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of June 17, 2017.
The Inflammatory versus Constitutive
Trafficking of Mononuclear Phagocytes into
the Alveolar Space of Mice Is Associated with
Drastic Changes in Their Gene Expression
Profiles
Mrigank Srivastava, Steffen Jung, Jochen Wilhelm, Ludger
Fink, Frank Bühling, Tobias Welte, Rainer M. Bohle,
Werner Seeger, Jürgen Lohmeyer and Ulrich A. Maus
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:1884-1893; ;
doi: 10.4049/jimmunol.175.3.1884
http://www.jimmunol.org/content/175/3/1884
The Journal of Immunology
The Inflammatory versus Constitutive Trafficking of
Mononuclear Phagocytes into the Alveolar Space of Mice Is
Associated with Drastic Changes in Their Gene
Expression Profiles1
Mrigank Srivastava,*¶ Steffen Jung,‡ Jochen Wilhelm,† Ludger Fink,† Frank Bühling,§
Tobias Welte,¶ Rainer M. Bohle,† Werner Seeger,* Jürgen Lohmeyer,* and Ulrich A. Maus2*¶
M
ononuclear phagocytes are known to contribute to
both acute and chronic inflammatory diseases of the
lung, including acute respiratory distress syndrome
(ARDS)3, bronchiolitis obliterans, and idiopathic pneumonia syndrome (1–5). In addition, we recently demonstrated that monocytes may act as regulators of the neutrophilic response in a mouse
model of acute lung inflammation (6, 7). However, the molecular
mechanisms and potential candidate genes that might regulate the
accessory function of mononuclear phagocytes in acute lung inflammation have not been characterized. The lack of potent purification protocols allowing the isolation of monocytic cells from
both peripheral blood and alveolar compartments may partially
explain the lack of currently available data addressing the changes
*Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine
and †Department of Pathology, Justus-Liebig-University, Giessen, Germany; ‡Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel;
§
Institute of Immunology, Otto-von-Guericke University, Magdeburg, Germany;
and ¶Department of Pulmonary Medicine, Hannover School of Medicine, Hannover,
Germany
Received for publication March 24, 2005. Accepted for publication May 9, 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 German Research Foundation Grant 547 “Cardiopulmonary Vascular System” and the National Network on Community-Acquired Pneumonia (CAPNETZ). S.J. is a Scholar of the Benoziyo Center for Biomolecular
Medicine.
2
Address correspondence and reprint requests to Dr. Ulrich A. Maus, Department of
Pulmonary Medicine, Hannover School of Medicine, Feodor-Lynen Strasse 21, Hannover 30625, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; BAL,
bronchoalveolar lavage; BALF, BAL fluid; FSC, forward scatter; DC, dendritic cell;
IP, inflammatory protein; PRR, pattern recognition receptor.
Copyright © 2005 by The American Association of Immunologists, Inc.
in gene expression profiles of these cells transmigrating from the
vascular into the alveolar compartment under baseline vs inflammatory conditions.
Previous studies from our laboratory made use of the lipophilic
intravital dye, PKH26-PCL, to discriminate resident alveolar macrophages (strongly PKH26 positive) from newly alveolar-recruited
monocytic cells (PKH26 dull) to study their recruitment pathways
during lung inflammation (6, 8, 9). However, this method did not
stain circulating blood monocytes to allow their subsequent purification for molecular and functional characterization. Therefore,
in the current study, we made use of a novel transgenic mouse
strain (CX3CR1⫹/GFP) that allows the identification and subsequent FACS of both circulating and alveolar recruited mononuclear phagocytes to determine changes in their gene expression
profiles during their recruitment into the alveolar compartment under both baseline and acute lung inflammatory conditions. In
CX3CR1⫹/GFP mice, one allele for the gene encoding CX3CR1,
the receptor for the membrane-tethered chemokine fractalkine
(CX3CL1, Fkn) is replaced by the gene encoding GFP (10). Because the transgene GFP is under the control of the CX3CR1 gene
promoter and because CX3CR1 is homogeneously expressed on
circulating monocytes but not on resident alveolar macrophages,
CX3CR1⫹/GFP mice were used in the current study to track, sort,
and genotype mononuclear phagocytes during their migration into
the alveolar air space and, at the same time, allowing their clearcut
discrimination from differentiated, resident alveolar macrophages
and most other inflammatory elicited leukocyte subsets.
DiVa-assisted FACS analysis of bronchoalveolar lavage (BAL)
fluid cells collected from CX3CR1⫹/GFP mice enabled us for the
first time to detect, sort, and transcriptionally profile constitutively
0022-1767/05/$02.00
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Mononuclear phagocytes enter the lungs both constitutively to maintain alveolar macrophage and dendritic cell homeostasis, as
well as during lung inflammation, where the role of these cells is less well defined. We used a transgenic mouse strain (CX3CR1ⴙ/
GFP) that harbors a GFP label in circulating monocytes to identify and sort these cells from the vascular and alveolar compartments under both constitutive and acute lung inflammatory conditions. Using nylon arrays combined with real-time RT-PCR for
gene expression profiling, we found that flow-sorted, highly purified mononuclear phagocytes recruited to acutely inflamed mouse
lungs showed strongly up-regulated mRNA levels of the neutrophil chemoattractants KC, MIP-2, and IP-10, which contrasted with
alveolar mononuclear phagocytes that immigrated in steady state. Similar observations were made for the lysosomal cathepsins
B, L, and K being strongly up-regulated in mononuclear phagocytes upon recruitment to inflamed lungs but not during constitutive alveolar immigration. Inflammatory elicited mononuclear phagocytes also demonstrated significantly increased mRNA
levels of the cytokine TNF-␣ and the PRR-associated molecules CD14, TLR4, and syndecan-4. Together, inflammatory elicited
mononuclear phagocytes exhibit strongly increased neutrophil chemoattractants, lysosomal proteases, and LPS signaling mRNA
transcripts, suggesting that these cells may play a major role in acute lung inflammatory processes. The Journal of Immunology,
2005, 175: 1884 –1893.
The Journal of Immunology
migrated alveolar mononuclear phagocytes under noninflammatory conditions. In addition, we demonstrate that alveolar mononuclear phagocyte recruitment in response to the monocyte chemoattractant, CCL2, is associated with profound changes in their
gene expression profiles. Finally, we provide evidence that mononuclear phagocytes recruited into the lungs of mice in response to
CCL2 in the presence of low endotoxin challenge exhibit an activated, highly proinflammatory genotype, characterizing these cells
as powerful cellular contributors of the lung inflammatory response. The current technical approach may help to identify candidate genes regulating the functional role of mononuclear phagocytes in both acute and chronic lung inflammatory conditions.
