Circulating Progenitor Epithelial Cells
Traffic via CXCR4/CXCL12 in Response to
Airway Injury
This information is current as
of June 15, 2017.
Brigitte N. Gomperts, John A. Belperio, P. Nagesh Rao,
Scott H. Randell, Michael C. Fishbein, Marie D. Burdick and
Robert M. Strieter
J Immunol 2006; 176:1916-1927; ;
doi: 10.4049/jimmunol.176.3.1916
http://www.jimmunol.org/content/176/3/1916
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References
The Journal of Immunology
Circulating Progenitor Epithelial Cells Traffic via
CXCR4/CXCL12 in Response to Airway Injury1
Brigitte N. Gomperts,* John A. Belperio,† P. Nagesh Rao,*‡ Scott H. Randell,§
Michael C. Fishbein,‡ Marie D. Burdick,† and Robert M. Strieter2†
T
he airway epithelium is in direct contact with the environment and, as such, is at constant jeopardy of injury
from environmental toxins and pathogens. Therefore, an
efficient mechanism for regeneration and repair of the airway epithelium is essential to maintain this barrier and protect the host.
Our understanding of this repair process is limited and, until recently, was thought to occur only from resident tissue progenitor
cells. Basal resident progenitor epithelial cells are located in the
submucosal glands and ducts and have been shown to be capable
of differentiating into all airway epithelial cell types (1–3). They
possess the capacity for self-renewal, divide slowly, retain a BrdU
label, and express markers of early epithelial differentiation (i.e.,
cytokeratin 5(CK53 and CK14) (1– 6). CK5 and CK14 are type I
and type II intermediate filament proteins that are expressed together in basal regenerating cells of complicated epithelia. Injury
to the airway results in an initial increase in the number of
CK5⫹CK14⫹ basal cells, with a return to normal CK5⫹CK14⫹
*Department of Pediatrics, Mattel Children’s Hospital, †Department of Medicine, and
‡
Department of Pathology and Laboratory Medicine, University of California, Los
Angeles, CA 90095; and §Departments of Cell and Molecular Physiology and Medicine, Cystic Fibrosis Pulmonary Research and Treatment Center, University of North
Carolina, Chapel Hill, NC 27599
Received for publication April 28, 2005. Accepted for publication November
27, 2005.
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.
1
This work was supported by National Institutes of Health Grant K08 HL074229 and
Howard Hughes Medical Institute Award 76200-549702 (to B.N.G.), National Institutes of Health Grants CA87879, HL66027, P50 CA90388, and P50 HL60289 (to
R.M.S.), and National Institutes of Health Grant 1P50 HL084921-01.
2
Address correspondence and reprint requests to Dr. Robert M. Strieter, 900 Veteran
Avenue, 14-154 Warren Hall, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1786. E-mail address: [email protected]
3
Abbreviations used in this paper: CK5, cytokeratin 5; CISH, chromogenic in situ
hybridization; DAB, diaminobenzidine; FISH, fluorescent in situ hybridization.
Copyright © 2006 by The American Association of Immunologists, Inc.
resident progenitor cell numbers once the airway is repaired, suggesting a negative feedback mechanism (2).
Under homeostatic conditions, it is likely that there are sufficient
resident progenitor epithelial cells to replace the continuous loss of
airway epithelial cells. However, at times of airway injury a contemporary concept suggests that the resident progenitor cells may
be overwhelmed and that circulating progenitor epithelial cells
contribute to repair. Evidence of this recruitment comes from human sex-mismatched lung transplants where tissue biopsies have
shown pulmonary epithelial cells from the recipient contributing to
the repair of donor lungs, implying that circulating progenitor epithelial cells do contribute to regeneration of the airway epithelium
(7, 8). Furthermore, the circulating progenitor epithelial cells likely
originate from the bone marrow, because serial transplantation of
a single GFP⫹ bone marrow cell into lethally irradiated mice results in GFP expression in the bronchi as well as other tissues (9).
However, these cells represent a very small population of cells and
are not yet well understood. Therefore, identifying and characterizing these circulating progenitor epithelial cells and their mechanisms of trafficking is essential for our understanding of the repair
process and for harnessing their potential therapeutic applications.
We hypothesized that circulating progenitor epithelial cells traffic to the injured airway in response to the CXCL12/CXCR4 biological axis and contribute to repair. To test this postulate, we
created a mouse model of airway injury and used specific biomarkers to track the recruitment of circulating progenitor epithelial
cells. Our studies demonstrated that circulating progenitor epithelial cells contributed to repair of the injured airway in a model of
tracheal transplantation. Furthermore, the CXCR4/CXCL12 biological axis played a critical role in the trafficking of these cells to
the site of airway injury. In addition, neutralizing Abs to CXCL12
resulted in a phenotype of squamous metaplasia in the regenerated
epithelium, which was devoid of circulating progenitor epithelial
cells. These findings may ultimately result in the use of circulating
progenitor epithelial cells as a novel therapy for airway injury and
0022-1767/06/$02.00
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Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor
epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms
for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone
marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor
CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5ⴙ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence
of CXCL12 in tracheal transplants provided a mechanism for CXCR4ⴙ circulating progenitor epithelial cell recruitment to the
airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the
resident tissue progenitor epithelial cells. Our findings demonstrate that CK5ⴙCXCR4ⴙ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor
epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings
support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic
strategies for circulating progenitor epithelial cells in airway diseases. The Journal of Immunology, 2006, 176: 1916 –1927.
The Journal of Immunology
further our understanding of the pathogenesis of airway squamous
metaplasia.
Materials and Methods
Mouse tracheal transplant model
We used a well established, reproducible murine model of tracheal epithelial regeneration using syngeneic s.c. tracheal transplants from C57BL/6
females into C57BL/6 males for our sex-mismatched model (10, 11). Male
tracheas transplanted into male mice and female tracheas transplanted into
female mice served as our sex-matched controls. For the experiments with
GFP mice, wild-type C57BL/6 female tracheas were transplanted into
C57BL/6 male GFP⫹ mice (The Jackson Laboratory). For the experiments
with CK5-GFP mice, wild type C57BL/6C/C3H tracheas were transplanted
into C57BL/6/C3H CK5-GFP mice.
CXCL12 mobilization and enrichment of /bone marrow
Depletion of CXCL12
F(ab⬘)2 of either neutralizing goat anti-mouse CXCL12 Ab or control
F(ab⬘)2 were injected i.p. 1 h before tracheal transplant and then every other
day until the animals were euthanized (13). Five milligrams of Ab were
injected per dose as described previously (13). All animal experiments for
these studies were approved by the Department of Laboratory Animal
Medicine at the David Geffen School of Medicine at the University of
California, Los Angeles, CA.
Quantitative real-time PCR
Quantitative real-time PCR was performed using the 7700 sequence detection system (Applied Biosystems). Quantitative real-time PCR was performed for CXCL12 and CXCR4 from tracheal transplant cDNA as described previously (10, 11).
Chromogenic in situ hybridization (CISH)
Tracheal transplant tissue was fixed in 4% paraformaldehyde overnight and
paraffin-embedded for sectioning. Deparaffinization was performed before
boiling in CISH tissue heat pretreatment solution and digesting in pepsin
per the manufacturer’s protocol (Zymed Laboratories). The biotinylated Y
chromosome probe (Zymed Laboratories) and the tissue were denatured at
94°C for 5 min, and hybridization was performed overnight at 37°C. Immunodetection was performed per the manufacturer’s protocol.
