KGF pretreatment decreases B7 and granzyme B expression and
hastens repair in lungs of mice after allogeneic BMT
Angela
Panoskaltsis-Mortari1,
David H.
Ingbar2,
Patricia
Jung2,
Imad Y.
Haddad2,
Peter B.
Bitterman2,
O. Douglas
Wangensteen3,
Catherine L.
Farrell4,
David
L.
Lacey4, and
Bruce R.
Blazar1
1 Department of Pediatrics, Division of
Hematology-Oncology and Bone Marrow Transplantation, and Departments of
2 Pulmonary Critical Care Medicine and
3 Physiology, University of Minnesota,
Minneapolis, Minnesota 55455; and 4 Amgen,
Thousand Oaks, California 91320
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ABSTRACT |
We investigated keratinocyte growth factor (KGF) as a
pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT)
period. B10.BR (H2k) recipient mice were transplanted with
C57BL/6 (H2b) BM with spleen cells after lethal
irradiation with and without cyclophosphamide conditioning with and
without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer
injured alveolar type II (ATII) cells at the time of BMT and exhibited
ATII cell hyperplasia at day 3 post-BMT. The composition of
infiltrating cells on day 7 post-BMT was not altered by KGF
pretreatment, but the frequencies of cells expressing the T-cell
costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin
granzyme B (usually increased in IPS) were decreased by KGF. Sera from
KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4,
IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at
the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2)
attenuation of immune-mediated injury as a consequence of failure to
upregulate cytolytic molecules and B7 ligand expression and the
induction of anti-inflammatory Th2 cytokines in situ.
bone marrow transplant; keratinocyte growth factor; type II
pneumocytes; cytokines; macrophages; costimulatory molecules
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INTRODUCTION |
IDIOPATHIC PNEUMONIA SYNDROME (IPS) is a significant
cause of non-graft-versus-host disease (non-GVHD) deaths after bone
marrow (BM) transplant (BMT) and accounts for the majority of
complications involving the lung in the early post-BMT period (9).
Intense conditioning regimens lead to a higher incidence of pulmonary toxicity in BMT recipients but are beneficial in preventing relapse and
promoting BM engraftment (11, 20, 26, 43, 47, 55). Greater severity of
GVHD post-BMT also increases the risk of developing IPS. In a mouse
model study evaluating the contributions of preconditioning with total
body irradiation (TBI) with and without cyclophosphamide (Cy) and
allogeneic T cells to the generation of IPS, we reported that the
severity of early post-BMT IPS injury was dependent on allogeneic T
cells and potentiated by Cy (42). IPS injury was associated with the
recruitment of host monocytes and donor T cells into the lung in
response to tissue injury (42). The association of IPS with host
monocyte infiltration and the dependence on allogeneic T cells has
since been confirmed independently by two other laboratories (see Refs.
10, 13). The manifestations of lung injury we previously described
included epithelial cell injury, increased wet and dry lung weights,
and decreased specific lung compliance and lung capacity. Because
almost all of the macrophages in the lung are host derived at this
early time point post-BMT (42), measures to hinder the activation of
immune effectors of the recipient may present alternative strategies
for the prevention of IPS.
In response to injury in the lung, type II alveolar epithelial (ATII)
cells proliferate and differentiate to replace dying type I epithelial
pneumocytes (27a). The degree of lung injury ultimately manifested is
to a large extent dependent on the ability of type II cells to
effectively carry out this process to reepithelialize the alveolar
membrane. Acute injury also releases intracellular contents that
trigger acute-phase reactants, cytokines, and chemokines to initiate
the inflammatory response and tissue repair process. This cascade can
involve the induction of selectins and adhesion molecules leading to
transmigration of inflammatory cells into the alveolar space. Acute
lung inflammation is correlated with increased levels of
proinflammatory cytokine mRNA in the lung (31, 44, 46, 53, 54, 58).
Studies of bronchoalveolar lavage (BAL) fluid in mice following BMT
across minor histocompatible differences demonstrated that increased
levels of tumor necrosis factor (TNF)-
and endotoxin
[lipopolysaccharide (LPS)] at 6 wk post-BMT were associated
with lung damage and IPS generation (12). In addition, we also
described that the frequency of cells expressing T-cell costimulatory
B7 molecules increased in the lung, as did the frequency of cells
expressing mRNA for transforming growth factor (TGF)-
(a monocyte
chemoattractant), IL-1
, and TNF-
(42). This sets the stage for an
immune-mediated attack on lung tissue by allospecific donor T cells,
since activation of monocytes and their subsequent increased expression
of B7 regulates the generation of cytotoxic T lymphocytes (CTL) that
express the cytolysins granzymes A and B and, therefore, may
contribute to the amplification of tissue injury.