Materials and Methods
Animals
Reagents
Atlas Mouse 1.2 II nylon array membranes, BD Supersmart mRNA amplification kit and BD Atlas SMART fluorescent probe amplification kit
were purchased from BD Biosciences. Nylon membrane processing was
performed according to the manufacturer’s instructions. The DNA isolation
and Qiaquick PCR purification kit were purchased from Qiagen. The murine CCL2 protein, the homologue of the human MCP-1 gene product
(JE/MCP-1), was purchased as a recombinant protein preparation from
PeproTech and was routinely ascertained to be free of endotoxin as analyzed with the Coatest amoebocyte lysate assay (detection limit ⬍ 10 pg/
ml; Chromogenix). Escherichia coli LPS (0111:B4) was purchased from
Sigma-Aldrich. Roti-Quick kit for total RNA isolation was purchased from
Carl Roth. SYBR green 1 kit was purchased from Eurogentec. Random
hexamer primers were purchased from Boehringer Mannheim. Moloney
murine leukemia virus-RT and recombinant RNase inhibitor were purchased from Promega. AmpliTaq polymerase, 10⫻ PCR buffer containing
15 mM MgCl2, and dNTPs were purchased from Applied Biosystems.
DTT was purchased from Invitrogen Life Technologies.
Treatment of animals
RNA profiling of trafficking mononuclear phagocytes was performed in
three different treatment groups: mice in group 1 were left untreated to
monitor the constitutive mononuclear phagocyte trafficking into the lungs
of mice in the absence of any previous manipulation. Mice in group 2
received intratracheal applications of CCL2 (50 ␮g) alone for 24 h to study
changes in RNA expression profiles occurring in mononuclear phagocytes
accumulating within the lungs of mice in response to the major monocyte
chemoattractant, CCL2. Mice in group 3 received combined intratracheal
applications of CCL2 (50 ␮g) in the presence of low doses of endotoxin
(10 ng) for 24 h. The combined application of CCL2⫹LPS has recently
been shown to induce an ARDS-like acute lung inflammatory response
with monocytes acting as facilitators of the developing neutrophilic alveolitis (7, 11). All treatment protocols were done essentially as described
recently (6 –9, 11). Briefly, CX3CR1⫹/GFP mice were anesthetized with
tetrazoline hydrochloride and ketamine, and the trachea was exposed by
surgical resection. Intratracheal instillation of CCL2 in the absence (group
2) or presence (group 3) of endotoxin (10 ng/mouse) was performed under
stereomicroscopic control using a 29 gauge Abbocath, which was inserted
into the trachea. After instillations, wounds were closed with sterile sutures. Mice were allowed to recover from anesthesia and subsequently
returned to their cages with free access to food and water.
Collection of blood and BAL fluid (BALF)
Twenty-four hours after intratracheal instillations, animals were sacrificed
with an overdose of isoflurane (Forene; Abbott Laboratories). Blood and
BALF were collected as described earlier (6 –9, 11).
Flow cytometry
A high-throughput FACSVantage SE flow cytometer (BD Biosciences)
equipped with a DiVa sort option and an argon ion laser operating at 488
nm excitation wavelength and a laser output of 200 mW were used for the
sorting of peripheral blood and BALF mononuclear phagocytes. Blood and
BALF specimen were filtered through a 40-␮m cell strainer (BD Biosciences) before cell sorting. Flow cytometric data of GFP-positive peripheral blood and alveolar mononuclear phagocytes from the various treatment groups were acquired on five-decade log-scale dot plots displaying
forward scatter (FSC) area vs side scatter area and fluorescence 1 area vs
fluorescence 2 area characteristics, respectively. First, hierarchy sort gates
were set in FSC vs side scatter dot plots to exclude lymphocytes; second,
hierarchy sort gates specific for GFP-expressing mononuclear phagocytes
were set according to FSC area vs FL1 (F525 ⫾ 15 nm; FITC/GFP) characteristics; and third, hierarchy sort gates were set according to FL1 vs FL2
characteristics (F575 ⫾ 25 nm), thus allowing the exclusion of both alveolar macrophages and neutrophils.
Total cellular RNA isolation, cDNA synthesis, and amplification
Total cellular RNA was isolated from sorted and highly purified (⬎98%)
GFP-positive mononuclear phagocytes from the various treatment groups
using a commercially available RNA isolation kit (Carl Roth). RNA quantification and purity was determined on an Agilent Bioanalyzer 2100 (Agilent Biosystems). Only those RNA preparations exceeding absorbance ratios of (A260/280 nm) ⬎ 1.90 were further processed for amplification and
real-time RT-PCR validation experiments. Because numbers of constitutively alveolar recruited GFP-positive mononuclear phagocytes contained
in BALFs of untreated CX3CR1⫹/GFP mice amounted to only ⬃0.3– 0.5%
of total BALF cellular constituents (⬃1500 cells/mouse), RNA samples
from mononuclear phagocytes of ⬃100 untreated group 1 CX3CR1⫹/GFP
mice (10 –15 mice of group 2 ⫹ 3 mice) were pooled together and subsequently used for RNA amplification before gene expression profiling using
a nylon cDNA array (12). Experimental details regarding synthesis of complementary DNA and real-time RT-PCR validation were performed according to recently described experimental protocols (13–15).
Statistical analysis
The data are presented as mean ⫾ SEM. Differences in outcome variables
between treatment groups were analyzed by one-way ANOVA followed by
Scheffe’s post hoc analysis. Values of p ⬍ 0.05 were considered to be
significant.
Results
CX3CR1⫹/GFP mice allow tracking of circulating blood
mononuclear phagocytes into the alveolar compartment under
baseline and inflammatory conditions
Because of their homogeneously expressed, intrinsic GFP label,
peripheral blood monocytes from CX3CR1⫹/GFP mice were easily
detectable by their increased fluorescence 1 characteristics in flow
cytometry, which is consistent with recently published reports
(Ref. 10; Fig. 1A). To confirm the specificity of the chosen sorting
gates, the gated GFP-positive peripheral blood leukocyte population was sorted by high-speed flow cytometry combining both FSC
area vs FITC area characteristics (Fig. 1A) and fluorescence 1
(FITC, GFP) vs fluorescence 2 characteristics (Fig. 1B). Subsequently, flow-sorted cells were reanalyzed by FACS to assess the
purity of the sorted cell populations, which was found to be consistently ⬎98% (Figs. 1C and 2C). Flow-sorted cells were also
subjected to Pappenheim staining, which clearly identified these
cells to consist almost exclusively of peripheral blood monocytes,
as judged by their typical monocytic morphology (Fig. 1D). In
addition, the sorted cells clearly stained positive for unspecific
esterase (data not shown).
FACS analysis of BALF cells collected from untreated
CX3CR1⫹/GFP mice consistently revealed a small but clearly identifiable GFP-expressing cell population amounting to 0.3– 0.5% of
total BAL cells that was easily distinguishable from GFP-negative
resident alveolar macrophages (Fig. 2, A and B). High-speed cell
sorting and subsequent Pappenheim and unspecific esterase staining of these cells again revealed a monocytic cell population and,
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Heterozygous (CX3CR1⫹/GFP) mice were generated on a mixed C57BL/
6 ⫻ 129/Ola genetic background. BALB/c CX3CR1⫹/GFP mice were derived from repeated backcrosses (N9) into the BALB/c background (10).
