophils in bronchial biopsies in stable atopic
asthma. Am Rev Respir Dis 1990;142:1407–1413.
22. Hamelmann E, Oshiba A, Paluh J, Bradley K, Loader J, Potter TA,
Larsen GL, Gelfand EW. Requirement for CD81T cells in the de-
velopment of airway hyperresponsiveness in a marine model of air-
way sensitization. J Exp Med 1996;183:1719–1729.
23. Isogai S, Taha R, Tamaoka M, Yoshizawa Y, Hamid Q, Martin JG.
CD81alphabeta T cells can mediate late airway responses and airway
eosinophilia in rats. J Allergy Clin Immunol 2004;114:1345–1352.
24. Miyahara N, Takeda K, Kodama T, Joetham A, Taube C, Park JW,
Miyahara S, Balhorn A, Dakhama A, Gelfand EW. Contribution of
antigen-primed CD81T cells to the development of airway hyper-
responsiveness and inflammation is associated with IL-13. J Immunol
25. Miyahara N, Swanson BJ, Takeda K, Taube C, Miyahara S, Kodama T,
Dakhama A, Ott VL, Gelfand EW. 2004. Effector CD81T cells me-
diate inflammation and airway hyper-responsiveness. Nat Med 10:865–869.
26. Croft M, Carter L, Swain SL, Dutton RW. Generation of polarized
antigen-specific CD8 effector populations: reciprocal action of inter-
leukin (IL)–4 and IL-12 in promoting Type 2 versus Type 1
cytokine profiles. J Exp Med 1994;180:1715–1728.
27. Seder RA, Boulay JL, Finkelman F, Barbier S, Ben-Sasson SZ, Le Gros
G, Paul WE. CD81T cells can be primed in vitro to produce IL-4.
J Immunol 1992;148:1652–1656.
28. Coyle AJ, Erard F, Bertrand C, Walti S, Pircher H, Le Gros G. Virus-
specific CD81cells can switch to interleukin 5 production and induce
airway eosinophilia. J Exp Med 1995;181:1229–1233.
29. Takeda K, Miyahara N, Kodama T, Taube C, Balhorn A, Dakhama
A, Kitamura K, Hirano A, Tanimoto M, Gelfand EW. S-
carboxymethylcysteine normalises airway responsiveness in
sensitized and challenged mice. Eur Respir J 2005;26:577–585.
30. Lo ´pez-Ramos M, Prudent R, Moucadel V, Sautel CF, Barette C, Lafa-
neche `re L, Mouawad L, Grierson D, Schmidt F, Florent JC, et al. New
potent dual inhibitors of CK2 and Pim kinases: discovery and struc-
tural insights. FASEB J 2010;24:3171–3185.
31. Yan Bin , Zemskova M, Holder S, Chin V, Kraft A, Koskinen PJ, Lilly
M. The Pim-2 kinase phosphorylates BAD on serine 112 and reverses
BAD-induced cell death. J Biol Chem 2003;278:45358–45367.
32. Fox CJ, Hammerman PS, Cinalli RM, Master SR, Chodosh LA,
Thompson CB. The serine/threonine kinase Pim-2 is a transcription-
ally regulated apoptotic inhibitor. Genes Dev 2003;17:1841–1854.
33. Takeda K, Shiraishi Y, Matsubara S, Miyahara N, Matsuda H, Okamoto
M, Joetham A, Gelfand EW. Effects of combination therapy with
montelukast and carbocysteine in allergen-induced airway hyper-
responsiveness and airway inflammation. Br J Pharmacol 2010;160:
34. Tomkinson A, Cieslewicz G, Duez C, Larson KA, Lee JJ, Gelfand EW.
Temporal association between airway hyperresponsiveness and air-
way eosinophilia in ovalbumin-sensitized mice. Am J Respir Crit Care
35. Oshiba A, Hamelmann E, Takeda K, Bradley KL, Loader JE, Larsen
GL, Gelfand EW. Passive transfer of immediate hypersensitivity and
airway hyperresponsiveness by allergen-specific immunoglobulin (Ig)
E and IgG1 in mice. J Clin Invest 1996;97:1398–1408.
36. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J,
Berns A. Mice deficient for all Pim kinases display reduced body size
and impaired responses to hematopoietic growth factors. Mol Cell
37. Chen LS, Redkar S, Bearss D, Wierda WG, Gandhi V. Pim kinase in-
leukemia cells. Blood 2009;114:4150–4157.
38. Hu XF, Li J, Vandervalk S, Wang Z, Magnuson NS, Xing PX. Pim-1–
specific mAb suppresses human and mouse tumor growth by de-
creasing Pim-1 levels, reducing Akt phosphorylation, and activating
apoptosis. J Clin Invest 2009;119:362–375.
39. Kicic A, Hallstrand TS, Sutanto EN, Stevens PT, Kobor MS, Taplin C,
Pare ´ PD, Beyer RP, Stick SM, Knight DA. Decreased fibronectin
production significantly contributes to dysregulated repair of asth-
matic epithelium. Am J Respir Crit Care Med. 2010;181:889–98.
40. Lack G, Renz H, Saloga J, Bradley KL, Loader JE, Leung DYM, Larsen
GL, Gelfand EW. Nebulized but not parenteral IFN-g decreases IgE
production and normalizes airway function in a murine model of al-
lergen sensitization. J Immunol 1994;152:25446–25454.
41. Flaishon L, Topilaki I, Shoseyov D, Hershkoviz R, Fireman E, Levo Y,
Marmor S, Shachar I. Anti-inflammatory properties of low levels of
IFN-g. J Immunol 2002;168:3707–3711.