KGF is a mediator of epithelial cell proliferation (24) and
mesenchymal-epithelial interactions (23) as well as a growth factor for
type II pneumocytes (39, 52). KGF is protective against chemotherapy-
and radiation-induced injury in various rodent models (22, 51). We
recently reported that in vivo administration of exogenous KGF,
completed before conditioning, ameliorated GVHD-induced weight loss and
mortality following allogeneic BMT in mice (41). In addition, KGF
diminished GVHD-induced lesions in the target organs, especially the
skin and lung, of long-term allogeneic BMT survivors. The protective
effect of KGF on GVHD also has been confirmed recently in another
murine model (34). In investigations targeting pulmonary injury, KGF
was protective in lethal models of radiation-, hyperoxia-, acid-, and
bleomycin-induced lung injury in rats (28, 60, 62), possibly by
facilitating repair of DNA damage in alveolar epithelial cells (50,
57). Furthermore, KGF induces increased lung surfactant levels (49), potentiates alveolar fluid clearance by increasing
Na+-K+-ATPase activity (6), decreases
hyperoxia-induced apoptosis of (ATII) cells, and may detoxify reactive
oxygen species generated by injured cells (reviewed in Ref. 56). Here,
we report the effects of KGF on the IPS-inducing inflammatory events in
the lung. The goal of this study is to investigate some potential mechanisms of KGF-mediated amelioration of lung injury in the peri-BMT
period. Our data indicate that KGF diminishes IPS injury, at least in
part, by dampening the immune system response to
chemoradiotherapy-induced lung damage and by accelerating repair of the
damaged tissue.
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MATERIALS AND METHODS |
Mice.
B10.BR (H2k) and C57BL/6 (H2b) mice were
purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed
in microisolator cages in the SPF facility of the University of
Minnesota and cared for according to the Research Animal Resources
guidelines of our institution. For BMT, donors were 8-12 wk of age
and recipients were used at 8-10 wk of age.
KGF production.
Recombinant human KGF produced in Escherichia coli was prepared
as previously described (52) at Amgen (Thousand Oaks, CA).
Pre-BMT conditioning.
B10.BR mice received PBS or KGF (5 mg · kg
1 · day
1
sc) on days
6,
5, and
4
pre-BMT. Mice were then segregated into those receiving either PBS or
Cy (Cytoxan; Bristol Myers Squibb, Seattle, WA), 120 mg · kg
1 · day
1,
as a conditioning regimen pre-BMT on days
3 and
2. All mice were lethally irradiated on the day
before BMT (7.5-Gy TBI) by X-ray at a dose rate of 0.41 Gy/min as
described (3).
BMT.
Our BMT protocol has been described previously (5). Briefly, donor
C57BL/6 BM was T cell depleted (TCD) with anti-Thy 1.2 monoclonal antibody (MAb) (clone 30-H-12, rat IgG2b, kindly
provided by Dr. David Sachs, Charlestown, MA) plus complement
(Nieffenegger, Woodland, CA). Recipient mice were transplanted via
caudal vein with 20 × 106 TCD C57BL/6
(H-2b) marrow with or without 15 × 106 NK
cell-depleted (PK136, anti-NK1.1 + complement) spleen cells (BMS) as a
source of IPS-causing T cells.
Electron microscopy.
This was performed as previously described (42). After inflation to
~20 cmH2O by hand, 2- to 3-mm3 pieces of lung
tissue were fixed in 2% glutaraldehyde for 1-2 days at 4°C
followed by postfixation in 1% osmium tetroxide (EM Sciences, Fort
Washington, PA) in 0.1 M sodium cacodylate buffer for 1 h, dehydrated
in graded ethanol and propylene oxide, and embedded in Epon 812 (EM
Sciences). Sections were cut at a thickness of 600 nm, stained with
uranyl acetate-lead citrate (EM Sciences), and examined with a Philips
301 electron microscope. A minimum of 17 prints (maximum 39) taken at a
magnification of ×4,200 from multiple sections from 2 representative mice of each group at day 0 and day 3 post-BMT were examined by three observers in coded fashion. The number
of type II cells present (based on morphological appearance and
presence of lamellar bodies) was expressed as a percent of total
nucleated cells.
Frozen tissue preparation.
After death of the mouse at either day 0, 3, or
7, a mixture of 0.5 ml of optimal cutting temperature compound
(Miles, Elkhart, IN) and PBS (3:1) was infused via the trachea into the
lungs. Lungs were snap-frozen in liquid nitrogen and stored at
80°C.
Immunohistochemistry.