Parent CX3CR1GFP/GFP and CX3CR1⫹/⫹ mice were bred to yield offspring
with the CX3CR1⫹/GFP genotype, which were then used in all the experiments at 8 –12 wk of age. All mice were bred and kept under specific
pathogen free conditions with free access to food and water. All animal
experiments were approved in accordance with the guidelines of our Institutional Animal Care and Use Committee.
1885
1886
GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES
thus, to the best of our knowledge, identifies these rare cells as
contributors to either alveolar macrophage and/or dendritic cell
(DC) homeostasis in the absence of lung inflammation (Fig. 2D).
The intratracheal application of recombinant murine CCL2 in
the absence (or presence) of endotoxin did not affect GFP expression levels and proportions of circulating mononuclear phagocytes,
as shown by flow cytometry (Fig. 1, A–C, middle and right columns) but induced a strong increase in alveolar accumulating GFPpositive leukocytes (Fig. 2, A–C, middle and right columns), which
is in agreement with recently published reports (6, 8, 9). As expected, sorting of these CCL2-elicited, GFP-positive mononuclear
phagocytes and subsequent Pappenheim-staining identified these
cells as highly purified (⬎98%) alveolar recruited monocytic cells
(Fig. 2D, middle and right photographs). These data expand previous observations and clearly demonstrate the feasibility to track
and purify GFP-positive mononuclear phagocytes from both intravascular and intra-alveolar compartments under baseline and acute
inflammatory conditions for subsequent gene expression profiling
in CX3CR1⫹/GFP mice.
Inflammatory vs constitutive trafficking of mononuclear
phagocytes into the lungs of mice is associated with drastic
changes in their gene expression profiles
In the current study, we compared changes in gene expression
profiles of circulating vs alveolar recruited mononuclear phago-
cytes from untreated, CCL2 alone- and CCL2⫹LPS-treated
CX3CR1⫹/GFP mice. Using nylon array technology covering a set
of 1176 spotted genes, RNA expression levels assessed in alveolar
recruited mononuclear phagocytes are presented as mean fold regulation relative to the corresponding RNA expression levels detected in circulating mononuclear phagocytes of the same treatment groups from three independent experiments. In addition,
RNA level changes for selected genes of interest were validated by
real-time RT-PCR.
Baseline alveolar mononuclear phagocyte recruitment observed
in the lungs of untreated CX3CR1⫹/GFP mice was associated with
only minor changes in gene expression profiles, and most of the
differentially regulated genes (n ⫽ 13) were found to belong to the
growth and transcription factor gene families (Table I; Fig. 3, A
and B). This finding clearly suggests that the homeostatic process
of constitutive mononuclear phagocyte extravasation into the alveolar compartment under noninflammatory conditions does not
lead to a significant activation of the recruited cells. In contrast,
when mice received a single intratracheal instillation of the monocyte chemoattractant, CCL2, which is the key factor eliciting inflammatory monocyte trafficking into the lung, we observed an
overall ⬃6-fold increase in the number of differentially regulated
genes in alveolar vs circulating mononuclear phagocytes (n ⫽ 66)
compared with constitutively migrated mononuclear phagocytes
(Table I; Fig. 3A). Interestingly, most of these genes belong to the
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FIGURE 1. Flow cytometric identification and flow sorting of peripheral blood mononuclear phagocytes of untreated, CCL2-treated, and CCL2⫹LPStreated CX3CR1⫹/GFP mice. Transgenic CX3CR1⫹/GFP mice were either left untreated (A–D, left column) or received intratracheal instillations of CCL2
(50 ␮g/mouse, 24 h) in the absence (A–D, middle column) or presence (A–D, right column) of LPS. Twenty-four hours later, mice were killed, and
peripheral blood was collected for sorting of circulating monocytes. Gating of circulating monocytes was done using three-hierarchy sort gates, as described
in Materials and Methods. Population 1 (P1) in A and B (left, middle, and right dot plot) identifies the GFP-positive circulating monocyte populations in
the various treatment groups. The dot plots in C (left, middle, and right) illustrate the postsort analysis of the respective monocyte populations gated by
P1. The photomicrographs in D depict Pappenheim-stained cytospin preparations of the respectively sorted mononuclear phagocyte populations. PB-Mo,
peripheral blood monocyte; const., constitutive.
The Journal of Immunology
1887
cell adhesion molecule families, including CD63 and CD68, as
well as the growth factor, cytokine, and chemokine families. Gene
expression of GRO-1 encoding the neutrophil chemoattractant KC,
monocyte chemoattractant CCL2, and platelet-derived growth factor-B were found to be up-regulated in CCL2-elicited alveolar
mononuclear phagocytes compared with their circulating progenitors. CCL2-elicited alveolar mononuclear phagocytes also demonstrated strongly elevated gene expression levels of the matrix
metalloproteinase MMP-10 and the lysosomal cathepsins K, L,
and D (Table I). In contrast, the cathepsin L-specific inhibitor,
cystatin F, was found to be strongly down-regulated in alveolar
compared with circulating mononuclear phagocytes of the CCL2
treatment group, demonstrating that CCL2-elicited alveolar mononuclear phagocytes exhibit a differentially regulated spectrum of
proteolytically active enzymes with established functions in extracellular matrix degradation, pathogen elimination, as well as Ag
processing.
As anticipated, most drastic changes in gene expression profiles
of alveolar vs circulating mononuclear phagocytes were observed
in CX3CR1⫹/GFP mice cochallenged with CCL2 in the presence of
low levels of endotoxin. This treatment regimen has been charac-
terized recently to induce an ARDS-like acute lung inflammation
(7, 16). A total of 70 genes was found to be differentially regulated
within this treatment group, out of which 34 genes overlapped with
CCL2 alone-treated mice (Table I; Fig. 3, A and B). Importantly,
CCL2⫹LPS elicited alveolar mononuclear phagocytes again
showed strongly up-regulated mRNA levels for neutrophil chemoattractants such as KC and inflammatory protein (IP)-10,
whereas mRNA levels of chemokine receptors CX3CR1, CXCR2,
and CXCR3 were down-regulated in CCL2⫹LPS-elicited alveolar
mononuclear phagocytes (Table I; Fig. 3, A and B). The lysosomal
cathepsins K, L, and D were strongly up-regulated in CCL2⫹LPSelicited mononuclear phagocytes, whereas the inhibitor cystatin F
was not detectable by nylon array analysis (Table I).
