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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2337-2345
IMMUNOBIOLOGY
Transgenic expression of granulocyte-macrophage
colony-stimulating factor induces the differentiation and
activation of a novel dendritic cell population in the lung
Jun Wang,
Denis P. Snider,
Bryan R. Hewlett,
Nick W. Lukacs,
Jack Gauldie,
Hong Liang, and
Zhou Xing
From the Department of Pathology and Molecular Medicine and Division
of Infectious Diseases, Centre for Gene Therapeutics,
McMaster University, Hamilton, ON, Canada; and the Department of
Pathology, University of Michigan, Ann Arbor, MI.
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Abstract |
The role of granulocyte-macrophage colony-stimulating factor
(GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of
GM-CSF transgene expression established with an adenoviral vector
(AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF
in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like
cells was observed in the lung receiving AdGM-CSF but not control
vector. By FACS analysis, the majority of these cells were identified
at an early time point as macrophages and later as mature/activated
myeloid DCs characterized by CD11bbright,
CD11cbright, MHC class IIbright, and
B7.1bright. In contrast, GM-CSF had a weak effect on a
small DC population that was found present in normal lung and was
characterized by CD11cbright and CD11blow. By
immunohistochemistry staining for MHC II, the majority of activated
antigen-presenting cells were localized to the airway epithelium and
peribronchial/perivascular areas in the lung. A concurrently enhanced
Th1 immune response was observed under these conditions. The number of
CD4 and CD8 T cells was markedly increased in the lung expressing
GM-CSF, accompanied by increased release of interferon (IFN) in the
lung. Furthermore, lymphocytes isolated from either lung parenchyma or
local draining lymph nodes of these mice but not the control mice
released large amounts of IFN on adenoviral antigen stimulation in
vitro. These findings reveal that GM-CSF promotes the differentiation
and activation of a myeloid DC population primarily by acting on
macrophages during pulmonary immune responses.
(Blood. 2000;95:2337-2345)
© 2000 by The American Society of Hematology.
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Introduction |
The lung is constantly exposed to the external
environment. Thus, the generation of pulmonary immune responses against
invading pathogens, including bacteria and viruses, is critical for
host defense. Dendritic cells (DCs) are the most potent
antigen-presenting cells (APCs) essential for the development of
primary immune responses against microbial pathogens.1
Pulmonary DCs are normally distributed within the airway mucosa,
alveolar septa, and perivascular
parenchyma.2,3 Like DCs from other nonlymphoid
tissues, pulmonary DCs are originated from either "myeloid" or
"lymphoid" precursors and are believed to be functionally
immature APCs in the tissue where they exert a sentinel
function.1,4,5 On encounter with antigens, DCs move to the
T-dependent areas of secondary lymphoid organs where they become mature
DCs to activate antigen-specific lymphocytes. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a well-known hematopoietic growth factor, is indispensable for DC differentiation and maturation from bone marrow progenitors or peripheral blood monocytes in a number of in vitro systems.6-11 It also has
regulatory effects on purified pulmonary DCs.8-10,12
However, relatively little is known about whether GM-CSF has such
effects on DCs in vivo. GM-CSF-transduced tumor cells were shown to
induce the most potent antitumor immunity compared with tumor cells
transduced with other cytokines,13 and GM-CSF transgenic
mice were found to have increased DCs in the peritoneal cavity and
lymphoid organs.11,14 Furthermore, GM-CSF gene-deficient
mice were more susceptible to lung infections by a range of
opportunistic microorganisms.15,16 Indeed, GM-CSF has been
found in increased amounts in a number of pulmonary immune and
inflammatory conditions.17 Although these lines of evidence
suggest a regulatory role of GM-CSF in host immune-inflammatory
responses in the lung, the precise role of GM-CSF in the
differentiation/activation of DCs in the lung during immune responses
has remained to be elucidated. Because GM-CSF is induced primarily
under immune-inflammatory conditions in the lung, its effect on APCs in
the lung may be best studied in a transgenic model in which GM-CSF is
expressed concurrently with an ongoing immune-inflammatory response.
In this study, we utilized a GM-CSF transgene model established by
local lung delivery of a replication-deficient adenoviral GM-CSF gene
transfer vector to examine the effect of GM-CSF on the differentiation
and activation of pulmonary APCs, particularly DCs. This viral-mediated
transgene approach targets GM-CSF transgene to the airway epithelial
cells and gives rise to a prolonged raised GM-CSF level in the lung and
thus allows us to study the effect of GM-CSF on APCs in the lung during
host immune responses to viral infection.