Fluorescent in situ hybridization (FISH)
Four-micrometer sections of paraformaldehyde-fixed, paraffin-embedded
tissue were deparaffinized, incubated in sodium thiocyanate at 80°C for 30
min, and digested with 0.1% pepsin in 0.1 N HCl for 20 min. Tissues and
probes were denatured at 60°C for 10 min and then hybridized at 37°C with
FITC-labeled mouse Y whole chromosome paint (Cambio) for 16 h per the
manufacturer’s protocol. Washes were performed at 37°C with formamide
and 2⫻ SSC, 2⫻ SSC alone, and 4⫻ SSC with 0.1% Tween 20. Tissues
were mounted in Vectashield hard mountant with 4⬘,6⬘-diamidino-2-phenylindole nuclear counterstain (Vector Laboratories). All images were obtained with a Zeiss Axioskop 2 Plus fluorescent microscope and an AxioCam HR camera and imaged with an AxioVision scanner and LE Real 4.2
software.
Immunohistochemistry
Tracheal tissue was fixed in 4% paraformaldehyde overnight and then embedded in paraffin and sectioned. Four-micrometer sections were deparaf-
finized in xylene and rehydrated in graded ethanols. For the anti-CXCL12
Ab only, the sections were boiled in Glyca-based Ag retrieval buffer (BioGenex Laboratories) for 20 min. Blocking was performed with Universal
blocking reagent (Biogenex). The primary Abs used were anti-GFP from
Rockland (anti-GFP Ab with minimum cross-reactivity to mouse; dilution
1/200) and anti-CXCL12 from BD Biosciences (dilution 1/500). Appropriate biotinylated secondary Abs were used, and the signal was amplified
with the ABC (avidin/biotin complex) kit (Vector Laboratories) per the
manufacturer’s protocol. Peroxidase staining was detected with diaminobenzidine (DAB) substrate (Vector Laboratories), and hematoxylin was
used as a counterstain.
Immunofluorescence
Tissue sections were deparaffinized, and Ag retrieval and blocking were
performed as described above for immunohistochemistry. The primary Abs
used were anti-mouse CK5 (dilution 1/200) (Covance Research Products),
anti-GFP (dilution 1/200) (Santa Cruz Biotechnology), and anti-mouse p63
(dilution 1/200) (Santa Cruz Biotechnology). Biotinylated secondary Ab
(Jackson ImmunoResearch Laboratories) followed by streptavidin-Cy3
(Jackson ImmunoResearch Laboratories) was used to amplify the signal.
For dual immunostaining, avidin and biotin (Vector Laboratories) blocking
steps were performed before repeating the above protocol with the second
primary Ab. Microscopy was performed with a Zeiss microscope as described above.
FACS analysis of buffy coat, bone marrow cells, and cells from
tracheal transplants
FACS staining was performed in 96-well disposable plates. Bone marrow
was isolated from mouse femurs by flushing with PBS through a 26-gauge
needle into RPMI 1640 medium. Collagenase A (Roche) and DNase
(Sigma-Aldrich) were added to the bone marrow, thoroughly mixed, and
incubated at room temperature for 20 min to create a single cell suspension.
The RBCs were lysed, and 1 ⫻ 106 cells were plated in each well of the
96-well plate for immediate FACS analysis. Peripheral blood was obtained
from mice by retro-orbital bleeding. Five hundred microliters of mouse
whole blood was collected from a single mouse in an Eppendorf tube with
50 ␮l of heparin to prevent clotting. Single mouse samples were not combined. The whole blood was centrifuged at 3,000 rpm for 10 min to separate out the fractions. The layer of nucleated cells between the plasma
layer and the RBC layer forms the buffy coat. The buffy coat was aspirated,
and RBC was lysed using ammonium chloride potassium lysis buffer. Cells
(1 ⫻ 105) were placed into each well of a 96-well plate for immediate
FACS analysis. The tracheal transplants were diced with a clean blade and
then incubated in collagenase A (Roche) and DNase (Sigma-Aldrich) at
37°C for 30 min to create a single cell suspension. Cells were plated immediately into FACS plates for analysis. The Abs used were FITC-conjugated anti-mouse-CK5 (Covance Research Products), PerCP anti-mouse
CD45 (BD Biosciences), PE-anti-mouse CXCR4 (BD Biosciences), PE
anti-MAC915 (BD Biosciences), PE anti-Ly6 (BD Biosciences), PE-antiNK1.1 (BD Biosciences), PE-anti-CD3 (BD Biosciences), PE anti-CD4
(BD Biosciences), PE anti-CD8 (BD Biosciences), PE anti-CD34 (BD
Biosciences), PE anti-Ly-6A/E(Sca-1) (BD Biosciences), and PE antiCD117 (c-Kit) (BD Biosciences). Cells were permeabilized with Cytofix/
Cytoperm (BD Biosciences) after surface staining but before CK5 staining
because CK5 is an intracellular protein. Isotype Ab controls were performed for all of the Abs used and were added either pre- or post-cell
permeabilization, depending on whether the Ag was intracellular or extracellular. We also used the purified rat anti-mouse CD16/CD32 (Fc␥III/II
receptor) mAb (mouse BD Fc block; 0.5 ␮g/well) in all of our conditions
to reduce nonspecific Ab binding to the Fc␥III/II receptor. Fluorescent cell
staining was analyzed with a flow cytometer and CellQuest software (both
from BD Biosciences). The CK5⫹ population of cells was gated on first,
followed by analysis of CD45 and CXCR4 expression.
Luminex protein analysis
Mouse CXCL12 expression in the tracheal transplants was measured using
a bead multiplex system (CXCL12; Upstate Biotechnology) as described
previously (14). Briefly, whole tissue extracts were made from tracheal
transplants at 21 days posttransplant and incubated in complete protease
inhibitor (Roche). Samples were incubated for 2 h at room temperature
with anti-CXCL12 beads in a 96-well plate. Biotinylated anti-CXCL12
Abs were added to each well, and the plate was incubated for 1.5 h. Streptavidin-PE was added to the wells, and the plate was incubated for 30 min.
Next, 0.2% paraformaldehyde was added to the wells, and the plate was
read on a Luminex100 IS instrument. The concentration of cytokine was
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The mobilization and enrichment of bone marrow was performed by s.c.
injection of CXCL12 (100 ␮g), as compared with control vehicle, in mice.
Six groups of mice (n ⫽ 5) were treated in the following manner. Groups
1 and 2 were treated with CXCL12 or control vehicle, respectively, at time
0 and were sacrificed in 1 h for analysis of the number of CK5 cells in both
the buffy coat and bone marrow. Groups 3 and 4 were treated with
CXCL12 or control vehicle, respectively, at time 0 and received a second
dose of CXCL12 or control vehicle, respectively, at 24 h followed by
sacrifice in 1 h for analysis of the number of CK5 cells in both the buffy
coat and bone marrow. Groups 5 and 6 were treated with CXCL12 or
control vehicle, respectively, at time 0, received a second dose of CXCL12
or control vehicle, respectively, at 24 h, and a third dose of CXCL12 or
control vehicle, respectively, at 48 h followed by sacrifice in 1 h after the
last dose for analysis of the number of CK5 cells in both the buffy coat and
bone marrow (12).
1917
1918
determined from a standard curve assayed at the same time with known
amounts of recombinant CXCL12.
Statistical analysis
Data were analyzed on a PC using the StatView 4.5 statistical
package (Abacus Concepts). Comparisons were evaluated by the
unpaired t test. Continuous data were expressed as mean ⫾ SEM.
Data were considered statistically significant if p values were
ⱕ0.05.
Results
FACS analysis of CK5⫹ cells from the bone marrow and
circulation
expressed Sca-1, and 31.7 ⫾ 1.8% of CK5⫹ cells expressed c-kit.
In the buffy coat, 28.2 ⫾ 2.5% of CK5⫹ cells expressed CD34,
22 ⫾ 1.7% of CK5⫹ cells expressed Sca-1, and 21.3 ⫾ 2.9% of
CK5⫹ cells expressed c-kit.
Male CK5⫹ circulating progenitor epithelial cells contribute to
regeneration of female tracheal airway epithelium
To test the hypothesis that CK5⫹CXCR4⫹ circulating progenitor
epithelial cells contribute to repair of the airway epithelium, female tracheas were transplanted into the flanks of syngeneic male
mice, and male cells were identified by Y chromosome expression
in the female trachea. This heterotopic tracheal transplant mouse
model has been well characterized, especially as an allograft model
system to study transplant rejection where syngeneic controls have
demonstrated normal regeneration of the pseudostratified airway
epithelium by day 21 posttransplantation (15, 16). The model is
associated first with tracheal ischemia, followed by reperfusion
from neovascularization posttransplantation. The airway injury is
associated with complete sloughing of the epithelium from the
basement membrane with gradual re-epithelialization starting by
day 3 posttransplantation. The sloughed epithelium and recipient
inflammatory cells form a luminal plug that organizes over time.
Normal re-epithelialization following this injury is seen as early as
day 7, and full regeneration of the pseudostratified columnar epithelium occurs by day 21 posttransplantation (10, 11, 15, 16). The
syngeneic tracheal transplant is associated with minimal inflammatory changes after day 7 posttransplantation (10, 11, 15, 16).
Our aim was to determine the contribution of circulating cells to
the repairing airway epithelium, we therefore performed both
CISH and FISH in situ hybridization for the Y chromosome in
sex-mismatched tracheal transplants. These two methods used biotin- and FITC-labeled DNA probes, respectively, for the murine
Y chromosome. Male cells were detected in the luminal epithelial
cells as well as the submucosal glands and ducts at day 3 posttransplant (Fig. 2A, i and ii). By day 7 posttransplant, male cells
were present in the submucosal gland ducts along with the basal
cells of the regenerating epithelium (Fig. 2A, iii and iv). By day 21
FIGURE 1. CK5⫹ cells are present in the bone marrow and buffy coat of mice. i, FACS analysis of bone marrow mononuclear cells revealed a population
of cells (⬃85%) that were CD45⫹. ii, Dual immunostaining demonstrated a population of CK5⫹CD45⫹ cells (R1) (5 ⫾ 0.5%; n ⫽ 5). iii, Gating on these
cells (R1) demonstrated that 90 ⫾ 3.5% of these cells expressed CXCR4 on their surface (n ⫽ 5). iv, Isotype Ab CK5 bone marrow control is representative
of all the control Abs stained. v, FACS analysis of mouse circulating buffy coat cells showed a population of CD45⫹ cells. vi, 8 ⫾ 1.5% of mononuclear
cells from the buffy coat were CK5⫹, and 76 ⫾ 6% of the CK5⫹ cells were also CD45⫹ (R2) (n ⫽ 5). vii, Gating on this population (R2) demonstrated
56 ⫾ 3.9% of these cells to be CXCR4⫹ (n ⫽ 5). viii, Isotype Ab CK5 buffy coat control is representative of all the control Abs stained.
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Our aim was to determine whether a population of CK5⫹ progenitor epithelial cells were detectable by FACS analysis in the bone
marrow and circulation of mice. We first gated on the CK5⫹ cells
and then determined the percentage of these cells that were CD45⫹
and CXCR4⫹. Under homeostatic conditions we found that 5 ⫾
0.5% of mononuclear cells isolated from the bone marrow expressed CK5, of which almost 100% were CD45⫹ (n ⫽ 5). Of
these cells, 90 ⫾ 3.5% were CXCR4⫹ (Fig. 1, i–iii). In the circulation, 8 ⫾ 1.5% of mononuclear cells expressed CK5. Of the
CK5⫹ cells, 76 ⫾ 6% were also CD45⫹ (R2); of this group of
CK5⫹CD45⫹ cells (R2), 56 ⫾ 3.9% were CXCR4⫹ (Fig. 1, v–vii;
n ⫽ 5). Isotype Ab controls were performed for CK5, CXCR4, and
CD45 on bone marrow and buffy coat cells, and Fig. 1, iv and viii,
are representative of all of the control Abs stained.
FACS analysis of bone marrow and buffy coat cells from CK5GFP-transgenic mice that express GFP under control of the CK5
promoter confirmed our findings for CK5⫹ cells in both the bone
marrow and buffy coat cells under homeostatic conditions. We also
assessed mobilization of these CK5⫹ cells under conditions of
tracheal transplantation; however, we were unable to identify any
marked increase in the circulating numbers of the CK5⫹ cells at
12 h, 24 h, and 3 days posttransplantation. Furthermore, FACS
analysis of bone marrow from naive mice revealed that 16.5 ⫾
0.9% of CK5⫹ cells expressed CD34, 17.7 ⫾ 0.7% of CK5⫹ cells
CXCR4/CXCL12 IN AIRWAY REPAIR
The Journal of Immunology
1919
posttransplant, male cells were present in the submucosal gland
ducts and the basal and apical epithelial cells (Fig. 2A, v). Morphometric analysis and quantitation of CISH-positive cells demonstrated 0% male cells in tracheal epithelia from female tracheal
transplants in female mice on days 3, 7, and 21 posttransplantation.
In comparison, morphometric analysis and quantitation of CISHpositive cells demonstrated 8.2 ⫾ 2.6, 12.9 ⫾ 3.8, and 18.4 ⫾
1.4% male cells in sex-mismatched tracheal transplants at days 3,
7, and 21 posttransplantation, respectively ( p values of 0.001,
0.015, and 0.004, respectively; Table I).
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FIGURE 2. A, CISH and FISH for the Y chromosome in female tracheas transplanted into male recipients. i, CISH, day 3 posttransplant with Y
chromosome-positive cells seen on the luminal surface (brown dots; arrows). ii, FISH, day 3 posttransplant with Y chromosome-positive cells in the luminal
surface, submucosal glands, and ducts (fluorescent green dots; arrows). iii, CISH, day 7 posttransplant with Y chromosome-positive cells contributing to
the regenerating epithelium, submucosal glands, and ducts (arrows). iv, FISH, day 7 posttransplant with Y chromosome-positive cells contributing to the
basal (white arrows), parabasal (yellow arrow), and apical cells (red arrow) of the regenerating epithelium, as well as submucosal cells (dashed white
arrows). v, CISH, at day 21 posttransplant with chimerism for Y chromosome-positive and -negative cells in the submucosal glands, ducts, and pseudostratified epithelium (arrows). B, i, Male control trachea with Y chromosome-positive cells (arrows). ii, Female control nontransplanted trachea with no Y
chromosome-positive cells. Scale bars, 10 ␮m. C, i–iv, Immunostaining and immunofluorescence of GFP in the tracheal epithelium. i, Wild-type female
trachea transplanted into a wild-type female mouse demonstrated no immunostaining for GFP. ii, Homozygous GFP⫹ male trachea transplanted into a male
GFP⫹ mouse showed GFP⫹ immunostaining in all cells at day 21 posttransplant. iii and iv, DAB staining (arrows) and FITC staining (white arrows) at
day 7 posttransplantation of a wild-type female trachea into a male GFP mouse and iv, v and vi, Immunostaining with DAB and FITC, respectively, at day
21 posttransplantation of a wild-type female trachea into a male GFP mouse and iv, demonstrated GFP⫹ cells (white arrows) contributing to regeneration
of the epithelium. Scale bars, 10 ␮m. For quantitation, see Table I. D, and iv, Immunohistochemistry for GFP in airway epithelium from wild-type and
CK5-GFP mice at day 21 post-tracheal transplantation. i, GFP expression by DAB and iv, staining (arrows) in wild-type tracheas transplanted into CK5-GFP
mice. ii, CK5-GFP tracheas transplanted into CK5-GFP mice demonstrate immunostaining in all and iv, the basal cells of the regenerating airway epithelium
(arrows). iii, Wild-type tracheas transplanted into wild-type mice demonstrated no immunostaining and iv, for GFP. Scale bars, 10 ␮m.