To validate gene expression data by an independent method, a
selected set of interesting genes was additionally evaluated by realtime RT-PCR without any further preamplification step. As shown
in Table II, differential gene expression analysis by real-time RTPCR of alveolar vs circulating mononuclear phagocytes largely
matched the data obtained by nylon array analysis, albeit at a much
higher sensitivity level when compared with the nylon array approach. This suggests that the array technique, although accurately
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FIGURE 2. Flow cytometric identification and flow sorting of alveolar recruited mononuclear phagocytes of untreated, CCL2-treated, and CCL2⫹LPStreated CX3CR1⫹/GFP mice. Transgenic CX3CR1⫹/GFP mice were either left untreated (A–D, left column) or received intratracheal instillations of CCL2
(50 ␮g/mouse, 24 h) in the absence (A–D, middle column) or presence (A–D, right column) of LPS. Twenty-four hours later, mice were killed, and BAL
was collected for sorting of alveolar recruited mononuclear phagocytes from the various treatment groups. Gating of alveolar mononuclear phagocytes was
done using three-hierarchy sort gates, as described in Materials and Methods. Population 1 (P1) in A and B (left dot plots) identifies the GFP-positive,
constitutively alveolar migrated mononuclear phagocyte populations amounting to ⬃0.3– 0.5% of the total BALF cellular constituents, whereas P1 in dot
plot A and B of the middle and right columns depict the CCL2- and CCL2⫹LPS-elicited alveolar-accumulating mononuclear phagocytes, respectively. The
P2 populations in A and B (left, middle, and right column) illustrate the resident alveolar macrophages with lower FITC-area (FITC-A) characteristics
compared with alveolar mononuclear phagocytes. The populations not gated in A and B (middle and right columns) depict the fraction of CCL2-elicited
alveolar lymphocytes (middle dot plot in A and B) and alveolar-recruited neutrophils (right dot plots in A and B). The dot plots in C (left, middle, and right)
illustrate the postsort reanalysis of the respective mononuclear phagocytes populations gated by P1. The photomicrographs in D show Pappenheim-stained
cytospin preparations of the respectively sorted mononuclear phagocytes populations. FSC-A, FSC-area.
1888
GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES
Table I. Nylon array gene expression profiling of alveolar mononuclear phagocytesa
GenBank
Accession no.
Gene Symbol
Gene Description
Mean Fold
Regulation
CCL2
Mean Fold
Regulation
CCL2 ⫹ LPS
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫺1.7
⫺3.3
⫺2.0
5.3
1.8
ND
⫺2.5
2.9
2.4
1.6
ND
ND
ND
3.5
ND
⫺10
⫺1.4
⫺3.3
⫺2.0
4.6
ND
1.8
ND
ND
ND
ND
⫺2.5
⫺3.3
⫺3.3
2.3
⫺2.0
⫺5
ND
ND
⫺2.5
ND
ND
ND
⫺2.5
ND
ND
⫺2.5
2.9
21.9
ND
4.2
4.8
ND
4.5
⫺1.7
ND
ND
3.3
2.7
ND
4.1
2.4
4.9
⫺2.0
ND
ND
ND
ND
ND
ND
ND
⫺3.3
ND
2.4
1.6
⫺3.3
⫺2.5
ND
⫺3.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.8
ND
ND
ND
ND
ND
ND
ND
ND
1.4
ND
2.6
3.5
3.2
5.3
ND
1.9
1.7
6.6
ND
2.3
ND
ND
ND
ND
ND
ND
ND
2.4
ND
ND
ND
2.4
1.5
ND
ND
5.6
⫺1.4
2.9
ND
8
⫺2.0
3.6
2.3
ND
⫺1.7
⫺5
⫺2.5
3.2
⫺3.3
3.5
1.5
1.5
ND
ND
ND
ND
ND
3.6
2.8
ND
ND
⫺1.4
4.8
1.7
⫺1.4
ND
ND
⫺2.5
ND
ND
8.1
1.6
ND
ND
ND
ND
⫺2.0
ND
ND
ND
ND
ND
ND
ND
ND
⫺3.3
⫺3.3
⫺5
⫺1.7
ND
(Table continues)
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Cell surface Ags and receptors
X14951
ITGB2
Integrin ␤-2
L23108
CD36
CD36 Ag
U18372
CD37
Leukocyte Ag 37
D16432
CD63
CD63 Ag
X68273
CD68
CD68 Ag
AF043445
CD84
CD84 Ag
X65493
ICAM2
Intercellular adhesion molecule-2
L15435
TNFSF9
Tumor necrosis factor superfamily member 9
M16367
FCGR2
IgG Fc R II ␤
M14215
FCGR3
IgG Fc R III
AF074912
CX3CR1
CX3C chemokine receptor 1
D17630
CXCR2
High-affinity interleukin-8 receptor B
AB003174
CXCR3 (CD183)
CXC chemokine receptor 3
D89571
SDC4
Syndecan-4
AB007599
MD-1
MD-1 protein
AJ223765
AR1
Activating receptor 1
Growth factors, cytokines, and chemokines
S65032
BMP4
Bone morphogenetic protein 4
M86736
GRN
Granulin
J04596
GRO1 (CXCL1)
Growth-regulated protein 1
M86829
SCYB10 (CXCL10)
Small inducible cytokine B10
X53798
MIP2 (CXCL2)
Macrophage inflammatory protein 2
J04467
MCP-1 (CCL2)
Monocyte chemotactic protein-1
X57413
TGFB2
Transforming growth factor ␤-2
M84453
PDGFB
Platelet-derived growth factor, B chain
L26349
TNFRSF1A
Tumor necrosis factor receptor superfamily member 1A
IL and IFNs
L12120
IL10R-␣
IL-10R ␣ chain
U64199
IL12R-␤2
IL-10R ␤2 chain
X53802
IL6RA
IL-6 RA IL-6R ␣ chain
X01450
IL1A
IL-1␣
M15131
IL1B
IL-1␤
K00083
IFNG
IFN-␥
Miscellaneous proteins
M24554
ANXA1
Annexin A1
AJ001633
ANX3
Annexin A3
D63423
ANX5
Annexin A5
X70100
KLBP
Keratinocyte lipid-binding protein
J05020
FCERG
Low-affinity IgE Fc R 1 ␥
X63535
AXL
Tyrosine-protein kinase receptor UFO
X12905
PFC
Properdin factor complement
X03479
SAA3
Serum amyloid A-3
M35186
Apob
Apolipoprotein
D49733
LMNA
Lamin A
M26251
VIM
Vimentin
Y07919
AP1B1
Adaptor protein complex AP-1 ␤-1 subunit
D00208
S100A4
S-100 calcium binding protein A4
M83219
S100A9
S-100 calcium binding protein A9
U65586
TRF1
Telomeric repeat binding factor 1
X93167
FN
Fibronectin
M14342
PTPRC
Protein tyrosine phosphatase receptor type C
M31811
MAG
Myelin-associated glycoprotein
M57470
LGALS1
Lectin galactose-binding soluble protein 1
Z31554
CCT4
Chaperonin subunit 4 (␦)
U27129
HSC70
Heat shock cognate 71-kDa protein
X16834
LGALS3
Lectin galactose-binding soluble protein 3
Transcription factors
AF077742
TCFEC
Transcription factor TFEC
X03039
EIF4A1
Eukaryotic initiation factor 4A-1
X52803
PPIA
Peptidyl-prolyl cis-isomerase A
M31418
IFI202A
IFN-activated gene 202A
U51992
ISGF3G
IFN-stimulated gene factor 3 ␥
M32489
ICSBP1
IFN concensus sequence binding protein
X65553
PABPC1
Poly A binding protein cytoplasmic 1
V00727
FOS
Cellular oncogene fos
Metabolism enzymes
J04060
THBD
Thrombomodulin
U11494
SNF1LK
SNF1-like kinase
Y07708
NDUFA1
NADH-ubiquinone oxidoreductase MWFE subunit
S80446
ALOX12
Arachidonate 12-lipoxygenase
Mean Fold
Regulation
Constitutive
The Journal of Immunology
1889
Table I. Continued
GenBank
Accession no.