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Material and methods |
Gene transfer vectors and antibodies
Construction and characterization of a replication-deficient
adenoviral gene vector expressing murine GM-CSF (AdGM-CSF) have been
previously described.18 An adenoviral vector Addl70-3
without transgene was used as a control.18 UV-inactivated
wild-type adenovirus was used as adenoviral antigens for an in vitro
antigen stimulation assay. Anti-mouse monoclonal antibodies, including FITC-labeled anti-CD8 (clone 53-6.7), PE-labeled anti-CD11c (clone HL3) and anti-CD4 (clone GK1.5), and biotinylated anti-CD3 (clone 145-2C11), anti-B7.1 (clone BB1), and anti-natural killer (NK) cell
(clone DX5) antibodies, were purchased from PharMingen (Mississauga, ON, Canada). FITC-labeled anti-CD11b (anti-Mac1, clone M1/70.15) was
purchased from Cedarlane Laboratories Limited (Hornby, ON, Canada).
Anti-MHC II monoclonal antibody (mAb) (clone M5/114) was produced,
purified on protein G, and labeled with biotin. The mAb 2.4G2 (anti-FcR
IIb/III) was produced as ascites and purified on protein G. Binding of
biotinylated antibodies was identified by streptavidin-conjugated
peridinin chlorophyll protein (Becton Dickinson, San Jose, CA).
Mice and GM-CSF gene transfer in the lung
Female Balb/c and C3H mice of 10- to 14-week age were purchased from
Harlan Laboratories (Indianapolis, IN) and housed under specific
pathogen-free conditions before use at McMaster University Central
Animal Facility. Mice were anesthetized and AdGM-CSF or Addl70-3 was
intranasally (i.n.) delivered to mouse lung by a standardized procedure
that we have previously described.19 Briefly, a dose of
0.6 × 109 plaque-forming units (pfu) of viral
vector was diluted with phosphate-buffered saline (PBS) to a total
volume of 30 µL and delivered into mouse lungs with a fine pipette
tip in 2 aliquots (15 µL each). Mice were killed at days 2, 4, 7, 12, or 19 postgene transfer. We have previously shown that, following i.n.
vector delivery, transgene is expressed primarily by bronchial
epithelial cells and, to a certain extent, by alveolar
macrophages.19
Bronchoalveolar lavage (BAL) and cytologic analysis
At each time point, BAL was performed as previously
described.20,21 A total of 450 µL of PBS was used to
lavage the lung, and usually 350 µL of BAL fluids were retrieved. BAL
fluids were then spun in a microcentrifuge at 5000 rpm for 5 minutes,
and supernatants were stored at  20°C until cytokine
measurements. Cell pellets were resuspended in PBS and total cell
numbers were counted on a hemocytometer. Cytospins were prepared by
cytocentrifugation (Shandon Inc, Pittsburgh, PA). Differential cell
counts were determined on Diff-Quik-stained (Baxter, McGaw Park, IL)
cytospins by randomly counting 400-500 cells per slide.
Flow cytometric analysis of T-cell and NK-cell subsets in BAL and
APCs in the lung
To phenotype immune cell subsets in the lung, BAL-derived cells were
pooled from three AdGM-CSF-treated mice or five Addl70-3-treated mice 7 days after infection. About 0.3 × 106 cells were
blocked by an anti-FcR antibody 2.4G2 for 15 minutes and then labeled
with a combination of mAbs of biotinylated anti-CD3, PE-anti-CD4 and
FITC-anti-CD8, or FITC-anti-CD3 and biotinylated anti-NK DX5. The basic
staining procedure was carried out as previously described.22 A FACScan instrument was used (Becton
Dickinson, Sunnyvale, CA) to collect list mode data (10 000 total
events) for analysis. Analysis was carried out using Lysys II software (B-D) by first setting a forward- and side-scatter gate that included lymphocytes but excluded dead cells and debris.
In separate experiments, mice were killed 5 or 12 days after delivery
of AdGM-CSF, Addl70-3, or PBS. The lungs were removed from the chest
with the heart and a portion of the trachea intact. Pulmonary
vasculature was perfused with 5 mL of warm calcium and magnesium-free 1 X HBSS containing 5% fetal calf serum (FCS), 100 U/mL penicillin, and
100 g/mL of streptomycin via the right ventricle of the heart. Total
lung mononuclear cells were isolated by collagenase digestion, followed
by discontinuing gradient centrifugation as previously
described.22 Approximately 0.3 × 106
cells were labeled with mAbs in a combination of FITC-CD11b, PE-CD11c,
and a biotinylated-antibody to a surface molecule of interest (MHC
class II or B7.1) or biotinylated-isotype control antibody rat
immunoglobulin (Ig)G2a. List mode data (20 000 total events) were
collected on FACScan and analysis was performed on cells gated in high
forward and scatter region (R1 region), distinct from regions defined
for lymphocytes and debris (see Figure 1A). More than 90% of CD11c-positive cells were found within R1 region by
back-gating analysis. The cells from R1 region were further divided
into three subpopulations (R2-R4) on the basis of CD11b and CD11c
expression (Figure 1B). The unstained cells gated on R1 region were
used as negative controls, and the background was approximately 2% and
1.2% for PE-CD11c and FITC-CD11b, respectively. The absolute numbers
of cell subsets based on CD11c and CD11b were calculated on the basis
of the total number of cells recovered per mouse, multiplied by the
fraction of cells in the high scatter region, and then multiplied by
the fraction of cells with a given CD11c, CD11b phenotype.