After fixation in acetone, cryosections (4 µm) were immunoperoxidase
stained using biotinylated MAbs with avidin-biotin blocking reagents,
ABC-peroxidase conjugate, and diaminobenzidine chromogenic substrate purchased from Vector Laboratories (Burlingame, CA) essentially as described (4). The biotinylated MAbs used were as
follows: anti-CD4 (clone GK1.5), anti-CD8 (clone 2.43), anti-Mac-1 (clone M1/70), anti-Gr-1 (clone RB6-8C5), anti-I-Ak
(clone 11-5.2), anti-B7.1 (clone 1G10), and anti-B7.2 (clone GL1),
all purchased from PharMingen (San Diego, CA). Representative sections
from each tissue block were stained with hematoxylin and eosin for
histopathological assessment. The number of positive cells in the lung
was quantitated as the percent of nucleated cells under ×200
magnification (×20 objective lens). Four fields per lung were evaluated.
In situ hybridization.
This procedure has been described in detail elsewhere (40).
Cryosections (4 µm) were hybridized with digoxigenin-labeled antisense RNA probes. The corresponding ribonucleotide sequences used
were 80-910 bp for granzyme A and 239-775 bp for granzyme B. Immunological detection of digoxigenin-labeled RNA duplexes was
accomplished with anti-digoxigenin antibody (alkaline phosphatase conjugated; Boehringer Mannheim). After color development, sections were mounted in Crystalmount (Biomeda, Foster City, CA). Positive cells
were quantitated as described above.
Serum cytokine level determination.
At the time of death of the mouse, blood was collected by cardiac
puncture and placed immediately at 4°C; the serum was
separated at 4°C and stored at
80°C. Serum levels of
IL-1
, IL-4, IL-6, IL-10, IL-13, interferon (IFN)-
, and TNF-
were determined by ELISA using commercial kits (R&D Systems,
Minneapolis, MN).
Statistical analysis.
Data were analyzed by ANOVA (Dunnett's test) or Student's
t-test. Probability (P) values less than or equal to
0.05 were considered statistically significant.
 |
RESULTS |
KGF preconditioning preserves ATII cells in the lung after
allogeneic BMT.
To determine whether KGF administration (subcutaneous on days
6,
5, and
4 pre-BMT) would
affect early lung injury parameters documented previously by this
laboratory (42), experiments were set up to compare the effect of KGF
in the context of TBI or Cy/TBI conditioning as well as with
(BMS) or without (BM) the addition of a 100% lethal dose of allogeneic
spleen cells (see Fig. 1). B10.BR
recipients were infused with C57BL/6 cells and killed on either
day 0 (pre-BMT) or day 3 post-BMT, and lungs were
examined by electron microscopy. We chose days 0 and 3 for two reasons: 1) because the allogeneic cells are infused on
day 0, they would be exposed to early manifestations of
conditioning-induced injury and any potential tissue-altering effects
of KGF at this time; and 2) endothelial and epithelial cell
injury are evident at least as early as day 3 by electron
microscopy and represent the end of the first wave of host monocyte
infiltration (42).

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Fig. 1.
Keratinocyte growth factor (KGF) treatment and conditioning schedule
for bone marrow (BM) transplant (BMT) experiments. KGF (5 mg · kg 1 · day 1)
or PBS was administered subcutaneously to B10.BR recipients on
days 6, 5, and 4.
Mice were then further segregated into those receiving cyclophosphamide
(Cy; 120 mg · kg 1 · day 1)
or PBS intraperitoneally on days 3 and 2.
All recipients were irradiated (7.5 Gy) on the day before transplant
(day 1). Groups were segregated into those receiving
C57BL/6 BM alone or BM with allogeneic spleen cells (BMS)
intravenously. Lungs were harvested for analysis on days 0 (before BMT), 3, and 7 post-BMT. EM, electron
microscopy; IHC, immunohistochemistry; ISH, in situ hybridization; TBI,
total body inrradiation.
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At the time of BMT (day 0), the percentage of type II cells in
the alveolar areas, as a percent of nucleated cells counted by light
microscopy of hematoxylin and eosin-stained lung sections, was not
changed significantly by subcutaneous KGF pretreatment for any
treatment group (see Figs. 2 and
3). This was also confirmed by counting
ATII cells from electron micrographs (data not shown). Occasional small
foci of ATII cell clusters and diffuse cuboidal growth were seen in
hematoxylin and eosin-stained sections of lungs from KGF-pretreated
mice (see Fig. 3, blue and green arrows), but because these features
were sparse, they did not amount to a significant increase in ATII
number. However, because evidence of injured ATII cells could be seen
to some degree by light microscopy (shown in Fig. 3,
PBS/TBI and PBS/Cy/TBI photos as arrows
pointing to swollen-looking ATII cells), we decided to determine
whether KGF pretreatment affected the ratio of noninjured to injured
ATII cells by examination of electron micrographs from which injured cells are better discerned from noninjured cells. Figure 2 shows that,
from the representative mice we examined (n = 2/group), the
percent of intact, noninjured type II cells increased by 70% with KGF
pretreatment regardless of conditioning regimen, consistent with the
target cell specificity of KGF and its known cytoprotective effects
(52, 62). Morphological criteria used to identify injured ATII cells
included decreased density of intracellular components (edema), swollen
mitochondria, vacuolation, and lack of continuous discrete membranes
(representative examples are shown in Fig.
