Long-term exposure of chemokine CXCL10 causes bronchiolitis-like inflammation.
ABSTRACT Chemokines and chemokine receptors have been implicated in the pathogenesis of bronchiolitis. CXCR3 ligands (CXCL10, CXCL9, and CXCL11) were elevated in patients with bronchiolitis obliterans syndrome (BOS) and chronic allorejection. Studies also suggested that blockage of CXCR3 or its ligands changed the outcome of T-cell recruitment and airway obliteration. We wanted to determine the role of the chemokine CXCL10 in the pathogenesis of bronchiolitis and BOS. In this study, we found that CXCL10 mRNA levels were significantly increased in patients with BOS. We generated transgenic mice expressing a mouse CXCL10 cDNA under control of the rat CC10 promoter. Six-month-old CC10-CXCL10 transgenic mice developed bronchiolitis characterized by airway epithelial hyperplasia and developed peribronchiolar and perivascular lymphocyte infiltration. The airway hyperplasia and T-cell inflammation were dependent on the presence of CXCR3. Therefore, long-term exposure of the chemokine CXCL10 in the lung causes bronchiolitis-like inflammation in mice.
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ABSTRACT: The most common cause of late death after lung transplantation has been a progressive and unrelenting deterioration in pulmonary allograft function related to the development of obliterative airway disease. In the transplantation literature this entity has been referred to as bronchiolitis obliterans, obliterative bronchiolitis, chronic rejection and, most recently, bronchiolitis obliterans syndrome. Although the etiology of bronchiolitis obliterans remains unclear, it most likely represents a manifestation of chronic lung rejection. The current approaches to the diagnosis and management of bronchiolitis obliterans are reviewed below.08/2007: pages 547-555;
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ABSTRACT: Extended survival after lung transplantation is primarily limited by progressive airflow obstruction and fibrotic obliteration of the small airways, termed bronchiolitis obliterans syndrome (BOS) and bronchiolitis obliterans (BO), respectively. BO is thought to represent the pulmonary-specific manifestation of chronic allograft rejection and the end result of a spectrum of different immunological insults to the allograft. Historically, research has focused on the adaptive immune system and its cellular-based rejection as the driving factor in the development of BO. Recent research in animal lung transplant models and human lung transplant recipients has identified that chemokines, humoral immunity, autoimmunity, and innate immunity also contribute to lung allograft rejection and BO. This review explores the complex immunological mechanisms that promote the high rate of pulmonary allograft failure and significantly impair survival after lung transplantation. We also identify areas for further research critical to improving transplant outcomes.Seminars in Respiratory and Critical Care Medicine 11/2006; 27(5):534-43. · 2.75 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To study the role of T cells in diffuse panbronchiolitis (DPB), we investigated T-cell subsets in bronchoalveolar lavage fluid (BALF) or 33 patients with DPB, nine patients with bronchiectasis, and 20 healthy volunteers. BALF from DPB patients contained a higher percentage of neutrophils than that from patients with bronchiectasis or healthy volunteers, whereas the percentage of lymphocytes was similar in the three groups. DPB patients, however, had a higher number of lymphocytes and a reduced CD4/CD8 ratio compared with the other subjects. A two-color analysis of T-cell subsets in peripheral blood and BALF revealed a significant increase in the percentage and number of CD8+HLA-DR+ cells and in the number of CD4+HLA-DR+ cells in BALF of DPB patients. The expression of the adhesion molecules CD 11a and CD18 on lung CD3+ cells was enhanced over that on blood CD3+ cells in DPB patients. However, there was no significant difference in the expression of these antigens in peripheral blood or BALF among the groups. There was no significant relationship between BALF interleukin (IL)-8 and lymphocyte accumulation in the lungs of the DPB patients, whereas a significant correlation between the percentage of neutrophils and IL-8 levels in BALF of DPB patients was observed. After treatment with macrolide antibiotics, a significant reduction in the number of lymphocytes and activated CD8+ cells and an elevation in the CD4/CD8 ratio in BALF of DPB patients was observed. Our findings suggest an activation of CD8+ cells in the airway lumen of DPB patients, supporting the hypothesis that lymphocytes are important cellular components of bronchial inflammation in DPB.American Journal of Respiratory and Critical Care Medicine 09/1995; 152(2):613-8. · 11.04 Impact Factor
Long-Term Exposure of Chemokine CXCL10 Causes
Dianhua Jiang1, Jiurong Liang1, Rishu Guo1, Ting Xie1, Francine L. Kelly1, Tereza Martinu1,
Ting Yang1,2, Alysia K. Lovgren1, Jessica Chia1, Ningshan Liu1, Yoosun Jung1, Scott M. Palmer1,
and Paul W. Noble1
1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Duke University School of Medicine, Durham, North Carolina;
and2Department of Respiratory Medicine, Capital Medical University, Beijing, China
Chemokines and chemokine receptors have been implicated in the
pathogenesis of bronchiolitis. CXCR3 ligands (CXCL10, CXCL9, and
CXCL11) were elevated in patients with bronchiolitis obliterans
syndrome (BOS) and chronic allorejection. Studies also suggested
thatblockageof CXCR3 or itsligandschangedtheoutcomeof T-cell
recruitment and airway obliteration. We wanted to determine the
role of the chemokine CXCL10 in the pathogenesis of bronchiolitis
and BOS. In this study, we found that CXCL10 mRNA levels were
significantly increased in patients with BOS. We generated trans-
genic mice expressing a mouse CXCL10 cDNA under control of the
rat CC10 promoter. Six-month-old CC10-CXCL10 transgenic mice
sia and developed peribronchiolar and perivascular lymphocyte
infiltration. The airway hyperplasia and T-cell inflammation were
dependent on the presence of CXCR3. Therefore, long-term expo-
sure of the chemokine CXCL10 in the lung causes bronchiolitis-like
inflammation in mice.
Keywords: bronchiolitis; chemokine CXCL10; inflammation; airway in-
Acute bronchiolitis is a disorder most commonly found in infants.
It is caused by viral lower respiratory tract infection characterized
by inflammation, edema, and necrosis of epithelial cells lining the
small airways; increased mucus production; and bronchospasm
(1). Chronic bronchiolitis is characterized by a hyperplasia of
lymphoid tissue along the airways (including the large and the
medial bronchi) and by the development of follicles and follicular
centers (2). A persistent peribronchiolar inflammation gives way
to airway fibrosis and obliteration, leading to bronchiolitis oblit-
erans syndrome (BOS). BOS is the major limitation to survival
after lung or bone marrow transplantation (3, 4). The follicles can
obstruct the bronchiolar lumen, and the obstruction leads to sec-
ondary infection and peribronchiolar pneumonia (2). There is an
increase in activated CD81cells in bronchoalveolar lavage
(BAL) fluid in patients with diffuse panbronchiolitis (5). T-helper
1 cytokines and chemokine expression are up-regulated in post-
transplant airway obliteration (6). Higher concentrations of IL-6
and IL-8 in bronchial and alveolar fractions of the BAL were
significantly associated with an increased risk of developing
Chemokines are released during tissue injury and play a crit-
ical role in regulating cytokine production and leukocyte recruit-
ment, in engendering the adaptive immune responses, and in the
pathogenesis of many human diseases (8). CXC chemokines
CXCL10 (IFN-g–induced protein 10-kD), CXCL9 (monokine
induced by IFN-g), and CXCL11 (IFN-inducible T cell a chemo-
attractant) bind to their receptor CXCR3. Their expression is
dramatically up-regulated by IFN-g (9). CXCR3 is preferen-
tially expressed on Th1 cells (10). CXCR3 and its ligands act
primarily on activated T and natural killer (NK) cells and have
been implicated in mediating the effects of IFN-g as well as of
T cell–dependent inflammatory responses (11). CXCR3 ligands
that attract Th1 cells can concomitantly block the migration of
Th2 cells in response to CCR3 ligands, thus enhancing the po-
larization of effector T-cell recruitment (11).
CXCL10 is inducedduring infectiousandnoninfectious tissue
injuries such as liver ischemia/reperfusion injury (12), respira-
tory syncytial viral infection (13), and chronic hepatitis C virus
infection (14). CXCL10 also plays a critical role in host defense
(15). Indeed, the blockage of CXCR3–CXCL10 interaction with
anti-CXCL10 antisera in mice led to increased mortality and
delayed viral clearance from the central nervous system as com-
pared with control mice when infected with mouse hepatitis
virus (16). Similarly, mice deficient in CXCL10 infected with
hepatitis virus had an impaired ability to control viral replica-
tion in the brain (17). The elevated levels of CXCR3 chemo-
kines in human BAL fluid were associated with the continuum
from acute to chronic rejection (18). CXCR3 and its ligand
CXCL10 are expressed by inflammatory cells infiltrating lung
allografts and mediate chemotaxis of T cells at sites of rejection
(19, 20). Furthermore, in vivo blockage of CXCR3 receptor–
ligand interactions with neutralizing antibodies to receptor
CXCR3 or to the ligands CXCL9 and CXCL10 decreased intra-
graft recruitment of CXCR3-expressing mononuclear cells and
attenuated BOS (18). In a mouse model, deletion of CXCR3,
but not deletion of CXCL9 or CXCL10, in recipients reduces
airway obliteration (21). We hypothesized that the chemokine
CXCL10 plays a causal role in the pathogenesis of bronchiolitis.
In the current study, we measured CXCL10 expression in hu-
man BOS and overexpressed chemokine CXCL10 in mice to
(Received in original form April 6, 2011 and in final form October 19, 2011)
This work was supported by National Institutes of Health grants R01-HL77291,
RO1-HL060539, and P50-HL084917 (P.W.N.).
Correspondence and requests for reprints should be addressed to Paul Noble,
M.D., Division of Pulmonary, Duke University School of Medicine, 106 Research
Drive, Durham, NC 27710. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2011-0116OC on December 8, 2011
Internet address: www.atsjournals.org
Vol 46, Iss. 5, pp 592–598, May 2012
The role for CXCR3 receptor–ligand interactions in allor-
ejection and obliterative airway disease is controversial. The
current study used a genetics approach to demonstrate that
long-term exposure of CXCL10 to the lung is sufficient to
cause airway inflammation characterized by airway epithe-
lial hyperplasia as well as peribronchial and perivascular
lymphatic infiltration in a CXCR3-dependent manner, of-
fering potential therapeutic targets to prevent the devel-
opment of bronchiolitis.
examine directly the role of CXCL10 in the pathogenesis of
MATERIALS AND METHODS
Total RNA was isolated from the lungs of patients with progressive BOS
undergoing pulmonary retransplantation and tissue trimmed from donor
lungs used for transplantation before implantation. The patients included
twowomenandonemenwhowerea meanof 1,490 days fromtheoriginal
transplantation and were a mean 23.5 years of age at the time of retrans-
plantation. The original native diseases that led to the first lung transplan-
tation werecystic fibrosis (n ¼ 2) and congenital heart disease (n ¼ 1). All
patients had BOS grade 3 at the time of retransplantation, with a mean
FEV1 of 0.85 (22.6% predicted). Histological examination of the
explanted transplanted lung confirmed extensive bronchiolitis obliterans
(BO) lesions in the absence of any other significant pathology (22, 23).
Donor lung tissue had no histopathological abnormalities. The human
study received institutional review board approval at Duke University.
CC10–CXCL10 transgenic mice were generated by cloning the mouse
CXCL10 cDNA (24) downstream of the rat CC10 promoter (25) and
upstream of human growth hormone polyadenylation and intronic se-
quence (26). Two copies of the chicken b-globin insulator sequence
were flanked on both ends of the transgene cassette (27). Transgenic
mice were generated in (C57Bl/6 X SJL/J) F2 eggs using standard
pronuclear injection and backcrossed onto C57Bl/6 background for
more than six generations before use. Several lines of the CXCL10
transgenic mice were generated. Lines 3, 5, and 9 were used in the
study. The transgene was genotyped via PCR with the following
transgene-specific primers: 59-ATGAACCCAAGTGCTGCCGTC-39
(forward) and 59-GAGCTGTTTGTTTTTCTCTCTCC-39 (reverse).
CXCR3-deficient mice (28) were crossed with CXCL10 transgenic
mice to generate CXCL101CXCR3–/–mice. Mice were housed and
cared for in a pathogen-free facility at Duke University, and all animal
experiments were approved by the Institutional Animal Care and Use
Committee at Duke University.
BAL fluid was collected as described (29) in the online supplement.
Levels of mouse chemokine CXCL10 in the BAL were measured with
commercial ELISA kits (R&D Systems, Minneapolis, MN) according
to the manufacturers’ instructions.
Chemotaxis assays (30) are described in the online supplement.
Histology and Immunohistochemistry Analysis
The mouse trachea was cannulated, and the lungs were inflated with 10%
formalin.The tissueswerefixedovernight,embeddedinparaffin,and sec-
tioned for staining with hematoxylin and eosin. Leukocytes in lung sec-
tions were stained with specific antibodies against CD3, F4/80, Gr-1, and
control IgG (BD Biosciences, San Diego, CA) and visualized with a Vec-
tastain ABC kit (Vector Laboratories, Burlingame, CA).
Flow cytometric analysis on whole lung single-cell homogenates and
BAL was performed as described (29) in the online supplement.
mRNA analysis was performed by PCR as described in the online
Cell Proliferation Assay
The MTT assay is described in the online supplement.
Differences in measured variables between genetically altered mice and
the control group, or between treatment groups, were assessed using the
Student’s t test with JMP software (SAS Institute, Cary, NC) or Prism 5
(GraphPad, La Jolla, CA). Data are expressed as the mean 6 SEM
where applicable. Statistical difference was accepted at P , 0.05.
Elevated CXCL10 in Patients with BOS
Previous studies implied that CXCR3–CXCL10 interaction
plays a role in acute rejection (19, 31) and airway obliteration
(18, 21). When comparing CXCL10 mRNA levels of lung tis-
sues between patients with BOS and control subjects, we found
that there was a significant increase in CXCL10 mRNA in
patients with BOS (Figure 1). This finding is consistent with
previous observations that the elevated levels of CXCR3 che-
mokines in human BAL fluid were associated with the contin-
uum from acute to chronic rejection (18). These data led us to
hypothesize that CXCL10 may play a causal role in the patho-
genesis of bronchiolitis.
CXCL10 Transgenic Mice Develop Bronchiolitis
We wanted to directly examine the role of CXCL10 in the path-
ogenesis of bronchiolitis by expressing CXCL10 specifically in
the lung. CXCL10-expressing plasmid was constructed where
the mouse CXCL10 cDNA was under the control of the rat
CC10 promoter, with two copies of the chicken b-globin insu-
lator sequence on both ends of the transgene cassette (Figure
2A). The insulator sequences were used to reduce potential
chromosomal position effects (27). The mice were viable and
fertile, with normal appearance and no obvious abnormalities.
The CC10–CXCL10 transgenic mice produced significant amounts
of CXCL10 in BAL and lung tissue (Figure 2B).
CXCL10 in BAL was biologically active, as demonstrated
with a chemotaxis assay (Figure 2C). Histologically, the lungs
Figure 1. Increased CXCL10 mRNA in the lungs of patients with bron-
chiolitis obliterans syndrome (BOS). Total RNA was isolated from the
lung tissues of patients with BOS and from control subjects. RT-PCR was
used to determine CXCL10 mRNA levels in the lung tissues. House-
keeping gene GusB mRNA levels were used a control (n ¼ 3; P , 0.05).
Jiang, Liang, Guo, et al.: CXCL10 Causes Bronchiolitis593
of CXCL10 transgenic mice were normal in the first 8 weeks of
age (Figure 3A). There was no noticeable lung inflammation in
the first 2 months. However, airway inflammation was seen in
mice after 6 months of age (Figure 3A).
Airway Epithelial Hyperplasia
Careful examination of the histology of 6-month-old CC10–
CXCL10 transgenic mice revealed airway thickening (Figure
3B). Airway epithelial hyperplasia was seen in large and medial
airways but more often in small airways (Figure 3B). In some
small airways, villus-like structures of epithelial lining were ob-
served (Figure 3B). However, we found no obliterans of airways
even in 1-year-old mice. Despite the significant airway inflam-
mation, there was no alveolar inflammation in CC10–CXCL10
transgenic mice (Figure 3C). The airway epithelial hyperplasia
may have resulted from persistent inflammation or from the
effect of CXCL10 on epithelial cell proliferation (32). We con-
firmed that CXCL10 protein increased proliferation of human
airway epithelial cells (BEAS-2B) as determined by MTT assay
(medium alone, 0.128 6 0.0025 in absorbance at 595 nm;
CXCL10 at 100 ng/ml, 0.148 6 0.0032; n ¼ 4; P , 0.01).
There was no change in mucus production. Periodic acid-
Schiff staining revealed no difference between CC10–CXCL10
transgenic mice and wild-type (WT) littermate control mice
(data not shown). There was no evidence of emphysema or
fibrosis. Functional study revealed that there was no difference
at baseline between CC10–CXCL10 transgenic mice and WT
littermate control mice (10 wk old) without any challenge. How-
ever, reduced airway responsiveness was found in 6-month-old
CC10–CXCL10 transgenic mice (see Figure E1 in the online
supplement). These data suggest that CXCL10-induced airway
abnormality affected airway responsiveness.
Figure 2. Overexpression of CXCL10 in the lung. (A) Transgenic con-
struct to show that the mouse CXCL10 cDNA was cloned downstream
of the rat CC10 promoter and upstream of human growth hormone
polyadenylation and intronic sequence. Two copies of the chicken
b-globin insulator sequence were flanked on both ends of the trans-
gene. (B) CC10–CXCL10 transgenic mice produced a large amount of
CXCL10 protein into bronchoalveolar lavage (BAL) and lung tissue.
CXCL10 levels in BAL and lung tissue in 8-week-old transgenic mice
and their wild-type (WT) littermates were measure with ELISA (n ¼ 6).
(C) CXCL10 in BAL was bioactive. Jurkat cells were plated onto a Boyden
chamber. BAL from wild-type or CXCL10 transgenic mice was added to
the bottom chamber. Recombinant CXCL10 was used as a positive con-
trol. Cells that migrated to the bottom chamber were counted after 4
hours. (n ¼ 3; ***P , 0.001).
Figure 3. Airway inflammation in CC10–CXCL10 transgenic mice. (A)
Lung micrographs (H&E staining) of CC10–CXCL10 transgenic mice
and wild-type (WT) littermate control mice at 3 weeks, 8 weeks, or 6
months. Airway inflammation was seen in CC10–CXCL10 transgenic
mice at 6 months of age. Scale bar, 100 mm. (B) Lung histology (H&E)
of CC10–CXCL10 transgenic mice and WT littermate control mice at 6
months. Upper panels, large airways; middle panels, small airways; lower
panels, terminal airway. Scale bar, 100 mm. (C) Lung histology (H&E) of
CC10–CXCL10 transgenic mice and WT littermate control mice at 6
months to demonstrate that there was no apparent alveolar inflamma-
tion. Scale bar, 100 mm.
594AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 462012
Perivascular and peribronchiolar infiltration of lymphocytic in-
flammation was apparent when CC10–CXCL10 transgenic mice
grew to 6 months and older (Figures 3B and 4A–4F). Inflam-
matory nodules can be seen around airway (Figures 4A–4F) and
blood vessels (Figures 4B, 4C, and 4E). In some severe cases,
inflammatory nodules can reach 1 mm in diameter (Figure 4A).
Inflammatory infiltration can also be diffused around airway or
blood vessels (Figure 3B). The lymphocyte infiltration was very
similar to those found in the lungs of transgenic mice expressing
IL-6 (25), TNF-a (33), and IL-1b (34).
tissue with antibodies specific to CD3 lymphocytes, F4/80 macro-
phages, or Gr-1 neutrophils and found that CD3 lymphocytes,
but not macrophages or neutrophils (Figure 4G), were present in
those nodules. We also performed conventional differential cell
count and flow cytometry analysis to determine the extent of in-
flammation in the lungs of CC10–CXCL10 transgenic mice. There
was no difference in total BAL cells between 8-week-old WT and
CC10–CXCL10 transgenic mice (Figure 5A), over 90% of the cells
were macrophages, and there was no difference in cell differential
count (data not shown). Consistent with histological demonstration
of the lack of alveolar inflammation, there was no difference in
total BAL cells even in older CC10–CXCL10 transgenic mice
(Figure 5B). However, total lung cells were increased over 6
months of age (Figure 5C), with a significant increase in lympho-
cytes (Figure 5D). There was no significant difference in macro-
phages and neutrophils between CC10–CXCL10 transgenic
mice and WT control mice. Flow cytometry confirmed that
there were more CD31T lymphocytes in CC10–CXCL10 trans-
genic mice than in WT control mice (Figure 5E). CD4 and CD8
T cells were more abundant in CC10–CXCL10 transgenic mice
than in WT control mice (Figure 5F). Next, we determined
whether CXCL10 expression affects the recruitment of regula-
tory T cells (Tregs) (CD41CD251FoxP31). CXCL10 did not
change the percentage of Tregs in CD31 cells (Figure 5G),
although CXCL10 mice showed a trend toward an increase in
Treg number in BAL (Figure 5H).
CXCL10-Induced Bronchiolitis Depends on the Presence
The CXCL10–CXCR3 interaction regulates activated T cells
and NK cells. We next determined whether CXCL10-induced
airway inflammation and leukocyte infiltration are dependent
on its receptor CXCR3. To this end, CC10–CXCL10 transgenic
mice were crossed with CXCR3-deficient mice to generate
CXCL101CXCR3–/–mice. When CC10–CXCL10 transgenic
mice were crossed with CXCR3-deficient mice, airway and al-
veolar structures were normal (Figure 6). There was no airway
epithelial hyperplasia, no leukocyte infiltration, no nodules, and
no diffuse infiltration (Figure 6). These data suggest that CXCL10-
induced bronchiolitis is dependent on the presence of its recep-
In this study, we demonstrate that patients with BOS expressed
higher levels of CXCL10 mRNA in the lung tissue than control
Figure 4. Lymphocytic infiltration. (A–F) Lung histology
(H&E staining) of CC10–CXCL10 transgenic mice at 6
months of age displayed lymphocytic nodules around air-
ways. (G) Lung sections of 6-month-old CC10–CXCL10
transgenic mice were stained with specific antibodies
against T-cell marker CD3, macrophage marker F4/80,
neutrophil marker Gr-1, or control IgG. Scale bar, 100 mm.
Jiang, Liang, Guo, et al.: CXCL10 Causes Bronchiolitis595
individuals did. We further demonstrated that overexpression of
chemokine CXCL10 under control of the CC10 promoter indu-
ces bronchiolitis-like inflammation in 6-month-old mice in a
CXCR3-dependent manner. Therefore, we provide evidence
to demonstrate that the CXCL10 pathway plays a causal role
in the development of bronchiolar inflammation.
Induction of CXCL10 occurs in immune-mediated and in
non–immune-mediated lung injury (29, 35, 36) and in several
models of liver injury and regeneration (12). CXCL10–CXCR3
interactions have been directly implicated in the pathogenesis of
immune-mediated tissue damage (18, 21, 28, 36). We and others
have shown that, after noninfectious lung injury, CXCL10 is
produced and has antifibrotic properties (29, 37–39). Further-
more, CXCR3 ligands are elevated during acute rejection (18,
19, 31, 36) and BOS (18). In addition, CXCL10 is up-regulated
during viral bronchiolitis (13, 40). We demonstrated that long-
term exposure of CXCL10 alone is able to induce bronchiolitis-
like airway inflammation. In adult CCL10–CXCL10 transgenic
mice at 6 months of age, airway inflammation is apparent, in-
cluding airway epithelial cell hyperplasia as well as perivascular
and peribronchiolar infiltration of lymphocytes, which resemble
the characteristics of chronic bronchiolitis. The airway epithelial
hyperplasia may have resulted from persistent airway inflamma-
tion, given the fact that there was significant perivascular
and peribronchiolar inflammation in CL10–CXCL10 transgenic
mice. The hyperplasia may also have resulted from the effect
of CXCL10 on epithelial cell proliferation because CXCR3
ligands promote airway epithelial proliferation (32). T-cell infil-
tration was evident in older CCL10–CXCL10 transgenic mice.
These leukocytes were mostly CD3-positive T cells. We did not
notice preferential recruitment to CD4 and CD8 because both
CD4 and CD8 T cells were increased in the CC10–CXCL10
Figure 5. Lung
(A) Total BAL cells from CC10–
CXCL10 transgenic mice and
WT littermate control mice at
8 weeks of age (n ¼ 4; P .
0.05). (B) Total BAL cells of
CC10–CXCL10 transgenic mice
and WT littermate control mice
at 6 months of age (n ¼ 4 or 5;
P . 0.05). (C) Total mouse
lung cells of CC10–CXCL10
transgenic mice and WT litter-
mate control mice at 6 months
of age (n ¼ 4 or 5; P , 0.01).
(D) Lymphocyte percentage of
cell differential count of total
mouse lung cells of CC10–
CXCL10 transgenic mice and
WT littermate control mice at
6 months of age (n ¼ 4 or 5;
P , 0.05). (E, F) Flow cytometry
to determine lymphocytes in
total mouse lung cells of CC10–CXCL10 transgenic mice and WT littermate control mice at 6 months of age (n ¼ 4 or 5; *P , 0.05; **P , 0.01).
(G) Flow cytometric analysis of regulatory T cells (CD41CD251FoxP31) in BAL (n ¼ 3 or 4; P . 0.05). (H) Total Treg cells in BAL (n ¼ 3 or 4; P . 0.05).
Figure 6. Histology of CXCL101CXCR3–/–
micrographs (H&E) of CXCL101CXCR3–/–mice, CXCR3–/–
mice, and WT littermate control mice at 6 months of age.
Normal airway structure (either large or small) was demon-
strated in CXCL101CXCR3–/–mice, and there was no leu-
kocyte infiltration and no apparent alveolar inflammation in
CXCL101CXCR3–/–mice. Scale bar, 100 mm.
596AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 462012
mouse lungs. These data are consistent with earlier observa-
tions that the CXCL10–CXCR3 interaction is critical for the
recruitment of T cells to the injury site (9, 11, 41, 42). We also
demonstrated that the effect of CXCL10 on airway inflamma-
tion required its receptor CXCR3. This is consistent with pre-
vious reports that effects of CXCL10 on T-cell recruitment are
dependent on CXCR3 (36). The lymphocyte infiltration was
similar to that found in transgenic mice expressing the cytokine
IL-6 (25), TNF-a (33), and IL-1b (34). Unlike those transgenic
mice, CC10–CXCL10 transgenic mice have no evidence of em-
physema or fibrosis. CXCL10 is induced by IFN-g (9, 24, 41, 43).
CC10–IFN-g transgenic mice have severe lung inflammation
and develop emphysema (44).
Despite significant airway inflammation induced by CXCL10
exposure, the CC10–CXCL10 mice did not spontaneously de-
velop BOS, even in 1-year-old mice. We did not observe measur-
able airway fibrosis. This may be because inflammation induced
by CXCL10 was not severe enough to disrupt basement mem-
brane. The bronchiolar basement membrane was disrupted in
lung allograft recipients who had multiple episodes of clinically
significant acute cellular rejection and later develop BO (45).
More importantly, there is no antigen presentation of mismatched
major histocompatibility complex antigens and no production of
antibodies to these MHC antigens in the inflammation induced by
CXCL10; several reports have suggested that T-cell recognition of
alloantigen is the central event that initiates graft rejection (46).
Studies have suggested that indirect allorecognition of donor ma-
jor histocompatibility complex–derived peptides by recipient CD4
T cells contributes to chronic rejection of skin, kidney, liver, and
heart allografts (46). Furthermore, CXCL10 plays a role in limit-
ing lung fibrosis by inhibiting angiogenesis (37) or by inhibiting
fibroblast migration (38, 39). Thus, CXCL10 may play dual roles
in these conditions.
Although allergic airway inflammation such as asthma is gen-
erally associated with Th2 activation, Th1 cytokines (e.g.,CXCR3
ligands) in asthma have been suggested to play a role. CXCL10 is
up-regulated in experimental asthma (47), and overexpression of
CXCL10 driven by the bovine keratin 5 on the FVB background
showed significantly increased airway hyperreactivity when im-
munized with ovalbumin (47). However, reduced airway respon-
siveness was found in CC10–CXCL10 transgenic mice without
allergy challenge at 6 months of age. This discrepancy may be
due to the age of mice, airway structural integrity, and the ex-
In summary, we successfully generated transgenic mice express-
ing mouseCXCL10 targetingCC10-expressing cells.Six-month-old
CC10–CXCL10 transgenic mice developed bronchiolitis charac-
terized by airway epithelial hyperplasia and T-cell infiltration
peribronchiolarly and perivascularly. The airway hyperplasia
and T-cell inflammation were dependent on the presence of
CXCR3. These data support our hypothesis that chemokine
CXCL10 plays a causal role in the pathogenesis of bronchiolitis.
However, long-term exposure to chemokine CXCL10 alone was
not sufficient to induce BO. Our study provides evidence to
support the critical role of chemokine CXCL10 in bronchiolitis
but also points to its limited role in the pathogenesis of BO.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgments: The authors thank Dr. J. Farber of National Institute of Allergy
and Infectious Diseases, NIAID, for providing mouse CXCL10 cDNA and Erin Potts
and Dr. Michael Foster for assistance with airway resistance experiments.
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