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| Fig 1.
Identification of pulmonary antigen-presenting cell (APC)
populations under different conditions.
Total mononuclear cells were isolated from mouse lungs that received
0.6 × 109 plaque-forming units adenoviral gene
vector expressing murine GM-CSF (AdGM-CSF) or Addl70-3, or 40 µL of sterile phosphate-buffered saline at day 12. Approximately
0.3 × 106 cells were immunostained with FITC-CD11b,
PE-CD11c and biotinylated-MHC II, B7.1, or rat immunoglobulin
(Ig)G2a antibodies. Data were collected from FACScan. (A) General
cellular profile for each group was shown on forward-side scatter,
macrophage/monocyte-like cell enriched population was gated as R1.
(B) R1 region cells were further divided into three
subpopulations in contour plot based on CD11b and CD11c expression. (C)
The absolute cell numbers for each population derived from single
mouse in different groups. Data are representative of three
individual experiments.
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T-cell enrichment from lung and draining lymph nodes and in vitro
stimulation with adenoviral antigen
Mice were killed 12 days after delivery of AdGM-CSF or Addl70-3. Total
lung mononuclear cells were isolated as above and were enriched for T
cells by using a mouse T-cell-enrichment column (R&D System,
Minneapolis, MN) according to manufacturer's instructions. The
resultant population contained more than 90% T lymphocytes with 5% to
10% antigen-presenting cells. Cell suspensions were cultured in
RPMI-10 medium (RPMI 1640, supplemented with 10% FCS, 100 U/mL
penicillin, 100 g/mL streptomycin, and 2 mmol/L of L-Glutamine). From
the same mice, mediastinal lymph nodes were collected and kept in HBSS,
then gently ground between two sintered glass slides. The resultant
lymphocyte suspension was filtered through one layer of nylon membrane
(55 µm) and centrifuged at 1200 rpm for 10 minutes at 4°C. The
cell pellets were washed once with PBS and resuspended in RPMI-10 medium.
Approximately 0.2 × 106 lung-derived or
0.3 × 106 lymph node-derived cells were seeded into
96-well plates and cultured with or without 1 × 109
pfu/well of UV-inactivated adenovirus at 37°C for 72 hours. Other wells received 8 g/mL of an irrelevant mycobacterial antigen (PPD). The
supernatants were taken at 72 hours and stored at 20°C until cytokine measurement.
Immunohistochemical staining for MHC II expression
Lung tissues were obtained at 12 days after delivery of
AdGM-CSF or Addl70-3 and fixed in 30 mL of 4% formaldehyde in 75 mmol/L phosphate buffer (pH 7.4) for 24 hours. Paraffin sections were deparaffinized in xylene followed by 100% ethanol and then placed in
freshly prepared methanol H2O2 solution for 30 minutes (200 mL methanol, 10 mL hydrogen peroxide, and 0.5 mL
concentrated HCl) to block endogenous peroxidase activity.
After rehydrolization, the slides were subjected to hot Hewlett's
Epitope Recovery Buffer (10 mmol/L citrate/phosphate buffer, pH 6.85)
for 10 minutes23 and then allowed to cool down to room
temperature. The slides were blocked with 1% BSA/PBS for 15 minutes,
followed by incubation with 1:100 rat anti-mouse biotinylated MHC II M5
mAb in 1% BSA/PBS overnight at room temperature. Following the
incubation with 1:300 secondary biotinylated rabbit anti-rat antibody
at room temperature for 1 hour and 1:1200 straptavidin/peroxidase
conjugate for 90 minutes, the slides were developed by a conventional
substrate/chromogen solution and counter-stained with 50% hematoxylin
for 2 minutes.