4). Formal morphometry was not performed
because we only wished to know whether there was a change in the ratio of noninjured to injured ATII cells. KGF pretreatment did not prevent
the minor focal irradiation-induced edema of the endothelium we
previously described (42). By day 3 post-BMT, all groups receiving allogeneic T cells exhibited increased cellularity of the
interstitium. Surprisingly at this time, KGF-pretreated mice additionally exhibited ATII epithelial cell hyperplasia compared with
non-KGF-pretreated mice, regardless of whether these mice also received
allogeneic spleen cells (day 3 Cy/TBI BMS mice
shown in Figs. 5 and
6). KGF pretreatment prevented the 25%
decrease in ATII cell number seen in BMS mice not given KGF (see Fig.
7). These findings suggest that KGF
mediated the preservation and, perhaps, proliferation of type II cells
that is necessary for their subsequent differentiation to replace
injured type I cells and maintain the integrity of the alveolar
epithelium.

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Fig. 2.
KGF pretreatment does not affect the number of alveolar type II (ATII)
cells by the day of BMT (day 0) but does preserve ATII cell
integrity. B10.BR recipient mice were preconditioned with TBI (day
1) with and without Cy (days 3 and
2). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 0 before BMT. Data
are expressed as percent of nucleated cells displaying ATII
characteristics (rounded nucleus, abundant cytoplasm, located at
alveolar septal vertices) as determined by counting hematoxylin and
eosin-stained lung cryosections under light microscope using ×40
objective lens with ×10 ocular. Mean values ± SD are for 3 mice
per group from 2 representative experiments. The proportion of
noninjured vs. injured ATII cells was determined by counting from
electron micrographs such as those shown in Fig. 4 that were generated
from 600-nm sections photographed under ×4,200 magnification.
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Fig. 3.
KGF-pretreated mice exhibit small focal clusters and diffuse cuboidal
proliferation of ATII cells on day 0. B10.BR recipient mice
were preconditioned with TBI (day 1) with and without Cy
(days 3 and 2). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 0 before BMT. Lung
cryosections (4 µm) were stained by hematoxylin and eosin, and black
arrows point to cells displaying ATII characteristics (rounded nucleus,
abundant cytoplasm, located at alveolar septal vertices). Blue arrows
point to small focal ATII cell clusters; green arrows point to diffuse
cuboidal proliferation. Resolution power is of ×20 objective lens
for wide-view image under light microscope.
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Fig. 4.
Representative electron micrographs of lungs from day 0 before
BMT of TBI or Cy/TBI conditioned mice with (B and
D) or without (A and C) KGF pretreatment.
KGF-pretreated mice exhibited fewer injured type II cells compared with
control vehicle recipients. Salient features and treatment groups are
indicated. a, Alveolar space; 1, type I epithelial cell; 2, type II
epithelial cell; m, macrophage (magnification ×4,200; bar = 5 µm). E and F are examples of injured and noninjured
ATII cells, respectively (magnification ×22,000; bar = 1 µm).
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Fig. 5.
KGF-pretreated mice exhibit hyperplasia of ATII cells on day 3 post-BMT. B10.BR recipient mice were preconditioned with TBI (day
1) with and without Cy (days 3 and
2) and given C57BL/6 BM with spleen cells (BMS) on
the day of BMT (day 0). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 3 post-BMT. Lung
cryosections (4 µm) were stained by hematoxylin and eosin, and black
arrows point to cells displaying ATII characteristics (rounded nucleus,
abundant cytoplasm, located at alveolar septal vertices). Resolution
power is of ×20 objective lens for wide-view image under light
microscope.
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Fig. 6.
Representative electron micrographs of lungs from day 3 post-BMT of Cy/TBI conditioned BMS recipient mice with
and without KGF pretreatment. Type II cell hyperplasia was apparent in
all mice pretreated with KGF (Cy/TBI BMS shown). Salient features and
treatment groups are indicated. a, Alveolar space; 1, type I epithelial
cell; 2, type II epithelial cell; m, macrophage. Magnification
×4,200, bar = 5 µm.