1920
CXCR4/CXCL12 IN AIRWAY REPAIR
Table I. Quantitation of circulating progenitor cell chimerism
Positive Cells (%)
p Value
8/97
12/93
35/189
85/200
0/200
8.2 ⫾ 2.6
12.9 ⫾ 3.8
18.4 ⫾ 1.4
42.5 ⫾ 2.5
0
0.001
0.015
0.004
⬍0.001
11/163
21/170
88/165
0/150
6.3 ⫾ 1.8
12.3 ⫾ 2.1
53 ⫾ 2.3
0
0.013
0.005
⬍0.001
25/125
132/271
200/200
0/200
20 ⫾ 3.8
48.7 ⫾ 8.5
100
0
0.006
0.005
⬍0.001
12/131
55/296
9.2 ⫾ 1.2
18.6 ⫾ 5.1
0.003
0.012
⫹
CISH Y
Day 3
Day 7
Day 21
Male
Female
FISH Y⫹
Day 3
Day 7
Male
Female
GFP⫹
Day 7
Day 21
GFP
Wild-type
CK5⫹/GFP⫹
Day 7
Day 21
The findings for CISH-positive male cells in sex-mismatched
tracheal transplants were confirmed by FISH. Morphometric analysis and quantitation of FISH-positive cells demonstrated 0% male
cells in female tracheal epithelia from sex-matched tracheal transplants. In comparison, morphometric analysis and quantitation of
FISH-positive cells demonstrated 6.3 ⫾ 1.8 and 12.3 ⫾ 2.1% male
cells in sex-mismatched tracheal transplants at day 3 and 7 posttransplantation, respectively ( p values of 0.013 and 0.005, respectively; Table I).
To further confirm the validity of our morphometric analysis, we
examined Y chromosome expression in male epithelia from sexmatched tracheal transplants. The Y chromosome was only detected in 42.5 ⫾ 2.5% of control male tracheal cells by CISH and
53 ⫾ 2.3% by FISH at day 21 posttransplantation (Fig. 2B, i, Table
I, and data not shown). Other authors have also shown that not all
male cells are positive for the Y chromosome by FISH, which may
reflect the plane of sectioning of individual nuclei (17, 18). Control
female tracheal sections from female mice did not demonstrate any
hybridization with the Y-labeled probe (Fig. 2B, ii).
To further demonstrate that male circulating progenitor epithelial cells contribute to tracheal epithelial repair, we transplanted
wild-type sex-mismatched tracheas into the flanks of homozygous
GFP transgenic mice, thereby using GFP as a marker of circulating
recipient cells. For controls, wild-type sex-matched tracheal transplants were transplanted into wild-type mice or male GFP⫹ tracheas were transplanted into GFP⫹ sex-matched mice. Morphometric analysis and quantitation of male GFP⫹ cells by
immunohistochemistry in sex-mismatched wild-type tracheal epithelium of tracheas transplanted into wild-type mice demonstrated
0% GFP⫹ cells at day 7 (not shown) and day 21 posttransplantation (Fig. 2C, i). All cells in the male GFP⫹ tracheal transplants in
sex-matched GFP⫹ mice were positive by immunohistochemistry
for GFP (Fig. 2C, ii). In comparison, morphometric analysis and
quantitation of GFP⫹ cells by immunohistochemistry and immunofluorescence in female wild-type epithelia of tracheas transplanted into GFP⫹ sex-mismatched mice demonstrated 20 ⫾ 3.8%
and 48.7 ⫾ 8.5% GFP⫹ epithelial progenitor cells in the submucosal gland ducts and basal and apical cells at days 7 (Fig. 2C, iii
and iv) and 21 (Fig. 2C, v and vi) posttransplantation, respectively
( p values of 0.006 and 0.005, respectively; Table I).
To prove that cells from the circulation are progenitor epithelial
cells, we performed tracheal transplants with wild-type tracheas
CXCL12 induces early mobilization and later enrichment of the
bone marrow with CK5⫹ progenitor cells
We hypothesized that CXCL12 may mobilize the CK5⫹ progenitor cells and enrich the bone marrow with CK5⫹ progenitor cells.
We therefore performed s.c. injection of mice with CXCL12 (100
␮g), as compared with control vehicle. Six groups of mice (n ⫽ 5)
were treated in the following manner. Groups 1 and 2 were treated
with CXCL12 or control vehicle, respectively, at time 0 and sacrificed in 1 h for analysis of the number of CK5 cells in both the
buffy coat and bone marrow. Groups 3 and 4 were treated with
CXCL12 or control vehicle, respectively, at time 0 and received a
second dose of CXCL12 or control vehicle, respectively, at 24 h
followed by sacrifice in 1 h for analysis of the number of CK5 cells
in both the buffy coat and bone marrow. Groups 5 and 6 were
treated with CXCL12 or control vehicle, respectively, at time 0,
received a second dose of CXCL12 or control vehicle, respectively, at 24 h, and received a third dose of CXCL12 or control
vehicle, respectively, at 48 h followed by sacrifice in 1 h after the
last dose for analysis of the number of CK5 cells in both the buffy
coat and bone marrow using a method described previously (12).
For group 1, we found 3.2 ⫻ 105 ⫾ 4.4 ⫻ 104 CK5⫹ cells in the
buffy coat and 9 ⫻ 105 ⫾ 1.8 ⫻ 105 CK5⫹ cells in the bone
marrow, as compared with 5.39 ⫻ 104 ⫾ 4.37 ⫻ 104 CK5⫹ cells
in the buffy coat and 4.7 ⫻ 105 ⫾ 9.4 ⫻ 104 CK5⫹ cells in the
bone marrow from group 2. This represented a 6-fold increase in
the mobilization of CK5⫹ cells in the circulation and a 2-fold
increase in the enrichment of the bone marrow CK5⫹ cells in
animals treated with CXCL12. For group 3, we found 2.7 ⫻ 105 ⫾
5.4 ⫻ 104 CK5⫹ cells in the buffy coat and 2.2. ⫻ 106 ⫾ 1.8 ⫻ 105
CK5⫹ cells in the bone marrow as compared with 6.0 ⫻ 104 ⫾
1.2 ⫻ 104 CK5⫹ cells in the buffy coat and 5.6 ⫻ 105 ⫾ 1.1 ⫻ 105
CK5⫹ cells in the bone marrow from group four. This represented
a 4.5-fold increase in mobilization of CK5⫹ cells in the circulation
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Positive Cells/Total
Cells Counted
transplanted into CK5-GFP mice. Immunohistochemistry of GFP
in the wild-type tracheas in CK5-GFP mice at day 21 posttransplantation demonstrated GFP⫹ cells in the regenerating basal cells
of the airway (Fig. 2D, i). CK5-GFP tracheas transplanted into
CK5-GFP mice demonstrated GFP expression in all basal cells
(Fig. 2D, ii), and wild-type tracheas transplanted into wild-type
mice did not demonstrate any GFP expression (Fig. 2D, iii).