Cytokine measurements
The level of cytokines in the BAL and culture supernatants
was determined by using mouse specific enzyme-linked immunosorbent assay (ELISA) kits. Interferon (IFN) and GM-CSF ELISA kits were purchased from R&D Systems. The sensitivity of detection for these ELISA kits was 2 pg/mL. MIP-1 and MCP-1 ELISAs were developed as
previously described.24
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Results |
GM-CSF transgene protein levels and cellular responses in the
lung post-GM-CSF gene transfer
To determine the level of GM-CSF transgene protein in the lung, BAL
fluids collected at various time points following lung GM-CSF gene
transfer were measured for murine GM-CSF by ELISA. The level of GM-CSF
markedly increased by day 2, peaked by day 4, still remained high at
day 7, and significantly decreased by day 12 in the lung of mice after
GM-CSF gene transfer (Table 1). In
contrast, little GM-CSF was detected in the BAL from mice receiving the
control vector Addl70-3. To evaluate cellular responses to GM-CSF in
the lung, total and differential cell counts in the BAL were determined
at various time points. The control vector induced only minimal
cellular responses throughout the entire experiment (Table
2). In contrast, GM-CSF induced an increase in total cell numbers that peaked at day 12, being 8 times as many as
in mice receiving Addl70-3 (Table 2). Macrophages/monocytes represented
a major cell type among increased leukocytes. The number of lymphocytes
also markedly increased. For instance, at day 12, there were
50.4 × 104, 24 × 104, and
13.2 × 104 of macrophages/monocytes, lymphocytes,
and neutrophils, respectively, in BAL from the lung of mice expressing
GM-CSF versus 10.8 × 104,
0.64 × 104, and 0.17 × 104,
respectively, in the lung of mice receiving Addl70-3 (Table 2). By day
19, although the cellular response almost completely resolved in the
lung of control mice, the number of macrophages/monocytes in the lung
of mice receiving AdGM-CSF still remained elevated. In addition to
macrophages, lymphocytes, and neutrophils, there was also a small, but
significant, increase in the number of eosinophils. The delayed peak
cellular response, as compared with the earlier peak time of GM-CSF
transgene product in the lung, suggests that such increased cell
responses are not just the effect of GM-CSF on cell influx and that it
is more likely a result of the enhancement by GM-CSF of cellular immune
responses to viral infection that normally takes 7 to 10 days to peak.
Consistent with the cellular profiles observed in the BAL, total
mononuclear cells isolated from the lung tissue of mice expressing
GM-CSF at day 12 were 10 times as high as that in control groups (data
not shown).
Phenotypes of APCs induced by GM-CSF transgene expression
in the lung
Having demonstrated that GM-CSF induced a marked cellular
response of primarily monocytic nature in the lung, we analyzed the
phenotype of this macrophage/monocyte population by FACS. To this end,
total mononuclear cells were isolated from mouse lung tissue receiving
AdGM-CSF, Addl70-3, or PBS at day 12 and stained with different
combinations of mAbs to various leukocyte surface markers, including
CD11b (Mac-1), CD11c, MHC II, and B7.1. A cell population (Figure 1A,
R1 region) showing higher forward- and side-scatter properties was
gated and subdivided into three populations, based on their relative
CD11c and CD11b expression. These populations were cells
expressing CD11blow and CD11cbright (R2
region), CD11bbright and CD11cbright (R3
region), or CD11bbright and CD11clow (R4
region), respectively (Figure 1B).
CD11blow/CD11cbright cells were lung
residential DCs, phenotypically similar to some DCs found in lymphoid
organs.11,25-27
CD11bbright/CD11cbright cells represented a
myeloid DC phenotype also found in lymphoid organs11,26 and
were a novel DC population induced by GM-CSF that we now identified in
the lung. CD11bbright/CD11clow cells were
macrophages.25 In PBS- or Addl70-3-treated animals, about
60%-80% of CD11c+ cells were
CD11blow/CD11cbright. In contrast, GM-CSF
expression resulted in a striking increase in the percentage of
CD11b/CD11c double-positive cells, which accounted for about 90% of
CD11c+ cells (Figure 1B). Thus, GM-CF induced an approximately 44-fold
increase in the number of
CD11bbright/CD11cbright DCs over that found in
PBS- or Addl70-3-treated groups (Figure 1C). GM-CSF also induced a
fourfold and threefold increase in the number of
CD11bbright/CD11clow cells and
CD11blow/CD11cbright cells, respectively
(Figure 1C). These results indicate that GM-CSF plays a major inductive
role in the differentiation of the CD11b/CD11c double-positive myeloid
DC population.