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Fig. 7.
KGF-pretreated mice have increased numbers of ATII cells on day
3 post-BMT. B10.BR recipient mice were preconditioned with TBI
(day 1) with and without Cy (days 3
and 2) and given C57BL/6 BM alone or with
spleen cells (BMS) on the day of BMT (day 0). Groups received
KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 3 post-BMT. Data
are expressed as percent of nucleated cells displaying ATII
characteristics (rounded nucleus, abundant cytoplasm, located at
alveolar septal vertices) as determined by counting hematoxylin and
eosin-stained lung cryosections under light microscope using ×40
objective lens with ×10 ocular. Mean values ± SD are for 3 mice
per group from 2 representative experiments. * P < 0.05 vs. non-KGF counterpart.
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KGF preconditioning attenuates B7 costimulatory molecule expression
and the induction of the cytolysin granzyme B in the lung on day 7 post-BMT.
In the initial description of our murine IPS model, we showed that the
pulmonary cellular infiltrate was composed of CD4+ and
CD8+ donor T cells, neutrophils, and host
monocytes/macrophages. Therefore, we sought to determine the effects of
KGF on the cellular inflammatory response to Cy-, TBI-, and T
cell-mediated lung injury on day 7 post-BMT. Day 7 was
chosen because we previously demonstrated that lung dysfunction and
inflammatory infiltrates were evident at this time (42). Although
conditioning caused a transient increase in host monocytes, the
prolonged second wave of host monocyte infiltration and induction of B7
expression was T cell dependent. Figure 8
shows that KGF pretreatment does not significantly affect the
composition of the cellular infiltrate in the lung at day 7 post-BMT as assessed by immunohistochemical staining of cryosections.
Regardless of conditioning regimen, KGF attenuated the expression of
B7.1 (CD80) and B7.2 (CD86), molecules, which are costimulatory for T
cells, while not affecting the frequency of cells expressing host major
histocompatibility complex (MHC) class II (I-Ak) as shown
in Fig. 9 (from the same pool of
experiments). KGF pretreatment did not affect the lack of donor MHC
class II (I-Ab) cells, characteristic of our IPS model at
this day 7 time point (42) (data not shown). Representative
photomicrographs of B7.1 staining are shown in Fig.
10.

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Fig. 8.
KGF does not affect the cellular composition of lung infiltrates
post-BMT. Expression of Mac-1, CD4, CD8, and Gr-1 was determined by
immunoperoxidase staining with biotinylated monoclonal antibodies.
B10.BR recipient mice were preconditioned with TBI (day
1) with and without Cy (days 3 and
2) and given C57BL/6 BM with spleen cells (BMS) on
the day of BMT (day 0). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 7 post-BMT. Data
are expressed as percent of nucleated cells expressing the surface
marker in the lung as determined by counting 4 fields per lung section
under light microscope. Mean values ± SE are for 3-5 mice per
group from 3 representative experiments.
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Fig. 9.
KGF attenuates expression of B7.1 and B7.2, while not affecting host
major histocompatibility complex (MHC) class II (I-Ak), as
determined by immunoperoxidase staining with biotinylated monoclonal
antibodies. B10.BR recipient mice were preconditioned with TBI (day
1) with and without Cy (120 mg · kg 1 · day 1,
days 3 and 2) and given C57BL/6 BM
with 15 × 106 spleen cells (BMS) on the day of BMT
(day 0). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4
pre-BMT. Lung tissues were harvested on day 7 post-BMT. Data
are expressed as percent of nucleated cells expressing the indicated
surface marker in the lung as determined by counting 4 fields per lung
section under light microscope. Mean values ± SE are for 4-6
mice per group from 3 representative experiments. Staining for donor
MHC class II at these time points in the lung was negative for all
groups. *P < 0.05 vs. non-KGF counterpart. ^P = 0.07 vs. non-KGF counterpart.
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Fig. 10.
Immunoperoxidase staining for B7.1 expression of lung tissue taken on
day 7 post-BMT of lethally irradiated B10.BR recipients
pretreated with PBS (A) or KGF (days 6, 5,
and 4; B) and transplanted with C57BL/6 BMS
cells. Frozen sections were incubated with biotinylated anti-B7.1
monoclonal antibody (clone 1G10) and developed with
peroxidase-conjugated avidin-biotin complex and diaminobenzidine
chromogen (methyl green counterstain). Positive cells are indicated by
solid arrows. Magnification ×100; resolution power is equivalent
to ×40 objective lens.
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We have described in a murine GVHD model that the cytolytic capacity of
circulating alloreactive T cells is increased as is the expression of
granzyme B that is induced upon activation of cytolytic cells (2).