To further confirm that the GFP⫹-labeled cells in the regenerating female tracheal epithelium were truly epithelial cells, dual
immunofluorescence for mouse CK5 and GFP was performed on
tracheal sections from female wild-type tracheas transplanted into
male GFP⫹ mice (Fig. 3). In female wild-type tracheas transplanted into sex-matched wild-type mice, we determined there was
no evidence for GFP⫹ cells (Fig. 3i, iii). The isotype Ab control
for CK5 demonstrated no staining for CK5 (Fig. 3, i). Dual CK5⫹
and GFP⫹ expression was seen in the basal cells of the GFP⫹ male
mouse tracheas transplanted into sex-matched GFP⫹ mice by yellow fluorescence, as demonstrated by colocalization of GFP-FITC
and CK5-Cy3 (Fig. 3, ii). As another control, CK5 expression was
seen in the basal cells of female wild-type tracheal epithelium
transplanted into sex-matched wild-type mice with Cy3 staining of
CK5 and no GFP-FITC staining (Fig. 3, iii). In wild-type female
tracheas transplanted into male GFP⫹ mice, CK5⫹ basal cells colocalized with circulation-derived GFP⫹ cells at both day 7 (9.2 ⫾
1.2%; p ⫽ 0.003; Fig. 3, iv, and Table I) and day 21 (18.6 ⫾ 5.1%;
p ⫽ 0.012; Fig. 3, v, and Table I) posttransplant. Cells that were
only positive for GFP represented other circulating cell populations that had trafficked to the transplant (Fig. 3, v, green arrow) as
well as apical cells of the regenerating airway epithelium (Fig. 3,
v, green arrows).
The Journal of Immunology
1921
and a 3.9-fold increase in enrichment of the bone marrow CK5⫹
cells in animals treated with CXCL12. For group 5, we found 2 ⫻
105 ⫾ 4.1 ⫻ 104 CK5⫹ cells in the buffy coat and 2.8 ⫻ 106 ⫾
5.5 ⫻ 105 CK5⫹ cells in the bone marrow as compared with 4.4 ⫻
104 ⫾ 8.7 ⫻ 103 CK5⫹ cells in the buffy coat and 5.3 ⫻ 105 ⫾
1.1 ⫻ 105 CK5⫹ cells in the bone marrow from group 6. This
represented a 4.6-fold increase in mobilization of CK5⫹ cells in
the circulation and a 5.2-fold increase in enrichment of the bone
marrow CK5⫹ cells in animals treated with CXCL12 (Fig. 4).
Temporal and spatial expression of CXCL12 during
regeneration of airway epithelium
Because the CXCR4/CXCL12 biological axis has been found to be
essential for adult stem cell homing and specific subpopulations of
leukocyte recruitment (12, 19 –21), we postulated that the CXCR4/
CXCL12 axis could play a role in the recruitment of circulating
progenitor epithelial cells. The justification for our hypothesis was
that CXCR4 expression has been detected on other epithelial
FIGURE 4. CXCL12 mobilization and enrichment of bone marrow. A, Time course of mobilization in response to multiple s.c. injections of 100 ␮g of
CXCL12 (groups 1, 3, and 5) or control vehicle (groups 2, 4, and 6) in C57BL/6 mice given on day 1, days 1 and 2, or days 1, 2, and 3 given 24 h apart
and analyzed 1 h after the last injection (five mice per group). Significant mobilization of CK5⫹ cells is seen at all time points measured in response to
CXCL12, although the most mobilization was seen at 1 h post the initial dose of CXCL12 (group 1). ⴱ, p ⬍ 0.01; ⴱⴱ, p ⬍ 0.001. B, Time course of expansion
of CK5⫹ cells in the bone marrow in response to multiple s.c. injections of 100 ␮g of CXCL12 (groups 1, 3, and 5) or control vehicle (groups 2, 4, and
6) in C57BL/6 mice given on day 1, days 1 and 2, or days 1, 2, and 3 given 24 h apart and analyzed 1 h after the last injection. (five mice per group).
Significant expansion of CK5⫹ cells in the bone marrow is seen at all time points measured in response to CXCL12, although the greatest expansion was
seen in the group of mice that received three doses of CXCL12 (group 5). ⴱ, p ⬍ 0.01; ⴱⴱ, p ⬍ 0.001.
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FIGURE 3. Colocalization of circulating male GFP⫹ cells with CK5 by dual immunofluorescence with primary Ab to CK5 labeled with Cy3 and GFP
labeled with FITC in a tracheal epithelium. i, Isotype Ab control demonstrated background Cy3 staining of the basement membrane but no staining of cells.
ii, Control nontransplanted homozygous male GFP⫹ trachea with colocalization of CK5-Cy3 and GFP-FITC resulting in yellow staining of the basal cells
(arrows). iii, Control nontransplanted wild-type female trachea with red CK5-Cy3 staining of basal cells (arrows). iv, Wild-type female trachea day 7
posttransplant into a male GFP⫹ mouse with colocalization of CK5-Cy3 and GFP-FITC (yellow staining) in basal cells (white arrows) and CK5-Cy3
staining only in other cells (red staining, dashed white arrows). v, Day 21 posttransplant with chimerism of basal cells with staining for CK5-Cy3 and
GFP-FITC (yellow staining, white arrows) and other cells expressing only CK5 (red staining, dashed white arrows). GFP-FITC staining is seen in apical
epithelial cells (FITC staining, green arrows), illustrating chimerism in the regenerating epithelium. Scale bars, 10 ␮m. See Table I for quantitation.
1922
CXCR4/CXCL12 IN AIRWAY REPAIR
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progenitor cell types, such as the crypts of intestinal epithelium
(19), and that CXCL12 expression occurs after hypoxic injury,
which appears to create a chemotactic gradient in ischemic tissue
for the migration of CXCR4⫹ cells (19, 21). Thus, CXCR4⫹ circulating progenitor epithelial cells would be expected to home to
the regenerating epithelium after tracheal transplantation.
Our aim was to demonstrate that the CXCR4/CXCL12 biological axis is important in the trafficking of these circulating progenitor epithelial cells. Therefore, we first compared CXCL12 mRNA
expression at 3 and 7 days posttransplant by quantitative real-time
PCR and found CXCL12 to be markedly increased over uninjured
tracheal levels (20-fold at day 3 ( p ⫽ 0.01) and 50-fold at day 7
( p ⬍ 0.001) posttransplant, respectively) (Fig. 5A). Next, we assessed the kinetic expression of CXCL12 protein in sex-mismatched tracheal transplants as compared with naive tracheas.
CXCL12 protein was measured from tracheal tissue homogenates
using the Luminex assay. We found that CXCL12 protein levels
were the highest at day 3 and were persistently elevated from day
3 out to day 21 posttransplantation (Fig. 5B). We next performed
immunohistochemistry of CXCL12 in tracheal transplant tissue to
examine its temporal and spatial expression. CXCL12 was expressed in the lumen of the submucosal glands and ducts at day 3
posttransplant (Fig. 5C, i and ii). The CXCL12 staining was
present in the mucus glands within the mucus vacuoles, consistent
with the ability of CXCL12 to bind to glycosaminoglycans present
in mucus. When CXCL12 is bound to glycosaminoglycans, its
biological half-life is long-lived and is cleared by binding to the
CXCR4 receptor (19). By day 7 posttransplant the expression of
CXCL12 changed spatially from the submucosal glands and duct
epithelium to the basal cells (Fig. 5C, iii). By day 21 posttransplant, CXCL12 expression spatially moved to the apical surface of
the pseudostratified columnar epithelium (Fig. 5C, iv). The spatial
expression of CXCL12 in the pseudostratified epithelium at day 21
posttransplant was similar to its location seen in uninjured tracheal
epithelium. Morphometric analysis of epithelium from proximal to
distal tracheal sections demonstrated similar patterns of CXCL12
expression for all of the tracheal transplantations at each time
point. The temporal and spatial change in the pattern of expression
of CXCL12 directly correlated with that of the spatial orientation
of progenitor epithelial cells as they formed a pseudostratified
epithelium.