Activation of DCs and macrophages by GM-CSF transgene expression in
the lung
To examine the activating effect of GM-CSF on each APC population
during viral infection, we also examined expression of phenotypic activation markers MHC class II and costimulatory molecule B7.1 that
are expressed in high intensity by activated APC
populations.28,29 CD11blow/CD11cbright DCs in the PBS group
expressed very low levels of MHC class II and moderate levels of B7.1
(Figure 2A). This type of DC in Addl70-3 control vector group expressed slightly enhanced expression of MHC
class II and B7.1, which was likely a response to viral infection. In
comparison, although there were 60% of
CD11blow/CD11cbright cells in the GM-CSF group
that expressed bright MHC II, about 40% of this type of cell
translated from moderate to bright B7.1 expression, in contrast to the
cells in control groups most of which only expressed moderate density
of B7.1 (Figure 2A). In terms of absolute cell number, GM-CSF induced
an approximately 14-fold and 7-fold increase in the number of DCs
expressing bright MHC class II and B7.1, respectively, compared with
control groups (Figure 2B).
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| Fig 2.
Effect of granulocyte-macrophage colony-stimulating
factor (GM-CSF) on activation of R2 region
CD11blow/CD11cbright population during
pulmonary adenoviral infection.
Following the procedure described in Figure 1, (A) MHC II and
B7.1 expression were examined on R2 region for each group. Open
histograms represent the background staining with the isotype control
antibody, whereas solid histograms indicate staining with relevant
monoclonal antibody against the indicated surface molecule. M1 markers
represent the limit defining the expression at high levels of the
corresponding marker. In the case of B7.1 expression, the majority of
cells from R2 region in control groups expressed at dull level that
labeled as M8. (B) The absolute cell numbers for double positive for
CD11c and MHC II or B7.1 derived from single mouse in different
groups.
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The majority of CD11bbright/CD11cbright DCs in
the PBS group did not express MHC II and B7.1 (Figure
3A). There was only a moderately increased
percentage of these cells from the Addl70-3 group expressing bright MHC
II and B7.1, which likely represented a response to viral infection. In
contrast, almost 100% of CD11b and CD11c double-positive cells from
the GM-CSF group expressed high intensity of MHC II and B7.1 (Figure
3A). Because a very small number of CD11b and CD11c double-positive
cells was present in the lung of mice receiving PBS or Addl70-3, in
contrast to that induced by GM-CSF (Figure 1C), the number of activated
DCs coexpressing high density of CD11b, CD11c, MHC II, and B7.1 in the
lung of the GM-CSF group was at least 60-fold greater than in the
control groups (Figure 3B). These findings highlight a potent inductive
effect of GM-CSF on this myeloid DC population in the lung during viral
infection.
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| Fig 3.
Effect of granulocyte-macrophage colony-stimulating
factor (GM-CSF) on activation of R3 region
CD11bbright/CD11cbright population during
pulmonary adenoviral infection.
Following the procedure described in Figure 1, (A) MHC II and
B7.1 expression on R3 region was examined for each group. Open
histograms represent the background staining with the isotype control
antibody, whereas solid histograms indicate staining with relevant
monoclonal antibody against the indicated surface molecule. M1 markers
represent the limit defining the expression at high levels of the
corresponding marker. (B) The absolute cell numbers for triple
positive for CD11b, CD11c, and MHC class II or B7.1 derived
from single mouse in different groups.
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GM-CSF also had a significant effect on MHC class II and B7.1
expression on CD11bbright/CD11clow macrophages
in R4 region (Figure 4A). In comparison,
macrophages from the PBS group were not activated and few of them
expressed MHC II and B7.1. There was a moderate increase in the
percentage of these cells expressing MHC II and B7.1 in the Addl70-3
group, likely again as a result of viral infection (Figure 4A). Similar to the effect on lung residential
CD11blow/CD11cbright DCs, GM-CSF induced about
9-fold and 7-fold increases in the number of macrophages expressing
bright MHC class II and B7.1, respectively, compared with control
groups (Figure 4B).
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| Fig 4.
Effect of granulocyte-macrophage colony-stimulating
factor (GM-CSF) on activation of R4 region
CD11bbright/CD11clow population during
pulmonary adenoviral infection.
Following the procedure described in Figure 1, (A) MHC II and
B7.1 expression was examined on R4 region for each group. Open
histograms represent the background staining with the isotype control
antibody, whereas solid histograms indicate staining with relevant
monoclonal antibody against the indicated surface molecule. M1 markers
represent the limit defining the expression at high levels of the
corresponding marker. (B) The absolute cell numbers for double positive
for CD11b and MHC II or B7.1 derived from a single mouse in
different groups.