Mean Fold
Regulation
CCL2
Mean Fold
Regulation
CCL2 ⫹ LPS
NAD -dependent 15-hydroxyprostaglandin dehydrogenase
Lipoprotein lipase
Carbonic anhydrase IV
Hephaestin
Methenyltetrahydrofolate cyclohydrolase
Arginase II
2,3-bisphosphoglycerate mutase
Cytochrome c oxidase polypeptide V
Cytochrome c oxidase polypeptide VIa
Cytochrome c oxidase polypeptide VIII
ND
ND
ND
⫺2.0
ND
ND
ND
ND
ND
ND
ND
1.8
2.6
ND
ND
6.8
⫺2.0
ND
⫺2.0
⫺1.7
⫺5
ND
2
ND
2.7
4.7
ND
1.5
1.4
ND
A disintegrin-like & metalloproteinase domain with
thrombospondin type 1 motif 1
Matrix metalloproteinase 10
Matrix metalloproteinase 8
Calpain 5
Cathepsin K
Cathepsin L
Cathepsin S
Cathepsin H
Cathepsin D
Legumain
Phospholipase D3
Vitamin K-dependent protein C
Kallikrein 11
PDE4D
⫺5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.8
ND
3
2
ND
4.7
3.7
1.3
1.6
1.8
2.5
1.9
ND
ND
ND
ND
ND
3.4
4.6
2.3
ND
ND
4.1
2
2.1
2.8
ND
⫺3.3
ND
ND
ND
ND
ND
⫺2.5
4.8
ND
1.9
⫺3.3
ND
3.3
4
2.2
⫺3.3
5-Hydroxytryptamine 3 receptor
Platelet-activating factor receptor
Galanin receptor 2
Adenosine A1 receptor
Vacuolar ATP synthase
Cellular nucleic acid binding protein
Intelectin
IFN-activatable protein 203
IFN-inducible protein 49
IL-18R 1
Palate lung and nasal epithelium clone protein
Tektin 1
Voltage-dependent N type calcium channel ␣ 1B subunit
ND
ND
ND
ND
ND
ND
ND
6.1
ND
ND
ND
⫺3.3
ND
ND
2.4
ND
⫺5
2.4
ND
⫺5
ND
ND
⫺3.3
ND
ND
ND
⫺3.3
2.4
4.5
ND
1.9
⫺2.0
ND
ND
3.3
⫺3.3
2.4
ND
3.6
Ubiquitin
Protein kinase C inhibitor protein 1
Glyceraldehyde-3-phosphate dehydrogenase
Beta-actin
Ribosomal protein S29
⫺1.4
ND
ND
ND
ND
⫺2.0
⫺1.4
⫺1.3
⫺2.0
⫺2.0
⫺1.4
ND
ND
ND
⫺1.4
Gene Symbol
U44389
HPGD
M60847
LPL
U37091
CA4
AF082567
HEPH
J04627
MTHFD2
U90886
ARG2
M22867
BPGM
X53157
COX5B
L06465
COX6A1
U37721
COX8
Proteases and kinases
D67076
ADAMTS1
X76537
MMP10
U96696
MMP8
Y10656
CAPN5
X94444
CTSK
X06086
CTSL
AJ223208
CTSS
U06119
CTSH
X52886
CTSD
AJ000990
LGMN
AF026124
PLD3
D10445
PROC
X13215
KLK11
M94541
cAMP-specific PDE4D
Protease inhibitors
AF031825
CST7
U59807
CSTB
U07425
HCF2
M65736
MUG1
U96700
SPI6
Functionally unclassified proteins
M74425
HTR3A
D50872
PAFR
AF077375
GALR2
U05671
ADORA1
M64298
ATP6VOC
L12693
CNBP
AB016496
ITLN
AF022371
IFI203
L32974
IFI49
U43673
IL18R1
U69172
PLUNC
AF081947
TEKT1
AF042317
CACNA1B
Housekeeping genes
X51703
UBB
D78647
KCIP1
M32599
G3PDH
M12481
ACTB
L31609
RPS29
⫹
Cystatin F
Cystatin B
Heparin cofactor II
Murinoglobulin 1
Serine protease inhibitor 6
a
The table shows the summary of genes differentially expressed in alveolar mononuclear phagocytes of different treatment groups. Positive values in the columns indicate
mean fold up-regulation of that particular gene in alveolar mononuclear phagocytes in comparison to peripheral blood monocytes, whereas negative values indicate mean fold
down-regulation of the respective gene in alveolar mononuclear phagocytes as compared with peripheral blood monocytes of the same treatment group. Lack of a given value
(indicated as ND) either indicates lack of regulation or regulation on a level too low to allow its quantification.
detecting trends in altered gene expression profiles, may not be
suitable to accurately quantify low levels of gene expression in
unstimulated cells, such as constitutively alveolar recruited mononuclear phagocytes. In fact, real-time RT-PCR analysis confirmed
that alveolar recruited compared with circulating mononuclear
phagocytes of CCL2-treated CX3CR1⫹/GFP mice expressed significantly elevated mRNA levels of the neutrophil chemoattractants KC, MIP-2 (not included in the nylon array analysis), and
IP-10. Particularly, the mRNA level of the major neutrophil chemoattractant, MIP-2, was found significantly up-regulated in alveolar vs circulating mononuclear phagocytes of CCL2⫹LPS-treated
mice. In line with the findings of the nylon array analysis, real-time
PCR confirmed a consistent decline in the mRNA levels of chemokine receptor CX3CR1 in alveolar compared with circulating
mononuclear phagocytes from untreated, CCL2 alone-, and
CCL2⫹LPS-treated CX3CR1⫹/GFP mice. In contrast, drastically
increased mRNA levels of the lysosomal cathepsins B, L, D, and
K together with down-regulated cystatin F mRNA levels were observed only in alveolar mononuclear phagocytes of the CCL2 and
CCL2⫹LPS treatment groups.