CXCR4/CXCL12 biological axis mediates recruitment of
circulating progenitor epithelial cells and regeneration of
pseudostratified airway epithelium
Because the temporal and spatial expression of CXCL12 was coincident with the regeneration of sex-mismatched chimeric
pseudostratified epithelium in female tracheas transplanted into
male mice, we hypothesized that the CXCR4/CXCL12 biological
axis is important for the recruitment of circulating progenitor epithelial cells. Therefore, our aim was to determine whether the
CXCR4/CXCL12 biological axis is important in recruiting the circulating progenitor epithelial cells. We thus performed passive immunization experiments with either neutralizing anti-CXCL12 or
control F(Ab⬘)2 Abs in male GFP⫹ mice that had received a female
wild-type trachea by using a modification as described previously
(13). Based on the fact that complete regeneration of pseudostratified epithelium was noted by day 21 post-tracheal transplantation,
we used this time point to assess the effect of neutralizing Abs on
circulating progenitor epithelial cells during the regeneration of the
tracheal epithelium. Regeneration of normal pseudostratified columnar epithelium was seen consistently throughout the tracheal
transplants from mice treated with control Ab (Fig. 6, i and iii).
Whereas re-epithelialization of the tracheal epithelium was seen in
FIGURE 5. CXCL12 is temporally and spatially expressed in the airway during re-epithelialization of the pseudostratified epithelium. A, Quantitative real-time PCR of CXCL12 mRNA expression. CXCL12 expression
was increased in the tracheal transplants ⬎20-fold above that of uninjured
control trachea at day 3 posttransplant (p ⫽ 0.01) (n ⫽ 3). CXCL12 mRNA
increased further in the tracheal transplants, ⬎50-fold above uninjured trachea at day 7 posttransplant (p ⬍ 0.001) (n ⫽ 3). B, Kinetic analysis of
CXCL12 protein levels of sex-mismatched tracheal transplants as compared with naive control tracheas. CXCL12 protein was measured from
tracheal tissue homogenates using the Luminex assay. C, Immunohistochemistry of CXCL12 in tracheal transplants. i and ii, CXCL12 is expressed in the mucus vacuoles in the lumen of the submucosal glands and
ducts of the trachea at day 3 posttransplant (arrows). iii, CXCL12 expression changed spatially to the submucosal gland cells, ducts, and basal cells
at day 7 posttransplant. iv, In tracheas at day 21 posttransplant, CXCL12
expression was always present on the apical surface of the pseudostratified
columnar epithelium (arrow) and was similar to that in naive tracheas (not
shown). Scale bars, 10 ␮m.
The Journal of Immunology
1923
the anti-CXCL12-treated group at day 21, the phenotype of the
epithelium was squamous metaplasia (Fig. 6, ii and iv). Morphometric analysis of tracheal transplant sections of the entire transplant showed squamous metaplasia throughout the epithelium in
the anti-CXCL12-treated group. The mean percentage of squamous metaplasia of the tracheal circumference was 71.2 ⫾ 4.1%
( p ⬍ 0.0001). To assess the epithelium for GFP⫹ male cells, we
assessed both immunohistochemistry and immunofluorescence of
GFP⫹ cells. The areas of squamous metaplasia of sex-mismatched
wild-type tracheas transplanted into GFP⫹ mice had re-establishment of the epithelium derived solely from female resident progenitor epithelial cells, with no contribution of male GFP⫹ circulating progenitor epithelial cells by immunohistochemistry and
immunofluorescence for GFP⫹ cells, respectively (Fig. 7, i and ii).
In contrast, the regeneration of normal pseudostratified epithelium
in sex-mismatched tracheas transplanted into GFP⫹ mice treated
with control Ab demonstrated the presence of both resident (female) and circulating GFP⫹ (male) progenitor epithelial cells (Fig.
7, iii). FACS analysis showed a decrease in the CK5⫹CXCR4⫹
population of cells in the anti-CXCL12-treated mouse tracheal
transplants, with the absolute CK5⫹CXCR4⫹ cell numbers
decreasing from 11 ⫻ 105 ⫾ 2.3 ⫻ 105 to 4 ⫻ 105 ⫾ 0.9 ⫻ 105,
respectively ( p ⫽ 0.03; Fig. 8). FACS analysis of leukocyte subpopulations showed no significant difference in the numbers of
polymorphonuclear cells, lymphocyte populations, and NK cells in
the tracheas from either the control or anti-CXCL12 Ab-treated
groups at day 21 posttransplantation. However, there was a statistical decrease in the number of macrophages in the anti-CXCL12treated tracheal transplants ( p ⫽ 0.014; Fig. 9).
p63 is a member of the p53 family of proteins and is a recognized marker of the reserve cell hyperplasia reflective of the basal
regenerative cells of many epithelial tissues as well as squamous
FIGURE 7. Squamous metaplasia
of transplant airway epithelium is derived solely from female resident tissue progenitor epithelial cells at day
21inmicetreatedwithneutralizingantiCXCL12 Abs. i, Lack of peroxidase
staining for GFP⫹ cells in areas of
squamous metaplasia, with a recipient
cell staining for GFP in the submucosal area (arrow) in female tracheal
transplants from GFP⫹ mice treated
with anti-CXCL12 Abs. ii, Lack of
GFP⫹ cell immunofluorescence in areas of squamous metaplasia, with a
few recipient cells staining for GFP in
the submucosal area (arrow) in female
tracheal transplants from GFP⫹ mice
treated with anti-CXCL12 Abs. iii,
Immunofluorescence for GFP⫹ cells
in female tracheal transplants from
GFP⫹ mice treated with control Abs.
The trachea demonstrates chimerism
for GFP⫺ and GFP⫹ cells in the regenerated pseudostratified epithelium.
Scale bars, 10 ␮m.
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FIGURE 6. Depletion of CXCL12
inhibits the recruitment of CXCR4⫹
circulating progenitor epithelial cells
and leads to squamous metaplasia of
the regenerating airway epithelium in
response to injury, as seen by H&E
staining. i and iii, Representative example of regeneration of an airway
pseudostratified columnar epithelium
day 21 posttransplant in mice treated
with control Ab (magnifications,
⫻200 and ⫻400 for i and iii, respectively). ii and iv, Representative example of squamous metaplasia of the regenerated airway epithelium at day 21
posttransplant in animals treated with
neutralizing Abs to CXCL12 (magnifications, ⫻200 and ⫻400 for ii and
iv, respectively). Scale bars, 10 ␮m.
1924
FIGURE 8. FACS analysis of CK5⫹CXCR4⫹ cells in tracheal transplants. There is a significant reduction of CK5⫹CXCR4⫹ cells in the tracheal transplants at 21 days posttransplant (p ⫽ 0.03).
Discussion
Our findings demonstrate that there is a population of progenitor
epithelial cells in the bone marrow that is CK5⫹CD45⫹CXCR4⫹.
Other groups have shown the presence of mature cytokeratins such
as pancytokeratin and CK19-expressing populations in the bone
marrow (23, 24). However, to our knowledge this is the first report
of CK5⫹ progenitor epithelial cells present in the bone marrow.
This population of cells was also identified in the circulation under
homeostatic conditions. Moreover, these cells can be found recruited to the airway epithelium in response to injury, implying
that the cells may be mobilized into the circulation in response to
tissue injury and repair. This finding is supportive of the hypothesis that repair and regeneration of the airway epithelium may be
enhanced by circulating progenitor epithelial cells derived from
the bone marrow. Our FACS analysis studies identified a higher
percentage of CK5⫹ cells in the bone marrow and circulation of
mice than we would have predicted under homeostatic conditions.
This may be a phenomenon unique to mice, because we have detected a much smaller percentage of CK5⫹ cells in the buffy coat
of humans (0.5–1%) under homeostatic conditions. We propose
that the circulating progenitor epithelial cells may represent a precursor population of cells for a number of epithelial tissues that
would include the airway, lung parenchyma, skin, and prostate,
with CK5 basal cells representing an important component of the
regenerating epithelium of these tissues. The possibility exists in
the future that culturing these cells in vitro will allow us to further
characterize these CK5⫹ progenitor cells. Furthermore, we cannot
exclude the possibility that the higher number of CK5⫹ cells in
circulation of mice, as compared with humans, may represent turnover of fur.
Our explanation for the detection of more progenitor epithelial
cells in the circulation than in the bone marrow may be related to
FIGURE 9. FACS analysis of leukocyte subpopulations in tracheal
transplants. No significant difference is seen between the percentages of
CD3, CD4, and CD8 lymphocytes, NK cells, or polymorphonuclear cells at
day 7 posttransplant in the tracheal transplants between control-treated and
anti-CXCL12-treated mice. (p ⫽ 0.081, 0.52, 0.06, 0.64, and 0.453, respectively). There was a significant decrease in the percentage of macrophages (MAC) found in the CXCL12-treated tracheas compared with control (p ⫽ 0.014).
the fact that these cells are most likely in a state of transition as
they mobilize and traffic with gain of CK5 expression. In addition,
a previous study by Phillips et al. (13) demonstrated with another
circulating progenitor cell population, namely fibrocytes, that they
gain the more differentiated marker of ␣-smooth muscle actin expression as they traffic to a bleomycin-injured lung.
We have demonstrated that in the bone marrow the
CD45⫹CK5⫹ cells are uniformly positive for CXCR4, but in the
circulation approximately half of the cells lose CXCR4. CXCR4 is
down-regulated after transition from bone marrow to the peripheral blood because of the change in oxygen tension and protein
degradation. In addition, a previous study by Phillips et al. (13)
demonstrated that differentiation of a fibrocyte to an ␣-smooth
muscle actin-positive cell was associated with reduced expression
of CXCR4 under normoxic conditions. Therefore, we hypothesize
that maturation of a progenitor cell may contribute to the loss of
CXCR4 expression. In the tracheal transplants the number of
CK5⫹CXCR4⫹ cells may be underestimated, because CXCR4
positivity would be expected to decrease as the cells bind to their
CXCL12 ligand and the chemokine receptor is internalized and
degraded.
The CK5⫹ cells in the bone marrow and circulation express
CD45 and CXCR4. Whereas CXCR4 is expressed by neutrophils,
CXCR4 is the salient chemokine receptor found on several leukocytes and progenitor cells, and, therefore, these progenitor cells
may use CXCR4 to traffic into tissue. CXCR4 is included in membrane lipid rafts to more efficiently interact with other surface receptors such as downstream proteins of signal transduction pathways and, therefore, to induce chemotaxis. The receptor-like
protein tyrosine phosphatase CD45, the common leukocyte Ag,
can also be found in lipid rafts (25) and, therefore, may be important in facilitating the interaction of CXCL12 with the CXCR4
receptor, allowing membrane-proximal signaling events to proceed. Therefore, the expression of CD45 on progenitor cells that
traffic via the CXCL12/CXCR4 pathway is not unexpected.
Bone marrow mesenchymal cells have been shown to form pulmonary epithelial cells of both proximal and distal airways (9, 26,
27). These studies used markers of bone marrow-derived cells such
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metaplasia (22). Therefore, our aim was to determine whether the
areas of squamous metaplasia expressed p63. We thus examined
p63 expression by immunofluorescence in the tracheal transplants.
In mice treated with neutralizing anti-CXCL12, p63 expression
was not only found in the basal nuclei but in nuclei throughout the
areas of squamous metaplasia of the regenerated epithelium at day
21 posttransplant, compatible with reserve cell hyperplasia of female resident basal cells (Fig. 10, i). In contrast, mice treated with
control Abs only demonstrated p63 expression in the nuclei of the
basal cells at day 21 posttransplant (Fig. 10, ii). An isotype control
Ab showed no immunofluorescence (Fig. 10, iii).
CXCR4/CXCL12 IN AIRWAY REPAIR
The Journal of Immunology
1925
FIGURE 10. p63 expression in the
female tracheal epithelium of tracheal
transplants in GFP⫹ male mice treated
with neutralizing CXCL12 or control
Abs. i, p63 expression at day 21 posttransplant in areas of squamous metaplasia from female tracheal transplant
in male GFP⫹ mice treated with specific neutralizing anti-CXCL12 Abs.
p63 is seen associated with all squamous metaplastic cells (arrows). ii,
p63 expression at day 21 posttransplant in pseudostratified epithelium
from female tracheal transplants in
GFP⫹ mice treated with control Abs.
p63 is seen associated with only the
basal cells. Immunofluorescence of
p63 is seen by anti-p63-Cy3 (arrows).
iii, Isotype control Ab with no p63
staining of basal cells (arrows). Scale
bars, 10 ␮m.
stromal cells in vivo. The findings from these two studies have
implications for therapy using bone marrow-derived epithelial
cells in lung diseases such as cystic fibrosis. Our data suggests that
isolation of CK5⫹ progenitor epithelial cells from the bone marrow and/or circulation could be useful for identifying cells ideal
for ex vivo manipulation before autologous transplantation.
Our study demonstrated a mechanism for recipient circulating
progenitor epithelial cell trafficking to the injured airway through
the CXCL12/CXCR4 biological axis. This biological axis has been
found to be critical for the regulation of trafficking of other adult
stem cells in response to injury and repair of tissues such as the
liver bile duct epithelium (33). We found significant and early
mobilization of CK5⫹ cells in the circulation of mice treated with
CXCL12, whereas enrichment of CK5⫹ cells in the bone marrow
appeared to occur later. However, we cannot fully exclude the
possibility that these circulating progenitor epithelial cells may be
derived from a source other than the bone marrow and could just
be “hiding out” in the bone marrow as other authors have suggested (23, 34). Other tissue sources are possible, including the
possibility of peripheral differentiation of leukocyte subpopulations. However, given the protected environment of the bone marrow and its high levels of CXCL12, it is feasible that the bone
marrow could be the home of both hemopoietic and nonhemopoietic progenitor cells.
The presence of CXCR4 on undifferentiated epithelial cells in
tissues such as pulmonary and intestinal epithelia suggests a wider
role for CXCL12/CXCR4 in the regulation of cell homing (13, 19,
20). In addition, the CXCL12/CXCR4 biological axis is critical in
the normal development of several organs, suggesting that this
biological axis may play a role in the migration of most adult
progenitor cells (19). We demonstrated a temporal and spatial increase in the expression of CXCL12 in the epithelium after airway
injury related to tracheal transplantation, which provided a gradient for infiltration of CXCR4⫹ recipient circulating progenitor epithelial cells (21). The presence of CXCR4 on CK5⫹ bone marrow- and circulation-derived progenitor epithelial cells further
supports the role of this receptor in the recruitment of circulating
progenitor epithelial cells. In addition, the temporal/spatial change
of CXCL12 expression during the regeneration of the airway
epithelium mimicked the temporal and spatial recruitment and orientation of progenitor epithelial cells during re-epithelialization of
the basement membrane.
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as GFP or the Y chromosome. Using these same biomarkers, other
groups have also found bone marrow-derived chimerism in the
lung, especially in the setting of tissue injury (15, 24, 26 –28). In
general, these studies all specifically examined distal lung/parenchymal chimerism after lung injury due to bleomycin, radiation, or
LPS instillation. The donor chimerism ranged from ⬍1 to 14% of
pneumocytes. Our study is the first to evaluate circulating progenitor epithelial cells and the mechanism of their trafficking in the
setting of airway epithelial injury by using a mouse model of tracheal transplantation. The ischemia-reperfusion injury results in
complete sloughing of the airway epithelium from the basement
membrane, which may explain why we saw a relatively high percentage of circulation-derived progenitor epithelial cells in the epithelial repair process. This type of injury may appear severe; however, the injury is clinically relevant to lung transplantation.
During lung transplantation the airway is exposed to significant
ischemia and reperfusion injury, because the bronchial circulation
is usually not reanastomosed at the time of reimplantation. In addition, other forms of airway injury such as severe viral infections
related to respiratory syncytial virus or influenza may result in
significant epithelial injury and sloughing of the airway epithelium
and, therefore, may be relevant to our mouse model.
Using markers for both the Y chromosome and GFP⫹ recipient
cells, we saw similar numbers of recipient circulating progenitor
epithelial cells that contributed to regeneration of the normal airway pseudostratified epithelium. These cells were also found to
contribute to the resident progenitor epithelial cells seen in the
submucosal gland ducts. The recipient circulating progenitor epithelial cells were present and increased in number at day 21 posttransplantation. There is controversy as to whether cell fusion of
circulating progenitor epithelial cells occurs with local resident
progenitor cells during regeneration of the airway epithelium (29,
30). However, we failed to identify any binucleate cells in histologic sections of the chimeric tracheas and found only one Y chromosome per cell by either FISH or CISH techniques. These findings support the notion that stable cell fusion did not occur, but we
acknowledge that our experiments were not designed to detect reductive division of fused cells.
Wang et al. (31) recently demonstrated the ability of bone marrow cells to develop a pulmonary epithelial cell phenotype when
cocultured with pulmonary epithelial cells. These cells were amenable to retroviral transduction and gene therapy (31). Grove et al.
(32) found similar results with bone marrow-derived mesenchymal
1926
in the airway in the setting of chronic injury results in dysplasia
and subsequent carcinoma in situ. This possibility is currently being investigated in our laboratory.
The ⌬Np63␣ splice variant of p63 is the predominant form of
p63 in basal epithelial cells. p63 functions as a dominant-negative
inhibitor of the tumor suppressor gene p53. p63 counteracts the
apoptotic and cell cycle inhibitory role of p53 and contributes to
cell renewal of the pseudostratified epithelium. p63 has been
shown to be highly expressed in areas of abnormal cellular proliferation characterized by squamous metaplasia as well as squamous cell carcinomas and, therefore, may play an oncogenic role
in epithelial transformation. Whether p63 plays a causative role or
is merely temporally and spatially expressed during tumor development remains to be fully elucidated (22). Our finding of p63
expression in all cells associated with squamous metaplasia in
mice treated with an anti-CXCL12 Ab suggests that the abnormal
proliferation of donor resident progenitor epithelial cells in the
absence of recipient circulating progenitor epithelial cells may be
due to p63 inhibition of p53. Future studies will determine whether
the persistence of p63 expression will lead to oncogenic transformation of the squamous metaplastic epithelium in the absence of
the trafficking of circulating epithelial progenitor cells.
Our studies demonstrate the following sequence of events: 1) a
population of oriented progenitor cells expressing the epithelial
marker CK5 is harvested in the bone marrow; 2) these cells passing into the circulation provide a cellular pool able to repair damaged tracheal epithelium; 3) the CXCL12/CXCR4 axis is involved
in epithelial precursor mobilization and recruitment at sites of injury; 4) CK5⫹CXCR4⫹ cells have a crucial role in the re-epithelialization of tracheal transplants; and 5) CXCL12 blocking prevents precursor recruitment and appropriate epithelial repair and
favors squamous metaplasia. This sequence has major implications
for the therapeutic use of circulating progenitor epithelial cells in
a number of respiratory diseases where normal airway repair and
regeneration is required, such as infections like respiratory syncytial virus, severe acute respiratory syndrome, influenza, and inflammatory conditions such as cystic fibrosis, asthma, and chronic
obstructive pulmonary disease. Furthermore, our studies demonstrate that, if circulating progenitor epithelial cell recruitment is
impaired, the airway epithelium develops a squamous metaplastic
phenotype, creating a novel paradigm for the development of squamous metaplasia consisting of only resident epithelial progenitor
cells.
Disclosures
The authors have no financial conflict of interest.
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The depletion of CXCL12 with specific neutralizing Abs was
associated with the loss of the normal pseudostratified epithelium
and a change of the phenotype of the epithelium to squamous
metaplasia at day 21 posttransplant. Moreover, the areas of squamous metaplasia were completely devoid of sex-mismatched cells,
suggesting that recruitment of recipient CK5⫹CXCR4⫹ circulating progenitor epithelial cells is both necessary and sufficient to
re-establish the normal airway pseudostratified columnar epithelium. The areas of the tracheal transplant epithelium that had undergone squamous metaplasia under conditions of CXCL12 depletion represented 71.2 ⫾ 4.1% of the total epithelium by
morphometric analysis, which suggests that we were not able to
completely block all circulating CK5⫹CXCR4⫹ cells from contributing to repair of the epithelium. This is compatible with our
FACS data showing a significant decrease in the numbers of
CK5⫹CXCR4⫹ cells in the tracheal transplants after CXCL12 depletion but not complete abrogation.
Although we cannot fully exclude the possibility of an indirect
effect related to depletion of CXCL12 leading to squamous metaplasia, we would however argue in favor of a direct effect as follows: 1) CK5⫹ cells in circulation express CXCR4; 2) CK5⫹ cells
from the recipient (Y chromosome-positive, GFP⫹, and CK5GFP⫹) are present in the regenerating epithelium of the tracheal
transplant; 3) CXCL12 and CXCR4 are both temporally and spatially present during the regeneration of the airway epithelium in
the tracheal transplant; 4) depletion of CXCL12 leads to squamous
metaplasia with loss of recruited recipient CK5⫹ cells and, at the
same time, metaplasia of resident epithelial progenitor cells undergoing reserve cell hyperplasia with p63; and 5) depletion of
CXCL12 is directly related to a marked reduction in dual
CK5⫹CXCR4⫹ cells in the regenerating airway epithelium of the
tracheal transplant.
Squamous metaplasia results from reserve cell hyperplasia, and
continuous exposure to environmental stimuli subjects these cells
to epigenetic events and may predispose them to oncogenic transformation. The changes of squamous metaplasia in our model are
analogous to those seen in the airways of patients with chronic
bronchitis and may invoke the notion that repetitive airway injury
in these patients may be associated with inadequate recruitment of
circulating progenitor epithelial cells that could contribute to reestablishment of the normal pseudostratified epithelium. In support
of this concept, patients with congenital bone marrow failure syndromes have been found to be at increased risk for the development of squamous carcinomas (35). Although the etiology for the
epithelial cancers seen in these patients is presumed to be related
to a defect in DNA repair, it is plausible that these same patients
may lack bone marrow-derived progenitor epithelial cells. As a
consequence, these patients may have abnormal epithelial repair in
areas with high cellular turnover that may predispose the resident
cells to epigenetic events (35).
Houghton et al. (36) recently described a model of chronic gastric epithelial injury leading to gastric cancer. In this study, bone
marrow-derived cells repopulated the injured gastric mucosa and
developed metaplasia, dysplasia, and cancer over an extended period of time. In contrast to that study, we found the opposite findings, where inhibiting the recruitment of recipient circulating progenitor epithelial cells by blocking CXCL12 resulted in squamous
metaplasia, which was derived exclusively from resident female
donor progenitor epithelial cells. It is possible that various tissues
respond differently to injury, and in gastric mucosa, where very
few bone marrow-derived cells contribute to the regenerating epithelium, the signals for normal differentiation may be entirely different than those in the trachea. It is not known whether persistent
inhibition of the trafficking of circulating progenitor epithelial cells
CXCR4/CXCL12 IN AIRWAY REPAIR
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