Because B7 expression on antigen-presenting cells is needed for
efficient CTL generation and granzyme B is a mediator of CTL function,
we sought to determine whether the attenuated expression of B7 by KGF
would translate into attenuated induction of granzyme B in the lungs of
transplanted mice. In situ hybridization analysis (Fig.
11) showed that KGF reduced the frequency
of cells expressing mRNA for the induced granzyme B (P = 0.05 for BMS vs. BMS/KGF and P = 0.07 for BMS/Cy vs. BMS/Cy/KGF) while not affecting the frequency of cells positive for the
constitutively expressed granzyme A mRNA. Representative
photomicrographs of granzyme B mRNA staining are illustrated in Fig.
12. Therefore, although not affecting the
composition of the pulmonary cellular infiltrate, KGF pretreatment may
reduce its injury-inducing capacity by hindering the expression of
molecules necessary for cytolytic cell function, such as granzyme B.

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Fig. 11.
KGF decreases granzyme B expression as assessed by in situ
hybridization using antisense digoxigenin-labeled riboprobes for
granzymes A and B mRNA on lung cryosections taken day 7 post-allogeneic BMT. B10.BR recipient mice were preconditioned with TBI
(day 1) with and without Cy (120 mg · kg 1 · day 1,
days 3 and 2) and given C57BL/6 BM
with 15 × 106 spleen cells (BMS) on the day of BMT
(day 0). Groups received KGF (5 mg · kg 1 · day 1)
on days 6, 5, and 4 pre-BMT. Data
are expressed as percent of nucleated cells expressing the indicated
mRNA in the lung as determined by counting 4 fields per lung section
under light microscope. Mean values ± SE are for 2 or 3 mice per
group from 2 representative experiments. *P < 0.05 vs.
non-KGF counterpart. ^P = 0.07 vs. non-KGF
counterpart.
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Fig. 12.
In situ hybridization using anti-sense digoxigenin-labeled riboprobes
for granzyme B mRNA on day 7 after allogeneic BMT of TBI BMS
recipients pretreated with PBS (A) or KGF (B). Positive
cells are indicated by solid arrows and were detected with alkaline
phosphatase-conjugated antidigoxigenin antibody and
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
chromogen (no counterstain). Magnification ×100; resolution
power is equivalent to ×40 objective lens.
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KGF increases the expression of IL-4 and IL-13, suppressors of
monocyte differentiation.
Reduction of GVHD-induced lethality can be associated with the
induction of counterregulatory cells (Th2) producing anti-inflammatory cytokines (such as IL-4 and IL-10) that skew the T-cell response away
from inflammatory Th1 cytokine production (such as IL-2 and IFN-
)
(25). We wished to determine whether the beneficial effects of KGF
[i.e., amelioration of GVHD and lung pathology (41), lowered B7
and granzyme B expression in the lung] were related to effects on
circulating cytokine levels at the time of BMT when allogeneic donor T
cells are infused (i.e., day 0). If so, one may expect either
reduced levels of proinflammatory cytokines (IFN-
, TNF-
, and
IL-1
) or increased levels of anti-inflammatory cytokines (such as
IL-4, IL-10, and IL-13). Because the BM inoculum is administered
intravenously on day 0, the cells would be exposed immediately
to the cytokine milieu at this time point, which then may translate
into a subsequent effect on the cells infiltrating the lung. Table
1 (pooled data from 4 experiments) shows
the circulating levels of IL-4, IL-13, and IL-6 on day 0 immediately before BMT. One day postirradiation, TBI conditioning did
not significantly increase cytokine levels. Interestingly, KGF
pretreatment of TBI-conditioned mice did not affect IFN-
, TNF-
,
IL-1
, or IL-10 on day 0 (data not shown) but did
significantly increase levels of several Th2 (anti-inflammatory)
cytokines including IL-4, IL-6, and IL-13. The addition of Cy to the
TBI conditioning increased the level of IL-6 on day 0 and
decreased the level of IL-13 compared with control mice. KGF
significantly increased the level of IL-13 in Cy/TBI recipients
back to a level comparable to normal control mice. The increases in
IL-4 and IL-13 were due solely to KGF and not an interaction with
irradiation, since KGF-treated controls that were not irradiated had
equivalent, elevated levels of these cytokines. There was, however, a
multicomponent interaction to the increase in IL-6 seen in the TBI/KGF
group, since the increased IL-6 level was significantly higher than the
non-BMT KGF control group. These data indicate that at this day
0 time point, KGF pretreatment induced the production of Th2-like
(or anti-inflammatory) cytokines, especially IL-13. In addition to Th2
cells, the other major producer of IL-13 is the alveolar macrophage
(30); therefore, it is possible that these cells may have been the
source of this serum IL-13. However, analysis of BAL fluids harvested
on day 0 or day 3 post-BMT showed negligible levels of
IL-13 (data not shown). Therefore, it appears that KGF is inducing Th2
cells to produce IL-13 (and perhaps IL-4), which can serve to
downregulate macrophage differentiation and function.
 |
DISCUSSION |
We examined the use of KGF as a pretreatment therapy for IPS. IPS
results from a combination of tissue damage and allogeneic T
cell-dependent inflammatory events that occur in the early peri-BMT period in our established murine model. The effects of KGF pretreatment on lung repair entailed preservation of noninjured ATII cells on
day 0 and apparent type II cell hyperplasia on day 3 post-BMT. More strikingly, the effects of KGF pretreatment on the
immune response to lung injury were manifest as the attenuation of the expression of the T-cell costimulatory molecules B7.1 (CD80) and B7.2
(CD86) and a decrease in the frequency of cells transcribing mRNA for
the inducible cytolytic molecule granzyme B. These decreases were not
due to reduced cellular infiltration, since KGF did not change the
composition of the lung-infiltrating cells on day 7 post-BMT
and, furthermore, did not affect the frequency of cells transcribing
the constitutive granzyme A mRNA.
KGF-treated mice exhibited lower frequencies of injured ATII cells on
day 0, consistent with the cytoprotective effects of KGF on
these cells. Because this is the time of BMT, the infused donor cells
would be exposed to fewer injured cells, perhaps blunting their ensuing
activation that normally occurs in IPS. Because the ability to repair
the injured alveolar epithelium is dependent on the ability of type II
cells to proliferate, differentiate, and replace type I cells, an
epithelial cytoprotective agent such as KGF could potentially enhance
this process. The apparent ATII cell hyperplasia seen in the
KGF-pretreated mice at day 3 post-BMT is consistent with this
hypothesis and with the lack of evident pathology in the lungs of
long-term survivors of Cy/TBI-conditioned recipients of BMS
(usually exhibiting the most severe injury and mortality) that were
pretreated with KGF. We did not observe overt multifocal knobby
proliferation of alveolar epithelial cells as shown by others (52) by
light microscopy of hematoxylin and eosin-stained lung cryosections,
but we did see occasional small clusters and diffuse cuboidal growth of
ATII cells. These features have been demonstrated by Ulich et al. (52)
and have been described as the later stages of KGF-mediated ATII cell
proliferation by KGF given intratracheally. The route of KGF injection
is most likely the reason for the lack of this overt type II cell
sequela of KGF because we administered it subcutaneously as opposed to the intratracheal injection of KGF, which causes the formation of
knobby cuboidal cell growth along the alveolar septa composed of
proliferating type II cells. Consistent with our hypothesis, a recent
study by Guo et al. (28) has shown that intravenous administration of
KGF, while not causing overt type II cell proliferation, is still
effective at preventing bleomycin- and hyperoxia-induced lung injury
but not as potently as the intratracheal route. There is also recent
evidence by Borok et al. (7) that KGF may cause a reversion of type I
cells into cells with type II pneumocyte characteristics, making them
more resistant to injury, a process termed reversible
transdifferentiation. In vivo, this would translate into an increased
number of type II cells.
The relevance of the decreased B7 expression in KGF-pretreated BMS
recipient mice may relate to the decrease in cytolytic T-cell granzyme
B expression. In the absence of costimulatory signals such as those
mediated by B7 (on antigen-presenting cells) on binding to the CD28
counterreceptor (on T cells), antigen-triggered T cells become
nonresponsive or anergic (8, 45). It is well established that B7
expression augments CTL generation (35). In our KGF-pretreated IPS
model, the colocalization of donor T cells with monocytes expressing
low levels of B7 may lead to a failure of conducive costimulation for
an allo-MHC response resulting in the observed lower frequency of
cytolytic cells in situ. This is consistent with our recent
observations that preclusion of the CD28-B7 interaction by anti-B7 MAb
reduced the generation of granzyme B-positive cells and the in vitro
cytolytic activity of T cells obtained from MHC-disparate irradiated
recipients of allogeneic T cells (2). Because of the apparent
preservation of the type II cells by KGF, the lower level of B7 and
granzyme B expression may be sequelae of the reduced damage resulting
in less immune activation.