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Differentiation of macrophages to myeloid DCs by GM-CSF in the
lung
Having identified a potent effect of GM-CSF on the induction of a
novel myeloid DC population characterized by CD11bbright,
CD11cbright, MHC class IIbright, and
B7.1bright in the lung, we investigated whether this DC
population could have derived from a macrophage population expanded by
GM-CSF at an earlier time. To this end, a group of mice were killed 5 days post-AdGM-CSF gene transfer. Mononuclear cells were isolated, immunostained, and analyzed in the way we did at day 12. A comparison was made between 5 days and 12 days post-GM-CSF gene transfer. GM-CSF
markedly induced a
CD11bbright/CD11clow macrophage
population at day 5 after transgene expression (Figure 5). These cells accounted for 46.3% of
total analyzed cells as opposed to 16.3% found in PBS controls (Figure
1B) and had increased MHC II expression (data not shown). In
comparison, only 9% of analyzed cells were
CD11bbright/CD11cbright.. At day 12, however,
while macrophage population decreased dramatically to only 15.4% of
total cells, 57.6% of cells were
CD11bbright/CD11cbright. Of note, the
percentage of CD11blow/CD11cbright cells
remained similar to that found at day 5 (11.8% vs 9%). Thus, the
phenotypic shift from macrophages to myeloid DCs during the course of
GM-CSF transgene expression strongly suggests that CD11b
bright/CD11cbright DCs induced by GM-CSF were
primarily derived from a macrophage population activated earlier by
GM-CSF.
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| Fig 5.
Major effect of granulocyte-macrophage colony-stimulating
factor (GM-CSF) on macrophages at earlier times in the lung.
In a separate experiment, mononuclear cells were isolated from mice
receiving phosphate-buffered saline, Addl70-3, or adenoviral gene
vector expressing murine GM-CSF (AdGM-CSF) at day 5 and
immunostained in the exact same way as described in Figure 1. A
parallel comparison analysis was carried out between the AdGM-CSF day 5 group and the AdGM-CSF day 12 group. R1 region cells as defined in
Figure 1 were expressed in dot plot based on CD11b and CD11c
expression. The number marked on each corner represents the percentage
of a given phenotype out of total R1 region.
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Localization of APC in the lung by immunohistochemistry
We next examined the localization of APC in the lung by
immunohistochemistry with an anti-MHC II mAb. Few MHC II-positive cells
were found in the lung of mice receiving Addl70-3 (Figure 6A). In contrast, many MHC II-bearing cells
were localized to peribronchial and perivascular areas, many of which
could represent activated GM-CSF-induced DCs although morphologically
they appear to be macrophages (Figure 6B). In addition, some MHC
II-positive cells were also localized to the airway epithelium, and
because these cells possessed dendrites or projections, they were most likely intraepithelial dendritic cells (Figure 6B).
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| Fig 6.
Distribution of MHC II positive dendritic cells (DCs) and
macrophages in the lung.
Mice were killed 12 days after intrapulmonary injection of
adenoviral gene vector expressing murine granulocyte-macrophage
colony-stimulating factor (AdGM-CSF), Addl70-3, or phosphate-buffered
saline. Lung tissues were isolated and fixed in 10% formalin and 3-cm
sections were immunostained with monoclonal anti-MHC II M5 antibody and
50% hematoxylin counterstaining. (A) Addl70-3 control vector
treatment; (B) AdGM-CSF treatment. Many MHC II-positive cells are
seen in Figure 6B, some intraepithelial DC are marked with arrowheads.
Bronchial lumen is marked by b. (Magnification for both panels is
450.)
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Enhanced immune responses in the lung by GM-CSF
transgene expression
The primary function of DCs is to activate antigen-specific T cells,
enhancing their proliferation and cytokine responses during primary
immune responses.1,4,28,30 Having demonstrated that GM-CSF
transgene expression markedly promoted the differentiation and
activation of myeloid APC populations in the lung during immune responses to adenoviral infection, we further examined and compared the
level of immune responses with pulmonary viral infection in the lung of
mice receiving Addl70-3 or AdGM-CSF. To this end, we first examined by
FACS analysis the number of immune subsets including NK, CD4, and CD8 T
cells in the lymphocytic population present in BAL fluids recovered at
day 7 postgene transfer. In our previous studies, we have found that
the cellular profiles of lymphocytes, macrophages, and granulocytes in
BAL fluids always mirror those seen at the histopathologic
level.18-22 We have previously shown that T-cell responses
are an important aspect of host anti-adenoviral immune response in the
lung.31 The number of NK cells was found similar between
control and GM-CSF groups (Figure 7).
However, there were approximately 34-fold and 16-fold increases in the number of CD4 and CD8 T cells, respectively, in the lung of the GM-CSF
group (Figure 7).
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| Fig 7.