42. Yoshida M, Leigh R, Matsumoto K, Wattie J, Ellis R, O’Byrne PM,
Inman MD. Effect of interferon-g on allergic airway responses in
interferon-gamma–deficient mice. Amer J Resp Crit Care Med 2002;
43. Hessel EM, Van Oosterhout AJ, Van Ark I, Van Esch B, Hofman G,
Van Loveren H, Savelkoul HF, Nijkamp FP. Development of airway
hyperresponsiveness is dependent on interferon-gamma and inde-
pendent of eosinophil infiltration. Amer J Resp Cell Molec Biol 1997;
44. ten Hacken NHT, Oosterhoff Y, Kauffman HF, Guevarra L, Satoh T,
Tollerud DJ, Postma DS. Elevated serum interferon-g in atopic
asthma correlates with increased airways responsiveness and circa-
dian peak expiratory flow variation. Eur Resp J 1998;11:312–316.
45. Heaton T, Rowe J, Turner S, Aalberse RC, de Klerk N, Suriyaarachchi
D, Serralha M, Holt BJ, Hollams E, Yerkovich S, et al. An immu-
noepidemiological approach to asthma: identification of in-vitro T cell
response patterns associated with different wheezing phenotypes in
children. Lancet 2005;365:142–149.
46. Lopez AF, Sanderson CJ, Gamble JR, Campbell HD, Young IG, Vadas
MA. Recombinant human interleukin 5 is a selective activator of
human eosinophil function. J Exp Med 1988;167:219–224.
47. Laoukili J, Perret E, Willems T, Minty A, Parthoens E, Houcine O,
Coste A, Jorissen M, Marano F, Caput D, et al. IL-13 alters muco-
ciliary differentiation and ciliary beating of human respiratory
epithelial cells. J Clin Invest 2001;108:1817–1824.
48. Cohn L, Tepper JS, Bottomly K. IL-4–independent induction of airway
hyperresponsiveness by Th2, but not Th1, cells. J Immunol 1998;161:
49. Rosenberg HF, Phipps S, Foster PS. Eosinophil trafficking in allergy and
asthma. J Allergy Clin Immunol 2007;119:1303–1310.
50. Blanco-Aparicio C, Collazo AM, Oyarzabal J, Leal JF, Albaran MI,
Lima FR, Pequeno B, Ajenjo N, Becerra M, Alfonso P, et al. Pim 1
kinase inhibitor ETP-45299 suppresses cellular proliferation and syn-
ergizes with PI3K inhibition. Cancer Lett 2011;300:145–153, 2011.
51. van Lohuizen M, Verbeek S, Krimpenfort P, Domen J, Saris C,
Radaszkiewicz T, Berns A. Predisposition to lymphomagenesis in
Pim-1 transgenic mice: cooperation with c-myc and N-myc in murine
leukemia virus–induced tumors. Cell 1989;56:673–682.
52. Yan B, Zemskova M, Holder S, Chin V, Kraft A, Koskinen PJ, Lilly M.
The Pim-2 kinase phosphorylates BAD on serine 112 and reverses
BAD-induced cell death. J Biol Chem 2003;278:45358–45367.
53. Chen XP, Losman JA, Cowan S, Donahue E, Fay S, Vuong BQ, Nawijn
MC, Capece D, Cohan VL, Rothman P. Pim serine/threonine kinases
regulate the stability of SOCS-1 protein. Proc. Natl. Acad. Sci. USA
54. Maita H, Harada Y, Nagakubo D, Kitaura H, Ikeda M, Tamai K, Taka-
hashi K, Ariga H, Iguchi-Ariga SM. PAP-1, a novel target protein of
phosphorylation by Pim-1 kinase. Eur J Biochem 2000;267:5168–5178.
55. Koike N, Maita H, Taira T, Ariga H, Iguchi-Ariga SM. Identification of
heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-
1 kinase and the effect of phosphorylation on the transcriptional re-
pression function of HP1(1). FEBS Lett 2000;467:17–21.
56. Wang Z, Bhattacharya N, Meyer MK, Seimiya H, Tsuruo T, Tonani JA,
Magnuson NS. Pim-1 negatively regulates the activity of PTP-U2S
apoptosis inchronic lymphocytic
496 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 462012
phosphatase and influences terminal differentiation and apoptosis of Download full-text
monoblastoid leukemia cells. Arch Biochem Biophys 2001;390:9–18.
57. Rainio EM, Sandholm J, Koskinen PJ. Cutting edge: transcriptional
activity of NFATc1 is enhanced by the Pim-1 kinase. J Immunol 2002;
58. Patra AK, Drewes T, Engelmann S, Chuvpilo S, Kishi H, Hunig T,
Serfling E, Bommhardt UH. PKB rescues calcineurin/NFAT-induced
arrest of Rag expression and pre-T cell differentiation. J Immunol
59. Larche M, Robinson DS, Kay AB. The role of T lymphocytes in the
pathogenesis of asthma. J Allergy Clin Immunol 2003;111:450–463.
60. Mullarkey MF, Blumenstein BA, Andrade WP, Bailey GA, Olason I,
Wetzel CE. Methotrexate in the treatment of corticosteroid-
dependent asthma: a double-blind crossover study. N Engl J Med
61. Alexander AG, Barnes NC, Kay AB. Trial of cyclosporin in
corticosteroid-dependent chronic severe asthma. Lancet 1992;339:
Shin, Takeda, Shiraishi, et al.: Pim1 Kinase Activation in Development of AHR497