Sera from KGF-treated mice exhibited increased levels of the Th2
cytokines IL-4, IL-6, and especially IL-13 4 days after cessation of
KGF administration (day 0) even in the absence of irradiation or BMT. This argues that at least some of the effects of KGF may be
independent of the sequelae of reduced lung injury. Because day
0 is the time at which the BM inoculum is administered, these cells
would be immediately exposed to this Th2 type of cytokine environment
in the circulation into which the cells are infused. It has recently
been demonstrated that the functional response of bone marrow-derived
macrophages is determined by the first exposure to cytokine, thereby
rendering them unresponsive to subsequent exposure to alternate
cytokines (21). Several studies have demonstrated that IL-13 can
suppress many monocyte activities, similar to IL-4 (1, 14, 16-18, 36,
37, 48, 59). The downregulation of CD14 (LPS receptor) expression by
IL-13 (14) may, in part, explain the survival of KGF-pretreated BMT
recipients in the face of GVHD-induced colitis (41). Damage to other
GVHD target organs, particularly the gastrointestinal tract, induces
the systemic release of endotoxin (LPS) that primes monocytes to
release proinflammatory cytokines that exacerbate GVHD and IPS (12).
Because our data suggest that KGF pretreatment leads to decreased
monocyte activation, KGF also may protect the BMT recipient against
adverse consequences of monocyte activation that contribute to lung injury.
IL-13 does not affect the chemotactic properties of monocytes (61), and
we found no difference in the infiltration of monocytes into the lung
on day 7 post-BMT with KGF treatment. We do not yet know
whether the decreased expression of B7 molecules by these monocytes is
due to direct KGF effects or indirect effects via the inhibitory
effects of IL-13; the effect of IL-13 on monocyte-B7 expression has not
been addressed in the literature. Decreased monocyte function would
explain the decreased inflammatory (IFN-
, data not shown) and
cytotoxic (granzyme B) T-cell mediators we found in the lungs of
KGF-pretreated BMS recipients at day 7 post-BMT. These
recipients also had significantly lower levels of serum TNF-
than
non-KGF-treated counterparts on day 7 post-BMT (data not shown)
consistent with the recent observations of Krijanovski et al. (34)
using another murine GVHD model. The association of lower TNF-
and
LPS levels with less severe manifestations of GVHD and lung injury
post-BMT suggests that TNF-
and LPS contribute to IPS injury. The
source of the Th2-type cytokines found in the serum of KGF-treated mice
on day 0 just before BMT is most likely Th2 cells for the
following reasons: 1) aside from alveolar macrophages, Th2
cells are the only known major producers of IL-13, and BAL fluid IL-13
levels did not parallel serum levels; 2) IL-4 is produced primarily by activated Th2 cells; and 3) IL-1
and TNF-
,
monocyte products, were not increased at this time point. Although IL-6 also was elevated in response to KGF, it can be produced by numerous cells including keratinocytes, which likely were activated in response
to the subcutaneous injection of KGF. In addition, T cells costimulated
by activated keratinocytes preferentially produce IL-4 (27), and IL-4
was elevated in response to KGF pretreatment in our study. Of note,
IL-13 can also have proinflammatory properties depending on the target
cell (19, 29) and the timing of the exposure of the monocyte to IL-13
in relation to the stimulus (i.e., antigen) (15). In fact, high levels
of monocyte-derived IL-13 are found in the BAL fluid of asthmatics (32)
and patients with pulmonary fibrosis (30). Perhaps the latter are
examples of failed attempts at repair. Numerous reports have shown that type 2 alloreactive T cells, generally considered to be
anti-inflammatory, have a reduced capacity to induce GVHD (25, 33).
However, this may not be a universal finding in GVHD (38). We do not know whether the amelioration of IPS by KGF is Th2 dependent.
Taken together, these data suggest that KGF can hinder IPS by two
modes: 1) enhancement of alveolar epithelialization and 2) attenuation of B7 and cytolytic molecule (granzyme B)
expression in situ, possibly via induction of Th2 cytokines,
in particular IL-13, a potent macrophage downregulator, thus enabling
lung repair.
 |
ACKNOWLEDGEMENTS |
The expert technical assistance of John Hermanson, Sumiko Yoneji,
Claudia DeLlano, Chris Lees, Naomi Fujioka, Stacey Hermanson, and Kelly
Coffey is greatly appreciated. We also thank Dr. Patricia A. Taylor for
helpful discussions.
 |
FOOTNOTES |
This study was supported by National Heart, Lung, and Blood Institute
(NHLBI) Grant HL-55209; Morphology Core of the NHLBI Specialized Center
of Research in Acute Lung Injury Grant HL-50152; the Minnesota Medical
Foundation; and the Viking Children's Fund.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Panoskaltsis-Mortari, Dept. of Pediatrics, Div. of Hematology-Oncology
and Blood Marrow Transplantation, Univ. of Minnesota, Box 366 MAYO, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail:
panos001{at}tc.umn.edu).
Received 5 August 1999; accepted in final form 9 December 1999.
 |
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