Determination of the number of subtypes of lymphocytes
present in bronchoalveolar lavage (BAL) by FACS.
BALs were pooled out from three mice treated with adenoviral gene
vector expressing murine granulocyte-macrophage colony-stimulating
factor (AdGM-CSF) or Addl70-3, respectively, 7 days after gene
transfer. Cells were immunostained with different combinations of
monoclonal antibodies and examined by FACS analysis. Then, the absolute
cell numbers of CD4, CD8, and natural killer cells were calculated,
based on the total cell recovery and the percentage of each subtype.
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Further, we examined the level of an antiviral Th1 cytokine IFN in
the lung. The level of this cytokine was only marginally increased in
the lung of mice receiving the control viral vector. In sharp contrast,
there was a significant induction of IFN in the lung of the GM-CSF
group, which peaked at day 7 and remained high at day 12 and markedly
declined by day 19 (Figure 8). In addition,
we also observed increased levels of chemokines MIP-1 and MCP-1 in
the BAL in the AdGM-CSF group, particularly at early time points (data
not shown).
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| Fig 8.
Interferon (IFN) content in the bronchoalveolar lavage
(BAL) at various time points after delivery of adenoviral gene vector
expressing murine granulocyte-macrophage colony-stimulating factor
(AdGM-CSF) or Addl70-3.
BAL fluids were collected at days 4, 7, 12, and 19 postintranasal
delivery of AdGM-CSF or Addl70-3, and IFN content was determined by
enzyme-linked immunosorbent assay. The results are expressed as mean ± SEM for AdGM-CSF group (n = 3) and mean for Addl70-3 group
(n = 2).
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|
Furthermore, we isolated lymphocytes from both lung tissue and
mediastinal lymph nodes 12 days after administration of AdGM-CSF or
Addl70-3 and investigated the level of Th1-type lymphocyte response to
adenoviral antigen stimulation in vitro by measuring the release of
IFN. As shown in Figure 9, although
there was an antigen-specific recall IFN response by lymphocytes
isolated from the lung or mediastinal lymph nodes of mice infected with the control virus Addl70-3, such a response was enhanced many times in
lung- or mediastinal lymph node-derived lymphocytes from mice infected
with AdGM-CSF. These findings suggest that GM-CSF enhances a
Th1-type host immune response to viral infection.
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| Fig 9.
Interferon (IFN) level determined in ex vivo
antigen-recall responses.
Mediastinal lymph nodes and lungs were collected from the mice
receiving adenoviral gene vector expressing murine
granulocyte-macrophage colony-stimulating factor (AdGM-CSF)
or Addl70-3 at day 12. T lymphocytes were isolated and cultured at
various conditions as described in "Material and methods."
Supernatants of (A) mediastinal lymph node-derived T-cell culture and
(B) lung-derived T-cell culture were collected at 72 hours, and IFN
level was measured by enzyme-linked immunosorbent assay. The difference
in the level of IFN production between AdGM-CSF and control groups
is statistically significant (t test,
P  .01). Data are representative of three individual
experiments and expressed as mean ± SEM of triplicate samples.
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Discussion |
In this study, we have used a unique mouse model of
adenoviral-mediated GM-CSF transgene expression to investigate the role of GM-CSF in pulmonary DC differentiation and activation during an
immune response to viral infection in the lung. We have shown an
increased cellular response composed predominantly by
macrophage/monocyte-like cells following GM-CSF gene transfer in the
lung. By FACS analysis, two DC populations,
CD11blow/CD11cbright and
CD11bbright/CD11cbright, were identified with
the latter being highly inducible by GM-CSF. The potent effect of
GM-CSF on DC activation was demonstrated by up-regulation of both MHC
class II and the costimulatory molecule B7.1 expression on
CD11bbright/CD11cbright cells. Moreover, GM-CSF
also had an effect on expansion and activation of lung residential DCs
and macrophages characterized by surface expression of
CD11blow, CD11cbright, MHC
IIbright, and B7.1bright and of
CD11bbright, CD11clow, MHC
IIbright, and B7.1bright, respectively. These
potent effects on APC by GM-CSF were found associated closely with
enhanced Th1 immune responses, including increased CD4 and CD8 T cells,
IFN release in the lung, and viral antigen-specific IFN response
by lymphocytes from the lung or local draining lymph nodes.
GM-CSF is released by airway epithelial and endothelial cells,
fibroblasts, macrophages, and carcinoma cells in response to a number
of stimuli in vitro17,32-36 and has been found increased in
the lungs during a number of pulmonary immune conditions.17 However, the precise role of GM-CSF in the pathogenesis of these pulmonary conditions has remained poorly understood. Results from our
current study have not only demonstrated a potent effect of GM-CSF on
the differentiation and activation of DCs and macrophages but also
revealed the nature of a novel DC phenotype driven by GM-CSF during an
immune response to pulmonary viral infection. Our results suggest that
GM-CSF has a weak effect on the differentiation of a DC population that
is characterized by CD11blow and CD11cbright.