To gain further insight into changes in pattern recognition receptor (PRR) signaling molecules expression profiles of alveolar
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Gene Description
Mean Fold
Regulation
Constitutive
1890
GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES
Discussion
vs circulating mononuclear phagocytes of the various treatment
groups, we additionally determined mRNA expression levels of
TLR2, TLR4, CD14, MD-1, Ly78, and syndecan-4 in the corresponding mononuclear phagocyte populations. Interestingly, most
drastic changes in PRR mRNA levels were noted for CD14, showing highest up-regulation in CCL2-recruited and significantly less
increase in CCL2⫹LPS-elicited alveolar mononuclear phagocytes
when compared with circulating mononuclear phagocytes, which
is in agreement with recent data addressing CD14 cell surface
protein expression on freshly alveolar recruited mononuclear
phagocytes (8). Along the same line, we also found that PRRsignaling molecules other than CD14, such as TLR4 and syndecan-4, were significantly up-regulated in alveolar vs circulating
mononuclear phagocytes recovered from CCL2- and CCL2⫹LPStreated mice when compared with mononuclear phagocytes collected from untreated mice. Interestingly, TLR2 mRNA levels
were found to be slightly down-regulated in alveolar vs peripheral
blood mononuclear phagocytes of all three treatment groups,
whereas expression levels of Ly78 remained largely unchanged,
indicating differential regulation of various PRR molecules associated with the recruitment of mononuclear phagocytes.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. Distribution and schematic summary of genes expressed in
alveolar mononuclear phagocytes of the various treatment groups. A, Venn
diagram illustrating selective and overlapping spectra of genes expressed in
the different treatment groups, as determined by nylon array analysis. The
nonoverlapping areas represent the numbers of genes specifically expressed in the respective treatment group. B, Distribution pattern of differentially regulated genes belonging to different functional categories in
alveolar recruited mononuclear phagocytes of the various treatment groups,
according to nylon array analysis. f, CCL2⫹LPS treatment; 䡺, CCL2
alone treatment; and u, without treatment (constitutive (const.)).
In the current study, we exploited the endogenous GFP expression
characteristics of transgenic CX3CR1⫹/GFP mice to identify and
molecularly characterize mononuclear phagocytes before and after
their spontaneous, as well as inflammatory, trafficking from the
circulation into the alveolar compartment of the lung. Use of this
novel mouse strain enabled us to identify cells with a monocytic
phenotype within the alveolar compartment under baseline conditions, which may be involved in maintaining either alveolar macrophage and/or lung DC homeostasis. Combining DiVa-assisted
FACS with nylon array gene expression analysis and real-time
RT-PCR validation, we found that constitutively alveolar migrated
mononuclear phagocytes showed a basal gene expression profile
similar to circulating monocytes, including unchanged mRNA levels of major neutrophil chemoattractants, cell adhesion molecules,
lysosomal cysteine proteases, and PRRs. The same approach revealed that mononuclear phagocytes recruited into the lungs of
CX3CR1⫹/GFP mice in response to the major monocyte chemoattractant, CCL2 or CCL2, in the presence of low levels of endotoxin
demonstrated drastic changes in their gene expression profiles
compared with circulating mononuclear phagocytes, as reflected
by increased mRNA levels of major neutrophil chemoattractants,
LPS recognition molecules such as CD14 and TLR4, matrix metalloproteinases, and lysosomal cysteine proteases. The combination of flow cytometric and molecular approaches in the present
study reveals that the leukocyte recruitment stimulus within the
alveolar compartment strongly affects the gene expression profile
of the recruited leukocyte subsets.
Various studies in the past few years have identified peripheral
blood monocytes of mice not only to consist of different subpopulations, mainly depending on their Gr-1 expression profiles (Gr1high vs Gr-1low), but also to exhibit the plasticity to differentiate
into alveolar macrophages and/or pulmonary DCs upon their inflammatory recruitment to the lungs (6, 17–19). Because the rare
cell population of GFP-positive mononuclear phagocytes detected
in the lungs of untreated CX3CR1⫹/GFP mice showed a monocytic
morphology similar to what has been described elsewhere to reflect monocytes/small macrophages (18) and stained positive for
unspecific esterase (data not shown), it appears that these cells
might contribute to alveolar macrophage homeostasis. In contrast,
it cannot be excluded that these cells also contribute to the maintenance of pulmonary DCs, which are derived from the pool of
circulating monocytes as well. Because of its rarity, this alveolar
cell population was not extensively characterized with respect to
its immunophenotype in the current study, and clarification of
whether constitutively alveolar migrated monocytic cells primarily
differentiate into macrophages or DCs will be subject to future
investigations.
An important finding of the present study was that genes of the
key CXC chemokines KC, CXCL2 (MIP-2), and CXCL10 (IP-10)
were significantly up-regulated by a magnitude ranging from 10 to
33 in the CCL2⫹LPS treatment group, as compared with untreated
animals. These data clearly support the concept that CCL2⫹LPS
challenge provokes neutrophil chemotactic activities in alveolarrecruited mononuclear phagocytes, enabling them to act as facilitators in the development of neutrophilic alveolitis in acute lung
injury and, at least in part, contribute to the strong neutrophil chemotactic activities observed in the BALFs of CCL2⫹LPS-treated
mice (2, 6, 7, 16). This concept is further supported by the observation that S-100A9, a myeloid-related protein of the S100 protein
family known to be expressed by monocytes/macrophages that exerts neutrophil chemotactic activities in inflamed tissues (20 –25),
The Journal of Immunology
1891
Table II. Real-time RT-PCR profiling of alveolar recruited mononuclear phagocytesa
Mean Fold Gene Up-/Down-Regulation within
Treatment Group (Mean ⫾ SEM)
Gene Name/Transcript
Constitutive
CCL2
CCL2⫹LPS
Constitutive vs.
CCL2
Constitutive vs.
CCL2⫹LPS
CCL2 vs.
CCL2⫹LPS
⫺2.7 ⫾ 0.9
⫺7.5 ⫾ 2.0ⴱ
⫺2.8 ⫾ 0.7
2.7 ⫾ 0.8
⫺2.1 ⫾ 0.4
⫺3.1 ⫾ 1.2
⫺1.2 ⫾ 0.1
⫺3.9 ⫾ 1.8
1.7 ⫾ 0.2
6.2 ⫾ 0.8ⴱ
10.3 ⫾ 1.9ⴱ
⫺1.2 ⫾ 0.1
⫺2.5 ⫾ 1.1
⫺5.0 ⫾ 1.0ⴱ
⫺2.5 ⫾ 1.6
2.0 ⫾ 2.1
1.3 ⫾ 0.1
⫺1.6 ⫾ 0.1
2.2
2.0
4.7
2.3
21.5ⴱ
2.6
1.1
1.5
1.1
⫺1.4
2.7
2.0
⫺2.1
⫺1.3
⫺4.2
⫺3.1
⫺7.7ⴱ
⫺1.3
⫺3.3 ⫾ 0.6ⴱ
⫺2.3 ⫾ 0.2ⴱ
⫺1.6 ⫾ 0.5
⫺2.1 ⫾ 0.6
⫺5.0 ⫾ 0.8ⴱ
⫺2.4 ⫾ 0.7
⫺1.1 ⫾ 0.1
4.3 ⫾ 1.4
⫺1.5 ⫾ 0.1
5.0 ⫾ 0.6ⴱ
1.7 ⫾ 0.3
⫺1.3 ⫾ 0.1
⫺1.9 ⫾ 0.2
⫺1.7 ⫾ 0.1
10.0 ⫾ 3.7ⴱ
3.9 ⫾ 1.3
12.9 ⫾ 1.8ⴱ
⫺3.3 ⫾ 1.5
⫺4.6 ⫾ 0.9ⴱ
⫺2.6 ⫾ 1.0
⫺2.6 ⫾ 0.8
14.3
1.6
7.9ⴱ
3.5
3.9ⴱ
1.3
⫺1.5
33.3ⴱ
9.1ⴱ
20.5ⴱ
⫺1.7
1.1
⫺1.1
⫺2.5
2.3
5.9ⴱ
2.6ⴱ
⫺5.6
⫺3.4ⴱ
⫺1.4
⫺1.6
1.7 ⫾ 0.2
1.4 ⫾ 0.1
16.2 ⫾ 2.5ⴱ
4.6 ⫾ 0.9ⴱ
1.2 ⫾ 0.1
43.3 ⫾ 7.0ⴱ
5.2ⴱ
65.2ⴱ
12.2ⴱ
2.8ⴱ
2.5ⴱ
2.8
6.3ⴱ
4.5
5.4
⫺1.7
⫺1.7
3.4
1.2
⫺14.3ⴱ
⫺2.3
⫺4.7ⴱ
⫺4.2ⴱ
1.2
⫺3.7 ⫾ 0.8ⴱ
⫺3.2 ⫾ 1.3
3.0 ⫾ 1.1
7.8 ⫾ 2.2ⴱ
2.0 ⫾ 0.2
12.8 ⫾ 4.2ⴱ
1.4 ⫾ 0.2
20.2 ⫾ 3.2ⴱ
36.7 ⫾ 10.7ⴱ
22.1 ⫾ 4.4ⴱ
5.0 ⫾ 0.7ⴱ
35.7 ⫾ 12.1ⴱ
2.1 ⫾ 0.1ⴱ
⫺2.6 ⫾ 0.2ⴱ
3.4 ⫾ 1.0
⫺3.6 ⫾ 1.3
⫺2.5 ⫾ 0.6
⫺4.4 ⫾ 1.6
4.8 ⫾ 1.1ⴱ
⫺5.4 ⫾ 1.5ⴱ
47.6 ⫾ 4.8ⴱ
2.1 ⫾ 0.7
⫺1.6 ⫾ 0.1
9.4 ⫾ 0.4ⴱ
4.3 ⫾ 1.3ⴱ
⫺3.5 ⫾ 1.1
18.9 ⫾ 1.9ⴱ
⫺2.2 ⫾ 0.3
⫺2.2 ⫾ 0.6
3.1 ⫾ 0.3ⴱ
2.3
⫺2.0
14.0ⴱ
7.5ⴱ
1.6
40.8ⴱ
2.1
⫺1.3
5.6ⴱ
1.6
1.1
13.5ⴱ
⫺1.1
1.5
⫺2.5ⴱ
⫺4.8ⴱ
⫺1.4
⫺3.0ⴱ
⫺2.2 ⫾ 0.8
⫺2.6 ⫾ 0.3ⴱ
⫺3.0 ⫾ 1.0
⫺39.0 ⫾ 7.2ⴱ
346.8 ⫾ 25.4ⴱ
1.6 ⫾ 0.2
3.2 ⫾ 1.0
⫺1.8 ⫾ 0.8
61.1 ⫾ 8.3ⴱ
⫺1.7 ⫾ 0.5
3.5 ⫾ 0.5ⴱ
⫺4.3 ⫾ 1.5
770.7ⴱ
4.2ⴱ
9.7ⴱ
18.7ⴱ
135.8
1.6
10.6ⴱ
7.7ⴱ
⫺5.6ⴱ
⫺2.7ⴱ
1.1
⫺2.4
⫺11.0 ⫾ 1.5ⴱ
⫺6.7 ⫾ 1.7ⴱ
⫺2.4 ⫾ 0.1ⴱ
1.7
4.7ⴱ
2.8
⫺7.4 ⫾ 1.3ⴱ
⫺2.5 ⫾ 0.7
⫺2.0 ⫾ 0.1
2.9ⴱ
3.6ⴱ
1.3
a
Expression profiles of selected genes belonging to different functional categories were quantified in alveolar mononuclear phagocytes of the different treatment groups by
real-time RT-PCR, as outlined in Materials and Methods. The values are given as mean fold regulation within groups, i.e., gene expression level in alveolar compared with
circulating monocytes of the same treatment group set to 1 indicates no regulation, and values ⬍ 1 (⬎ 1) indicate respective down-regulation (up-regulation) of a given gene
in alveolar compared with circulating monocytes, columns 2– 4 from the left). In addition, values are also expressed as mean fold change between groups (comparative gene
expression level in alveolar mononuclear phagocytes of different groups, columns 5–7 from the left, as indicated). Positive values in the columns indicate up-regulation of that
particular gene, whereas negative values indicate the down-regulation of a given gene. The given values represent the mean ⫾ SEM from at least three independent experiments
performed. ⴱ indicates p at least ⬍0.05.
was also found to be significantly up-regulated in alveolar mononuclear phagocytes of CCL2 alone and CCL2⫹LPS-treated
CX3CR1⫹/GFP mice.
Our study also addressed changes in gene expression levels of
several PRR and associated signaling molecules, including TLR2
and TLR4, CD14, MD1, and syndecan 4. Most dramatic changes
were seen in mRNA levels of TLR4, syndecan-4, and CD14. The
strong up-regulation of CD14 (26, 27) by CCL2-elicited mononuclear phagocytes confirms earlier reports from our group and possibly reflects a priming of monocytes during the recruitment process with potential relevance for the pulmonary host defense (8).
Syndecan-4, a transmembrane heparan sulfate proteoglycan of the
syndecan family, was even more dramatically up-regulated in alveolar vs circulating mononuclear phagocytes of CCL2 and
CCL2⫹LPS-treated but not untreated mice. Recently, Muramatsu
and colleagues (28, 29) demonstrated that syndecan-4⫺/⫺ mice
lack susceptibility to endotoxic shock. Thus, the strong up-regulation of syndecan-4 together with other important LPS-signaling
molecules in alveolar mononuclear phagocytes supports the concept that freshly recruited mononuclear phagocytes may significantly contribute to the orchestration of acute lung inflammation.
In this regard, the observed weak changes in mononuclear cell
gene expression levels of the PRR MD-1 may well reflect the fact
that only a 24-h time point posttreatment was analyzed.
Interestingly, we found significantly increased alveolar mRNA
levels of lysosomal cathepsins together with decreased cystatin F
mRNA levels in mononuclear phagocytes of the CCL2 and
CCL2⫹LPS treatment groups compared with untreated mice.
Cathepsins play important roles in phagolysosomal degradation,
Ag processing, and tissue remodelling by professional phagocytes,
including mononuclear phagocytes. These proteolytic enzymes
have also been suggested to assist mononuclear cell transmigration
(30 –33). Cystatin F belongs to the superfamily of endogenous intracellular thiol proteinase inhibitors (34 –36). The reported findings of reciprocal expression patterns of cathepsins vs cystatins in
alveolar recruited mononuclear phagocytes of the CCL2 and
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Cell adhesion
CD18
CD36
CD37
CD63
CD68
ICAM-2
Cytokines, chemokines, chemokine
receptors, and growth factors
GRO-1 (KC)
MIP-2
CXCL 10 (IP10)
CCR2
CX3CR1
TGF␤1
TGF␤2
Proteases
MMP-10
Cathepsin B
Cathepsin L
Cathepsin D
Cathepsin S
Cathepsin K
PRR signaling
TLR4
TLR2
CD14
MD-1
Ly Ag 78
Syndecan-4
Proinflammatory
Serum amyloid A3
Cyclophilin A
TNF-␣
S-100A9
Protease inhibitors
Cystatin F
Miscellaneous
Aquaporin 1
Fold Difference in Gene Up-/Down-Regulation between
Treatment Groups
1892
In summary, the relative low proportion of circulating monocytes and the lack of sophisticated purification methods in mice are
the main reasons why the proinflammatory molecular phenotype of
this important leukocyte population is only poorly defined. In the
current study, we demonstrate the feasibility to track, sort to high
purity and transcriptionally profile peripheral blood and alveolar
mononuclear phagocytes of CX3CR1⫹/GFP mice without prior in
vivo manipulation. Based on this technique, this is the first study
to identify and sort constitutively trafficking alveolar mononuclear
phagocytes. Using gene array and real-time RT PCR we observed
that the gene expression patterns of these cells differs drastically
from that observed in CCL2-driven alveolar mononuclear phagocytes recruitment. These findings further support the concept that
constitutive mononuclear phagocyte trafficking is tightly controlled to avoid inflammatory activation and may be linked to different molecular pathways enabling migration processes. In addition, our gene expression profile analysis shows that inflammatoryelicited mononuclear phagocytes activate genetic programs that
render them potent contributors to the overall acute lung inflammatory response. Future studies using selective knockout mouse
models and gene silencing techniques, as well as protocols to specifically purify monocyte subpopulations, will help to further refine the role of distinct gene products in subsets of mononuclear
phagocytes and their relative contribution to the lung inflammatory
response to microbial challenge.
Acknowledgments
We are grateful to the excellent technical support by Regina Maus,
Petra Janssen, and Marlene Stein in preparing samples for flow cytometric
sorting of mononuclear phagocytes and in lung histology experiments.
Disclosures
The authors have no financial conflict of interest.
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CCL2⫹LPS treatment groups support the potentially important
contribution of these freshly immigrated cells in lung inflammatory remodeling processes. Interestingly, increased cathepsin B
and K mRNA levels were also found to be elevated in mononuclear phagocytes in bleomycin-induced lung injury in rats and
mice (37, 38). In addition, our study shows that the genes for
cathepsins L, D, and K were among those few genes that were
up-regulated even in alveolar mononuclear phagocytes compared
with peripheral blood monocytes of untreated mice, indicating that
constitutive trafficking of monocytic cells into the lungs to some
extent alters the cathepsin gene expression pattern in these cells.
We and others recently demonstrated that F4/80-positive peripheral blood monocytes of mice consist of two principal subpopulations, Gr-1high and Gr-1low (6). Because these subsets were found
to be equally recruited into the lungs of mice upon CCL2⫹LPS
treatment, the currently used treatment regimen to recruit mononuclear phagocytes into the lungs of CX3CR1⫹/GFP mice did not
allow us to specifically monitor subset-specific changes in gene
expression profiles between Gr-1high vs Gr-1low monocyte subsets.
However, a selective recruitment of Gr-1high but not Gr-1low
CX3CR1-expressing monocyte subsets was recently observed to
occur in a model of thioglycolate-induced peritonitis (19). These
data suggest that differences in experimental models and organs
investigated and/or inflammatory stimuli applied may possibly affect the recruitment profiles of Gr-1high vs Gr-1low monocyte subsets into tissues, which may have implications on the overall inflammatory response.
Various steps in the recruitment process may potentially contribute to the activation of genetic programs in recruited mononuclear phagocytes, particularly 1) the CCL2-CCR2 interaction itself
driving inflammatory monocyte recruitment, 2) additional molecular interactions during mononuclear phagocyte transendo/-epithelial migration, and 3) exposure of the recruited cells to an inflammatory altered alveolar microenvironment. In vitro exposure of
isolated monocytes to CCL2 is known to activate genetic programs
in monocytes but to a much more limited extent than that seen after
CCL2-driven monocyte recruitment to the alveolar space in vivo
(unpublished data). Therefore, we believe that additional activation signals that operate during the recruitment process are likely
to play a key role in determining the phenotype of alveolar recruited mononuclear phagocytes, particularly in the CCL2 alonetreated group, which showed no significant changes in BALF cytokine profiles compared with untreated animals (16). The fact that
several of the investigated genes, including cathepsins B and L,
were found to be less fold up-regulated in alveolar mononuclear
phagocytes of the CCL2⫹LPS as compared with the CCL2 alonetreated group might indicate a specific role of the alveolar micromilieu differentially modulating the expression kinetics of the
investigated genes, including the cathepsins. Indeed, LPS activation of alveolar mononuclear phagocytes (particularly monocytes)
may accelerate their transdifferentiation toward a “macrophage genotype” as is evident by the drastic up-regulation of cathepsin K
transcript levels in the CCL2⫹LPS treatment group. LPS activation is also known to down-regulate expression of cell surfacebound receptor molecules, such as CCR2 (39). In fact, chemokine
receptors such as CX3CR1 were found to be most strongly and
significantly down-regulated in alveolar mononuclear phagocytes
collected from CCL2⫹LPS compared with CCL2 alone-treated
mice. It is interesting to note that despite a strong down-regulation
of the CX3CR1 gene alveolar mononuclear phagocytes from all
treatment groups displayed a strong and easily detectable GFP label because of the characteristically long half-life of the GFP protein (⬎24h), according to our previous reports (10).
GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES
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