However, GM-CSF dramatically induces the emergence of a DC population characterized by CD11bbright, CD11cbright, MHC
IIbright, and B7.1bright, in addition to its
activating effect on macrophages characterized by
CD11bbright, CD11clow, MHC
IIbright, and B7.1bright. Recently, Suda et
al37 have reported that the number of DC precursors present
in the pulmonary vascular compartment is 76% greater than that in the
vena cava, and these precursors, on exposure to GM-CSF in vitro, have a
strong ability to activate alloreactive T cells. It is possible that
some of the GM-CSF-expanded DCs observed in our study derived from such
precursors. However, Palucka et al38 have demonstrated that
human macrophages could convert into DC in vitro in the presence of
GM-CSF. Our current study has provided the first in vivo evidence to
support such conversion. We found that macrophage population was
induced 5 days after GM-CSF transgene delivery and dramatically
decreased at the time when a myeloid DC population emerged at day 12. Thus, these findings, together with the fact that GM-CSF is a
well-known stimulator of macrophage proliferation,17,18,39
strongly suggest that GM-CSF-induced DCs derived primarily from
expanded macrophages. In further support of our findings, such
CD11b-expressing myeloid DCs have recently been identified in lymphoid
organs.11,25 Compared with lymphoid-derived DCs,
myeloid-derived DCs are believed to play a differential
immune-stimulatory role in host defense.30,40-42 This
selective effect on myeloid DCs by GM-CSF suggests that GM-CSF is a
proimmune cytokine in the lung. Indeed, we observed a markedly enhanced
immune response at both cellular and cytokine levels during adenoviral
infection in the lung. Our recent demonstration that airway allergic
sensitization to repeated airway ovalbumin challenges only
occurred in mice that expressed GM-CSF transgene has lent further
support to a key proimmune role of GM-CSF in the lung.43
In contrast to a pronounced effect of GM-CSF on macrophage-derived DCs,
the effect of GM-CSF on CD11blow/CD11cbright DC
population was minimal. Although GM-CSF transgene expression did not
markedly expand this cell population in the lung, it enhanced the level
of MHC II expression. Of note, GM-CSF did not markedly enhance B7.1
expression on this cell population. It is thus possible that enhanced
MHC II expression on these cells was an indirect effect from GM-CSF via
its effect on IFN release. This notion is supported by an in vitro
observation by Larsen et al44 that, different from GM-CSF,
IFN selectively induces MHC II but not B7.1 expression on murine DC
or even inhibits B7.1 expression on Langerhans cells.45
GM-CSF has been shown to be unable to induce the differentiation of DCs
of lymphoid origin.11,27 We also found that these lung
residential DCs expressed little CD11b but bright CD11c and a moderate
level of B7.1 and that GM-CSF had little effect on their
differentiation. Together, these findings suggest that these cells are
likely of lymphoid origin. However, consistent with the other
study,46 we have failed to detect any CD8 expression, a
lymphoid-lineage marker, on any of the DCs in the lung (data not
shown), suggesting a lack of such DCs in the lung. Nevertheless, it is
worthwhile to bear in mind that not all lymphoid DCs express
CD8.27
In summary, we have shown a potent effect of GM-CSF on induction of two
important APC populations, DCs and macrophages, during pulmonary viral
infection. We have also revealed that GM-CSF exerts its effect on a
myeloid-derived but not "lymphoid"-derived DC population. Our
findings not only suggest that GM-CSF is a potent immune enhancer
during pulmonary immune responses but also provide the rationale for
using GM-CSF as an immune adjuvant to prevent or treat
pulmonary infectious or malignant diseases, in particular, those
occurring in immune-compromised hosts.
| |
Acknowledgments |
The authors wish to thank Anna Zganiacz and Mary Jo Smith for their
excellent technical assistance.
| |
Footnotes |
Submitted February 25, 1999; accepted December 3, 1999.
Supported by grants from the Medical Research Council (MRC)
of Canada, McMaster University, Hamilton Health Sciences Corporation, and St Joseph's Hospital.
J.W. is an MRC-CLA (Canadian Lung Association) fellow. Z.X. is an MRC
scholar and holder of Ontario Premier's Research Excellence Award.
Reprints: Zhou Xing, Health Sciences Centre, Rm 4H19,
Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; e-mail: xingz{at}fhs.csu.mcmaster.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction