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Accumulation of CD11b+ Lung Dendritic
Cells in Response to Fungal Infection Results
from the CCR2-Mediated Recruitment and
Differentiation of Ly-6C high Monocytes
John J. Osterholzer, Gwo-Hsiao Chen, Michal A. Olszewski,
Jeffrey L. Curtis, Gary B. Huffnagle and Galen B. Toews
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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 © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:8044-8053; Prepublished online 23
November 2009;
doi: 10.4049/jimmunol.0902823
http://www.jimmunol.org/content/183/12/8044
The Journal of Immunology
Accumulation of CD11bⴙ Lung Dendritic Cells in Response to
Fungal Infection Results from the CCR2-Mediated
Recruitment and Differentiation of Ly-6Chigh Monocytes1,2
John J. Osterholzer,3*§ Gwo-Hsiao Chen,§ Michal A. Olszewski,†§ Jeffrey L. Curtis,*‡§
Gary B. Huffnagle, ‡§ and Galen B. Toews*§
M
igration of resident lung dendritic cells (DC)4 to regional lymph nodes is critical for the initiation of a
wide variety of immune responses to microbial pathogens but also to noninfectious inflammatory and allergic stimuli
(1–3). In concert with this efflux of DC from the lung, nonresident
DC and monocytes accumulate in the lung in response to local
microbial or host-derived “danger signals” (3–17). These cell types
are critically important for mediating host defense against microbial pathogens yet also contribute to deleterious immune responses
as seen in murine models of asthma (18, 19) or the lung injury
associated with influenza infection (8, 20). The biological mech-
*Pulmonary Section, Medical Service and †Research Service, Ann Arbor Veterans
Affairs Health System, Department of Veterans Affairs Health System, Ann Arbor,
MI 48105; and ‡Graduate Program in Immunology and §Division of Pulmonary and
Critical Care Medicine, Department of Internal Medicine, University of Michigan
Health System, Ann Arbor, MI 48109
Received for publication August 26, 2009. Accepted for publication October
16, 2009.
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 a Merit Review Award (to G.B.T.) and a Career Development Award-2 (to J.J.O.) from the Biomedical Laboratory Research and Development Service, Department of Veterans Affairs and by National Institutes of Health
Grants R01-HL065912 (to G.B.H.), R01-AI059201 (to G.B.H.), R01-HL051082 (to
G.B.T.), and R01 HL056309 (to J.L.C).
2
Portions of this work have been presented previously at the International Conference
of the American Thoracic Society, San Diego, CA, May 20, 2009.
3
Address correspondence and reprint requests to Dr. John J. Osterholzer, Pulmonary
and Critical Care Medicine Section (111G), Department of Veterans Affairs Medical
Center, 2215 Fuller Road, Ann Arbor, MI 48105-2303. E-mail address:
[email protected]
4
Abbreviations used in this paper: DC, dendritic cell; AF, autofluorescent; EdU,
5-ethynyl-2⬘-deoxyuridine; FL, fluorescence; FSC, forward scatter; PMN, polymorphonuclear leukocyte; SSC, side scatter.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902823
anisms responsible for the accumulation of these cells within inflamed lungs are not well understood; knowledge of these mechanisms might result in novel therapies designed to increase (or
diminish) lung DC numbers for the treatment of pulmonary
disease.
We previously demonstrated that CCR2 mediates the accumulation of large numbers of DC within the lungs of mice infected
with Cryptococcus neoformans (7). This encapsulated yeast, acquired by the respiratory tract, causes pneumonia and disseminated
infection in immunocompromised patients (21–23) and can also
circumvent intact host defenses (24 –26). A T1 immune response
and classical macrophage activation are essential to clear the organism (27–34). Our results suggest that T1 immune responses are
promoted by interactions between nonresident DC and CD4 T cells
specifically within newly formed bronchovascular infiltrates. In
contrast, mice deficient in CCR2 accumulate few DC within the
lung, do not develop T1 immune responses, and fail to clear the
organism (7, 35). Thus, this model system is conducive to studies
that enhance our understanding of pulmonary adaptive immune
responses.
The strong association between the accumulation of lung DC,
T1 polarization, and fungal clearance motivated us to further investigate the mechanism(s) responsible for this accumulation of
DC in mice infected with C. neoformans. In the prior study, DC
accumulation was preceded by an increase in the CCR2 ligands
CCL2 (MCP-1) and CCL7 (MCP-3) in the lungs of CCR2⫹/⫹
mice infected with C. neoformans (7). In concert with the profound
reduction in lung DC in infected CCR2⫺/⫺ mice, this finding
strongly suggests that the CCL2/CCL7/CCR2 axis mediates the
recruitment of a circulating DC precursor to the lung.
The circulating precursor that contributes to DC accumulation in
response to fungal infection in the lung has not been identified. We
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Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of
lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the
circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to
evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2ⴙ/ⴙ mice and CCR2ⴚ/ⴚ mice throughout a time
course following infection with C. neoformans. Results demonstrate that numbers of Ly-6Chigh monocytes increased in parallel in
the peripheral blood and lungs of CCRⴙ/ⴙ mice, whereas CD11cⴙ MHC class IIⴙ pre-DC were 10-fold less prevalent in the
peripheral blood and did not differ between the two strains. Accumulation of Ly-6Chigh monocytes correlated with a substantial
increase in the numbers of CD11bⴙ DC in the lungs of infected CCR2ⴙ/ⴙ mice. Comparative phenotypic analysis of lung cells
recovered in vivo suggests that Ly-6Chigh monocytes differentiate into CD11bⴙ DC in the lung; differentiation is associated with
up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that
Ly-6Chigh monocytes differentiate into CD11bⴙ DC. Accumulation of Ly-6Chigh monocytes and CD11bⴙ DC was not attributable
to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11bⴙ DC in the lungs of Cryptococcusinfected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6Chigh monocytes. The Journal of
Immunology, 2009, 183: 8044 – 8053.
The Journal of Immunology
Materials and Methods
Mice
Specific pathogen-free inbred female BALB/c (designated CCR2⫹/⫹) mice
purchased from Charles River Laboratories were used, except as specified.
CCR2⫺/⫺ mice (C57BL/6 ⫻ J129 (C57/J129) back-crossed eight times
onto a BALB/c background) were provided by W. Kuziel (Molecular Genetics and Microbiology, University of Texas, Austin, TX) (39) and were
bred on site. Mice were housed in the animal care facility at the Ann Arbor
Veterans Affairs Health System (Ann Arbor, MI) and cared for using a
protocol approved by the Veterans Administration’s Institutional Animal
Care and Use Committee. Mice were 8 –12 wk of age at the time of infection and there were no age-related differences in the responses of these
mice to C. neoformans infection.
Cryptococcus neoformans
C. neoformans strain 52D was obtained from the American Type Culture
Collection (strain 24067); this strain displayed smooth colony morphology
when grown on Sabouraud dextrose agar. For infection, yeast were grown
to the stationary phase (48 –72 h) at 37°C in Sabouraud dextrose broth (1%
neopeptone and 2% dextrose: Difco) on a shaker. Cultured C. neoformans
was then washed in nonpyrogenic saline, counted using trypan blue on a
hemocytometer, and diluted to 3.3 ⫻ 105 CFU/ml in sterile nonpyrogenic
saline before intratracheal inoculation. Unencapsulated, heat-killed C. neoformans strain 602 (ATCC strain 62072) was used for the stimulation of in
vitro cultures.
Surgical intratracheal inoculation
Mice were anesthetized by i.p. injection of ketamine (100 mg/kg; Fort
Dodge Laboratories) and xylazine (6.8 mg/kg; Lloyd Laboratories) and
restrained on a small surgical board. A small incision was made through the
skin over the trachea, and the underlying tissue was separated. A 30-gauge
needle was attached to a 1-ml tuberculin syringe filled with diluted C.
neoformans culture. The needle was inserted into the trachea, and a 30 ␮l
of inoculum (104 CFU) was dispensed into the lungs. The needle was
removed, and the skin was closed with a cyanoacrylate adhesive. The mice
recovered with minimal visible trauma.
Monoclonal Abs
The following mAbs purchased from BioLegend were used: N418 (antimurine CD11c, hamster IgG1); 2.4G2 (“Fc block”) (anti-murine CD16/
CD32, rat IgG2b); 30-F11 (anti-murine CD45, rat IgG2b); 16-10A1 (antimurine CD80, hamster IgG2); GL1 (anti-murine CD86, rat IgG2a); MIH5
(anti-murine B7-H1, rat IgG2a); TY25 (anti-murine B7-DC, rat IgG2a);
AMS-32.1 (“MHC class II”) (anti-murine I-Ad, mouse IgG2b); 145-2C11
(anti-murine CD3␧, hamster IgG1, k); 6D5 (anti-murine CD19, rat IgG2a);
and BM8 (anti-murine F4/80, rat IgG2a). The mAbs AL-21 (anti-murine
Ly-6C, rat IgM); M1/70 (anti-murine CD11b, rat IgG2b); and 3/23 (antimurine CD40, rat IgG2a) were purchased from BD Biosciences/BD
Pharmingen. The mAbs were primarily conjugated with FITC, PE, PerCPCy5.5, allophycocyanin, allophycocyanin-Cy7, or Pacific Blue. Isotypematched irrelevant control mAbs (BioLegend) were tested simultaneously
in all experiments.
Ab staining and flow cytometric analysis
Staining, including blockade of Fc receptors, and analysis by flow cytometry were performed as described previously (40, 41). Data was collected
on an LSR II or BD FACSVantage flow cytometer using FACSDiva software (both from BD Immunocytometry Systems) and analyzed using
FlowJo software (Tree Star). A range of cells (10,000 –100,000) were analyzed per sample.
Experimental design
We performed three types of experiments. First, we compared potential
precursors (in the peripheral blood, bone marrow, and lung) of lung DC in
CCR2⫹/⫹ mice and syngeneic CCR2⫺/⫺ mice at multiple time points
postinfection with C. neoformans. Uninfected CCR2⫹/⫹ mice and
CCR2⫺/⫺ mice served as additional controls (designated as day 0). We
used four- to six-color flow cytometric analysis on single cell suspensions
generated from peripheral blood, bone marrow, or enzymatically digested
lung tissue. Gating strategies were developed that distinguished subpopulations of potential DC precursors (in peripheral blood and bone marrow)
and monocytes and DC (in the lung). Gates were kept consistent in all
experiments. This approach permitted the simultaneous quantitative analysis of multiple monocytic cell populations throughout a kinetic time
course of C. neoformans infection. Second, we determined the capacity of
Ly-6Chigh monocytes to differentiate into DC in vitro. Third, we used a
5-ethly-2⬘-deoxyuridine (EdU) assay to assess lung and bone marrow tissue for the presence of proliferating DC or monocytes.
Tissue collection
PBMC were isolated from peripheral blood obtained from the retro-orbital
vein of deeply anesthetized mice and processed as previously described
(13). Lungs were perfused via the right heart using PBS containing 0.5 mM
EDTA until pulmonary vessels were grossly clear. Lungs were then excised, minced, and enzymatically digested and a single cell suspension of
lung leukocytes was obtained as previously described (7). Bone marrow
was harvested by removing the ends from both femurs with a no. 10 blade
scalpel (after removal of skin and muscle) and flushing the marrow with 2
ml of PBS (using a 3-cc syringe and a 25.5 gauge needle) into a sterile
100 ⫻ 15-mm dish. After erythrocyte lysis, cells were washed, filtered over
a 70-␮m mesh, and resuspended in complete medium (RPMI 1640, 10%
FCS, nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 ␮M 2-ME, 100 U/ml penicillin, and 100 ␮g/ml streptomycin
sulfate). Total numbers of viable lung, peripheral blood, and bone marrow
leukocytes were assessed in the presence of trypan blue using a hemocytometer. All cell preparations were washed twice in PBS before use for Ab
staining.
Flow cytometric analysis
Single cell suspensions were analyzed on an LSR II flow cytometer (BD
Biosciences) equipped with 488-nm blue, 405-nm violet, and 633-nm red
lasers. Data were collected using FACSDiva software with automatic compensation and analyzed using FlowJo software (Tree Star). Details of the
gating strategy are described in Results. A minimum of 10,000 CD45⫹
events were collected per sample.
To maintain complete consistency, cytometer parameters and gate position were held constant during analysis of all samples. We used the percentage of each cell population obtained from flow cytometry to calculate
the total number of each cell population within each tissue by multiplying
the frequency of the population by the total number of leukocytes (the
percentage of CD45⫹ cells multiplied by the original hemocytometer count
of total cells) identified within that sample.
In vitro culture of Ly-6Chigh monocytes
Bone marrow cells were harvested from flushed marrow cavities of femurs
and tibiae of uninfected CCR2⫹/⫹ mice under aseptic conditions. To obtain
Ly-6Chigh monocytes, we first depleted Ly-6G⫹ cells (granulocytes) using
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had found that lung DC accumulation in response to inhaled particulate Ag (in mice) is associated with a minor increase in
CD11c⫹MHC class II⫹ “pre-DC” in the peripheral blood (13).
However, the low percentage of such pre-DC did not readily explain the large increase in the numbers of lung DC. Evidence in
other models systems suggests that DC accumulation in infected
peripheral tissues can result from the differentiation of Ly-6Chigh
“inflammatory” monocytes (8, 36). In the mouse, these inflammatory monocytes do not express CD11c but strongly express Ly-6C,
Gr-1, CD11b, and CCR2. They are distinct (but may be derived
from) populations of “resident” or “patrolling” monocytes that express markedly less Ly-6C and CCR2 but strongly express the
fractalkine receptor CX3CR1 (37, 38). Thus, Ly-6Chigh inflammatory monocytes could differentiate into lung DC during cryptococcal infection.
The objective of the current study was to identify the circulating
precursor of DC recruited to the lungs of mice infected with C.
neoformans. Based on our prior results demonstrating that DC accumulation is CCR2 dependent, we predicted that the predominant
precursor of lung DC in CCR2⫹/⫹ mice would be diminished (or
absent) in CCR2⫺/⫺ mice. We conducted a thorough kinetic analysis identifying DC or their precursors in three tissues compartments to further localize the location of defects leading to impaired
lung DC accumulation. We further assessed whether in situ proliferation of DC or their precursors contributed to their overall
numbers in the lung.
8045
8046
Ly-6Chigh MONOCYTES DIFFERENTIATE INTO LUNG DENDRITIC CELLS
an EasySep PE-magnetic selection kit according to the manufacturer’s protocols (STEMCELL Technologies). Ly-6Chigh monocytes were subsequently positively isolated using EasySep FITC anti-Ly-6C-magnetic selection kit. Purified Ly-6Chigh monocytes (⬃90% purity) were cultured and
differentiated at 3 ⫻ 105 cells/ml in the presence of 20 ng/ml GM-CSF
(PeproTech) in complete medium (defined above). The resultant nonadherent cell populations were analyzed after days 3, 5, and 7 in culture (and
compared with freshly isolated cells, designated as day 0) by flow cytometry for expression of Ly-6C, CD11c, and CD11b.
In additional experiments, CD11c⫹CD11b⫹ cells were assessed for the
expression of MHC class II (I-Ad) and costimulatory molecules (CD40,
CD80, and CD86). These CD11c⫹CD11b⫹ cells resulted from the culture
of Ly-6Chigh bone marrow-derived monocytes (in complete medium; defined above) under one of three conditions: 1) GM-CSF alone for 7 days;
2) GM-CSF for 7 days plus 1 ␮g/ml LPS (Ultra Pure LPS; Escherichia coli
O111:B4, catalog no. 421; List Biological Laboratories) on day 6 of culture; or 3) GM-CSF for 7 days plus heat-killed C. neoformans strain 602
(HK-Cneo) at a 1:2 ratio of cells:HK-Cneo on day 6 of culture. Nonadherent cells from all cultures were removed for analysis on day 7.
Evaluation of monocyte and DC proliferation in vivo
Statistical analysis
All data were expressed as mean ⫾ SEM. Continuous ratio scale data were
evaluated by an unpaired Student t test (for comparison between two samples) or by ANOVA (for multiple comparisons) with post hoc analysis by
a two-tailed Dunnett test, which compares treatment groups to a specific
control group (44). Statistical calculations were performed on a Dell 270
computer using GraphPad Prism version 3.00 for Windows. Statistical difference was accepted at p ⬍ 0.05.
Results
CCR2 mediates an increase in the frequency of Ly-6Chigh
monocytes but not pre-DC within peripheral blood of mice with
cryptococcal lung infection
Our previous study demonstrated the accumulation of large numbers of DC in the lungs of CCR2⫹/⫹ mice, but not of CCR2⫺/⫺
mice, infected with C. neoformans (7). The first objective of this
study was to look throughout the course of infection for evidence
in peripheral blood of two potential DC precursors, pre-DC (defined as CD45⫹CD11c⫹I-Ad⫹) (45– 47) and Ly-6Chigh inflammatory monocytes (defined as CD45⫹CD11b⫹Ly-6Chigh), which are
known to express CCR2 (37, 38). Our gating strategy involved
initial exclusion of debris, RBC, and cell clusters using light scatter, followed by a forward scatter (FSC) vs fluorescence (FL)-4
scatter plot to select for CD45⫹ leukocytes (data not shown). To
identify pre-DC, we used a FL-8 (CD11c) vs FL-1 (MHC class II)
plot (Fig. 1, A and B), whereas analysis of the same samples using
a FL-1 (Ly-6C) vs FL-6 (CD11b) plot was used to identify Ly6ChighCD11b⫹ monocytes (Fig. 1, C and D).
In CCR2⫹/⫹ mice, the pre-DC population increased by only a
small amount by day 14 postinfection, relative to uninfected mice
(Fig. 1B). In contrast, numbers of Ly-6Chigh monocytes were increased by day 7 postinfection, peaked at day 14, and at all time
points were ⬃10-fold more prevalent than pre-DC (Fig. 1D). In
CCR2⫺/⫺ mice, the percentage of pre-DC was low and did not
differ significantly from the percentage of pre-DC identified in
CCR2⫹/⫹ mice at any time point. The percentage of Ly-6Chigh
monocytes in the blood of CCR2⫺/⫺ mice was low in uninfected
FIGURE 1. CCR2 mediates an increase in the frequency of Ly-6Chigh
monocytes but not pre-DC within peripheral blood of mice with cryptococcal lung infection. PBMC were obtained from CCR2⫹/⫹ mice and
CCR2⫺/⫺ mice at day 0 (uninfected) or days 7, 10, 14, 21, and 28 days
after intratracheal (IT) infection with C. neoformans (Days post Cneo IT).
A and C, Representative dot plots of PBMC from CCR2⫹/⫹ mice and
CCR2⫺/⫺ mice (day 7 postinfection) identifying either pre-DC
(CD45⫹CD11c⫹ I-Ad⫹) (A) or Ly-6Chigh monocytes (CD45⫹Ly6ChighCD11b⫹) (C). B and D, Frequency of pre-DC (B) and Ly-6Chigh
monocytes (D) in PBMC at specific time points. Black bars, CCR2⫹/⫹
mice; gray bars; CCR2⫺/⫺ mice. Data represent mean ⫾ SEM of 3– 6 mice
assayed individually per time point; ⴱ, p ⬍ 0.05 by ANOVA with Dunnet’s
post hoc analysis vs day 0 (uninfected) of mice of the same strain; ‡, p ⬍
0.05 by unpaired Student t test between CCR2⫹/⫹ and CCR2⫺/⫺ mice at
the same time point. These time courses were independently repeated once
in their entirety (n ⫽ 3 mice per time point) with similar results.
mice and did not increase following infection (with the exception
of a small but statistically significant increase at day 21). Ly-6Chigh
monocytes were significantly diminished in CCR2⫺/⫺ mice (relative to CCR2⫹/⫹ mice) at all time points. Collectively, these data
show that CCR2 mediates an increase in the frequency of circulating Ly-6Chigh monocytes, but not pre-DC, in mice with cryptococcal lung infection.
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A Click-iT EdU Pacific blue flow cytometry assay kit (catalog no. A10034)
was purchased from Invitrogen for the labeling and detection of DNA
synthesis. The labeling strategy used incorporation of the thymidine analog
EdU, followed by chemical coupling of the analog with an azide-conjugated fluorophore for detection. Mice were injected i.p. either with 100 ␮g
of EdU in PBS or PBS alone (negative control), and after 2 h the mice were
sacrificed and bone marrow cells (as positive control) and lung leukocytes
were identified by flow cytometric analysis (as described above) for EdU
uptake following the manufacturer’s protocols. Gates depicting positive
uptake were set based on the staining characteristics of control leukocytes
obtained from infected mice not receiving EdU (42, 43).
The Journal of Immunology
8047
and total number of Ly-6Chigh monocytes in the bone marrow of
CCR2⫺/⫺ mice. Thus, the decreased frequency of Ly-6Chigh
monocytes in the blood of CCR2⫺/⫺ mice with cryptococcal lung
infection appears attributable to impaired egress from the bone
marrow and not impaired generation.
CCR2 mediates the accumulation of large numbers of Ly-6Chigh
monocytes in the lungs of mice infected with C. neoformans
CCR2 mediates the egress of Ly-6Chigh monocytes, but not
pre-DC, from the bone marrow of mice with cryptococcal lung
infection
The relative paucity of Ly-6Chigh monocytes in the blood of
CCR2⫺/⫺ mice suggested that either their generation in bone marrow or their egress into peripheral blood might be impaired. We
therefore evaluated the bone marrow of infected CCR2⫹/⫹ and
CCR2⫺/⫺ mice for the presence of pre-DC and Ly-6Chigh monocytes 7 days postinfection. There was no difference between strains
in the numbers of CD45⫹ bone marrow leukocytes per femur (Fig.
2A). In both strains of mice, the percentages and total numbers of
pre-DC were low and did not differ significantly (Fig. 2, B and C).
In contrast, there were far greater percentages and total numbers of
Ly-6Chigh monocytes in the bone marrow of both strains. Importantly, there was a significant relative increase in the percentage
CCR2 mediates the accumulation of large numbers of CD11b⫹
DC in the lungs of mice infected with C. neoformans
Our next objective was to identify the predominant DC population
in the lungs of CCR2⫹/⫹ mice with cryptococcal infection by using flow cytometric analysis. We used a panel of Abs and gating
strategy (described in the preceding section) to first exclude cell
debris, lymphocytes, and eosinophils within a population of
CD45⫹ lung leukocytes (day 14 postinfection). Next, we identified
lung macrophages and DC as CD11c-expressing (CD11c⫹) cells
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FIGURE 2. Ly-6Chigh monocytes accumulate in the bone marrow of
CCR2⫺/⫺ mice with cryptococcal lung infection. Bone marrow cells were
obtained from both femurs of CCR2⫹/⫹ mice and CCR2⫺/⫺ mice 7 days
postinfection with C. neoformans. Flow cytometric analysis was used to
identify total CD45⫹ leukocytes (A) and the frequency (B) and total numbers (C) of pre-DC (CD45⫹CD11c⫹I-Ad⫹) and Ly-6Chigh monocytes
(CD45⫹Ly-6ChighCD11b⫹). Total numbers of cells were calculated by
multiplying the frequency of a cell population by the total number of cells
obtained from the bone marrow. Black bars, CCR2⫹/⫹ mice; gray bars,
CCR2⫺/⫺ mice. Data represent mean ⫾ SEM of 3– 4 mice per strain assayed individually per time point; ‡, p ⬍ 0.05 by unpaired Student t test.
These time courses were independently repeated once in their entirety (n ⫽
3 mice per time point) with similar results.
Our next objective was to identify Ly-6Chigh monocytes within the
lungs of infected mice. Unless otherwise indicated, all samples
were stained with the following Abs: either anti-MHC class II
(I-Ad) (FITC; FL-1) or anti-Ly-6C (FITC; FL-1); anti-F480 (PE;
FL-2); anti-CD3 plus anti-CD19 (PerCP Cy5.5; FL-3); anti-CD45
(allophycocyanin; FL-4); anti-CD11b (allophycocyanin-Cy7; FL6); and anti-CD11c (Pacific blue; FL-8). After excluding cell debris, lymphocytes, and eosinophils (based upon their unique
FSClow vs side scatter (SSC)high characteristics) and gating on
CD45⫹ leukocytes, we focused on the CD11c-negative (CD11c⫺)
population (FSC vs FL-8 plot) (Fig. 3A; gate R1). Use of a FL-6
(CD11b) vs FL-1 (Ly-6C) plot allowed us to distinguish
CD11c⫺Ly-6ChighCD11b⫹ inflammatory monocytes (gate “Ly6Chigh mono”, Fig. 3A) (8, 36) from neutrophils, which are
CD11b⫹Ly-6Cmoderate cells (Fig. 3A). Ly-6Chigh monocytes coexpressed F4/80 (Fig. 3B) and were medium-sized cells with scant
cytoplasm (Fig. 3C).
This gating strategy was then applied to leukocyte populations
obtained from the lungs of CCR2⫹/⫹ and CCR2⫺/⫺ mice at day 0
(uninfected) or days 7, 10, 14, 21, and 28 postinfection with C.
neoformans. Consistent with our prior studies (7, 35, 48), C. neoformans infection resulted in massive accumulation of CD45⫹ leukocytes in the lungs of CCR2⫹/⫹ mice (peaking at 1.7 ⫾ 0.2 ⫻ 108
leukocytes per lung at day 14 postinfection) relative to their accumulation in CCR2⫺/⫺ mice (5.9 ⫾ 0.9 ⫻ 107 leukocytes per
lung at day 14 postinfection). Ly-6Chigh monocytes were rare in
the lungs of uninfected CCR2⫹/⫹ mice but had significantly increased by day 7 postinfection and peaked at day 14 (6.0 ⫾ 0.1%
of total CD45⫹ leukocytes; 9.9 ⫾ 0.1 ⫻ 106 Ly-6Chigh monocytes
per lung). This change represents a 54-fold increase relative to
uninfected mice (Fig. 3, D and E). In CCR2⫺/⫺ mice, numbers of
lung Ly-6Chigh monocytes were markedly diminished, with the
maximal decrease (96%) occurring at day 14 postinfection (Fig.
3D and E).
To verify our findings, we also examined an alternative definition of lung monocytes as CD11c⫺ F4/80⫹CD11b⫹ cells (F4/80⫹
monocytes) (9, 14). Greater than 95% of F4/80⫹ lung monocytes
in CCR2⫹/⫹ mice expressed high amounts of Ly-6C at days 7, 14,
21, and 28 postinfection (data not shown). F4/80⫹ monocyte accumulation was reduced in CCR2⫺/⫺ mice relative to CCR2⫹/⫹
mice at all times postinfection. Overall, the pattern of F4/80⫹
monocyte accumulation was very similar to that shown in Fig. 3D
for Ly-6Chigh monocytes (data not shown). Collectively, these data
indicate that the kinetics of the CCR2-dependent accumulation of
Ly-6Chigh monocytes in the lungs during cryptococcal infection
closely parallels their increase in peripheral blood.
8048
Ly-6Chigh MONOCYTES DIFFERENTIATE INTO LUNG DENDRITIC CELLS
(FSC vs FL-8 plot) (Fig. 4A, left dot plot, gate R2). We then used
autofluorescence (FSC vs FL-3 plot) (Fig. 4A, middle dot plot) to
distinguish autofluorescent (AF)-positive (AF⫹) macrophages
from nonautofluorescent (AF⫺) DC (7, 49, 50). Within the nonautofluorescent gate (Fig. 4A, middle dot plot; gate R2a), a FL-6
(CD11b) vs FL-8 (CD11c) plot was used to identify a dense pop-
ulation of CD11c⫹AF⫺CD11b⫹ DC (Fig. 4A, right dot plot, gate
“DC”). CD11b⫹ DC expressed MHC class II and F4/80 (Fig. 4, B
and C) and CD11b⫹ DC sorted from lung leukocytes using this
gating strategy displayed characteristic cytoplasmic extensions and
ruffled cell membranes (Fig. 4D). They did not express CD103
(data not shown), distinguishing them from the small population of
AF⫺CD11b⫺CD103⫹ DC described in the airways of uninfected
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FIGURE 3. CCR2 mediates the accumulation of Ly-6Chigh monocytes
in the lungs of mice with cryptococcal infection. A, Gating strategy to
identify Ly-6Chigh monocytes in the lung using flow cytometric analysis
(representative plots of CCR2⫹/⫹ mice on day 7 postinfection): After exclusion of cell debris, lymphocytes, and eosinophils, a plot of CD11c
vs FSC identifies CD11c-negative cells (left dot plot; gate R1). Within this
population, Ly-6Chigh monocytes are identified as Ly-6ChighCD11b⫹ cells
(right dot plot; gate Ly-6Chigh mono). B, Ly-6Chigh monocytes express
F4/80 (shaded histogram, isotype control; open histogram, specific Ab
staining). C, Morphology of Ly-6Chigh monocytes (sorted using the above
gating strategy; H&E staining and ⫻1,000 original magnification). D and
E, Flow cytometric analysis of lung leukocytes from CCR2⫹/⫹ and
CCR2⫺/⫺ mice at day 0 (uninfected) or days 7, 10, 14, 21, and 28 days
after intratracheal (IT) infection with C. neoformans (Days Post Cneo IT).
D, Representative dot plots (day 7) identifying Ly-6Chigh monocytes within
lung leukocytes obtained from CCR2⫹/⫹ mice (left dot plot) and CCR2⫺/⫺
mice (right dot plot). E, Total numbers of Ly-6Chigh monocytes (calculated
by multiplying the frequency of Ly-6Chigh monocytes by the total number
of CD45⫹ lung leukocytes at each time point. Data in E represent mean ⫾
SEM of 3– 6 mice (black solid line, CCR2⫹/⫹ mice; gray dashed line,
CCR2⫺/⫺ mice) assayed individually per time point; ⴱ, p ⬍ 0.05 by
ANOVA with Dunnet’s post hoc analysis vs day 0 (uninfected) of mice of
the same CCR2 expression profile; ‡, p ⬍ 0.05 by unpaired Student t test
between CCR2⫹/⫹ vs CCR2⫺/⫺ mice at the same time point. These time
courses were independently repeated once in their entirety (n ⫽ 3 mice per
time point) with similar results.
FIGURE 4. CCR2 mediates the accumulation of CD11b⫹ DC in the
lungs of mice with cryptococcal infection. A, Gating strategy to identify the
predominant population of lung DC in mice infected with C. neoformans
(representative plots; CCR2⫹/⫹ mice on day 14 postinfection). After exclusion of cell debris, lymphocytes, and eosinophils, a plot of CD11c vs
FSC identifies CD11c-positive cells (the left dot plot gate is labeled R2 to
distinguish it from gate R1 in Fig. 3). A plot of FL-3 vs FSC is then used
exclude AF macrophages from non-AF DC (AF⫺; middle dot plot, gate
labeled R2a). Next, a dense population of cells expressing both CD11c and
CD11b were identified as CD11b⫹ DC (CD11c⫹AF⫺CD11b⫹; right dot
plot, gate labeled DC). B and C, Representative staining of CD11b⫹ DC for
isotype controls or MHC class II (I-Ad) (B) or F4/80 (B) (shaded histogram, isotype control; open histogram, specific Ab staining). D, Morphology of CD11b⫹ lung DC (sorted using the above gating strategy; H&E
staining with ⫻1000 original magnification). Note the numerous cytoplasmic extensions and ruffled cell membrane. E, Total numbers of CD11b⫹
DC (calculated by multiplying the frequency of CD11b⫹ DC by the total
number of CD45⫹ lung leukocytes at each time point). Data in E represent
mean ⫾ SEM of 3– 6 mice (black solid lines, CCR2⫹/⫹ mice; gray dashed
lines, CCR2⫺/⫺ mice) assayed individually per time point; ⴱ, p ⬍ 0.05 by
ANOVA with Dunnet’s post hoc analysis vs day 0 (uninfected) of mice of
the same CCR2 expression profile; ‡, p ⬍ 0.05 by unpaired Student t test
between CCR2⫹/⫹ mice vs CCR2⫺/⫺ mice at the same time point. These
time courses were independently repeated once in their entirety (n ⫽ 3
mice per time point) with similar results. Days post Cneo IT, days after
intratracheal (IT) infection with C. neoformans.
The Journal of Immunology
8049
mice (51). CD11b⫹ DC also did not express B220, distinguishing
them from plasmacytoid DC (data not shown).
We applied this gating strategy to assess the frequency and total
number of CD11b⫹ DC within the lungs of individual CCR2⫹/⫹
mice or CCR2⫺/⫺ mice at baseline and at multiple time points
following infection with C. neoformans (Fig. 4E). The percentages
and total numbers of lung CD11b⫹ DC increased significantly in
the lungs of infected CCR2⫹/⫹ mice, peaking at day 14 postinfection (10.7 ⫾ 0.1% of total CD45⫹ leukocytes, 17.7 ⫾ 0.1 ⫻ 106
CD11b⫹ DC per lung; Fig. 4E). In CCR2⫺/⫺ mice, total numbers
of lung CD11b⫹ DC were diminished (96%), most notably at day
14 postinfection (Fig. 4E). These data demonstrate the CCR2 dependence of CD11b⫹ DC accumulation during cryptococcal lung
infection.
Comparative phenotypic analysis of Ly-6Chigh monocytes and
CD11b⫹ lung DC in vivo
The strong temporal correlation between the accumulation of Ly6Chigh monocytes and CD11b⫹ DC in CCR⫹/⫹ mice suggested
that Ly-6Chigh monocytes might differentiate into CD11b⫹ DC
within the lungs. To assess this hypothesis, we compared the two
cell types for their scatter properties and expression of MHC class
II and costimulatory molecules. Ly-6Chigh monocytes were smaller
(FSClow) than CD11b⫹ DC and had less granular cytoplasm
(SSClow) (Fig. 5A). Ly-6Chigh monocytes expressed moderate
amounts of MHC class II and minimal amounts of CD40, CD80,
CD86, and B7-H1. They did not express B7-DC (Fig. 5B, top
histograms). In contrast, CD11b⫹ DC strongly expressed MHC
class II and a modest amount of the costimulatory molecules
CD40, CD80, and CD86 (Fig. 5B, bottom histograms). Furthermore, CD11b⫹ DC express moderate to high amounts of B7-H1
and B7-DC.
Reports suggest that differentiation of Ly-6Chigh monocytes results in down-regulation of Ly-6C (52, 53). Therefore, we assessed
CD11b⫹ lung DC for their expression of Ly-6C on days 7, 14, and
21 postinfection (in CCR2⫹/⫹ mice) (Fig. 5C). CD11b⫹ lung DC
expressed high amounts of Ly-6C at day 7 postinfection, whereas
expression was lower on CD11b⫹ DC in the lung at days 14 and
21. Collectively, our data suggested that Ly-6Chigh monocytes differentiate into CD11b⫹ DC within the lung; their differentiation is
associated with the following phenotypic changes: 1) up-regulation of CD11c; 2) an increase in size and in the cytoplasm:nuclear
ratio; 3) enhanced expression of MHC class II and costimulatory
molecules; and 4) down-regulation of Ly-6C.
Ly-6Chigh monocytes differentiate into CD11b⫹ DC in culture
To verify the capacity of Ly-6Chigh monocytes to differentiate into
CD11b⫹ DC, we purified Ly-6Chigh monocytes from the bone
marrow of uninfected CCR2⫹/⫹ mice and examined the nonadherent population at various times during in vitro culture for up to
7 days in GM-CSF (20 ng/ml) (Fig. 6). Freshly isolated Ly-6Chigh
monocytes were small cells with little cytoplasm. Following 7 days
in culture the cells were larger, contained more cytoplasm, and
displayed cytoplasmic extensions and ruffled cell membranes consistent with DC morphology. Flow cytometric analysis confirmed
that cell size (FSC) and granularity (SSC) increased over time
(data not shown). Ly-6C expression decreased over time, whereas
by day 7 the majority of cells coexpressed CD11c and CD11b (Fig.
6A). These CD11c⫹ CD11b⫹ cells expressed MHC class II (I-Ad)
and costimulatory molecules (CD40, CD80, and CD86; Fig. 6B,
top histograms). Expression of these molecules increased further
in response to stimulation with heat-killed C. neoformans (Fig. 6B,
middle histograms) or LPS (Fig. 6B, bottom histograms). These
results demonstrate that Ly-6Chigh monocytes can differentiate in
vitro into DC displaying a phenotype similar to that identified in
vivo for CD11b⫹ DC in the lungs of mice with cryptococcal lung
infection.
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FIGURE 5. Comparative phenotypic analysis of Ly-6Chigh monocytes and CD11b⫹ DC in the lungs of
CCR2⫹/⫹ mice infected with C. neoformans. A and B, Ly-6Chigh monocytes and CD11b⫹ DC were identified (using flow cytometric analysis)
within lung leukocyte populations
from CCR2⫹/⫹ mice at day 14 after
intratracheal (IT) infection with C.
neoformans (days after intratracheal
(IT) infection with C. neoformans).
Cells were evaluated for their FSC
and SSC properties (A) and expression of MHC class II (I-Ad) and the
costimulatory molecules CD40,
CD80, CD86, B7-H1, and B7-DC).
C, Expression of Ly-6C on CD11b⫹
DC recovered from the lungs of
CCR2⫹/⫹ mice on days 7, 14, and 21
after intratracheal (IT) infection with
C. neoformans (Days post Cneo IT).
Note that expression of Ly-6C is decreased on CD11b⫹ DC present in the
lung on days (D) 14 and 21 vs day 7.
Shaded histograms, isotype control;
open histograms, specific Ab staining.
These experiments were performed
four times with similar results.
8050
Ly-6Chigh MONOCYTES DIFFERENTIATE INTO LUNG DENDRITIC CELLS
(Fig. 7B, representative histograms; Table I, cumulative data). Collectively, these data suggest that the substantial accumulation of
CD11b⫹ DC that occurs within the lungs of Cryptococcus-infected
mice is not attributable to the proliferation of these cells (or Ly6Chigh monocytes precursors) within the lung.
Discussion
CD11b⫹ DC and Ly-6Chigh monocytes are not proliferating in
the lungs of mice infected with C. neoformans
To test the possibility that accumulation of CD11b⫹ DC could also
result in part from the proliferation of DC (or their precursors)
within the lung, we treated CCR2⫹/⫹ mice with the thymidine
analog EdU to identify proliferating cells by flow cytometric analysis (42, 43, 54). We evaluated leukocytes obtained from the bone
marrow and lungs 10 days postinfection (Fig. 7). This time point
was chosen because our kinetic analysis revealed that CD11b⫹ DC
accumulation (in the lung) was maximal between days 7 and 14
postinfection. Bone marrow and lung leukocytes were obtained 2 h
post-EdU administration to minimize the potential for cells to migrate from bone marrow to lung during the assay. We used total
bone marrow leukocytes as positive controls and background
staining of leukocyte populations from infected mice not receiving
EdU as negative controls. The assay was further validated by measuring EdU incorporation in polymorphonuclear leukocytes
(PMN; defined as Ly-6Cmoderate CD11b⫹), as prior studies have
demonstrated that PMN proliferate in bone marrow but not upon
arrival in the lung (55).
Within bone marrow, we clearly identified a population of proliferating cells, including a significant percentage of Ly-6Chigh
monocytes and PMN (Fig. 7A, representative histograms; Table I,
cumulative data). Within lung leukocyte populations, neither
CD11b⫹ DC, Ly-6Chigh monocytes, nor PMN were proliferating
This study is the first to define the mechanisms responsible for the
sizeable accumulation of CD11b⫹ DC in the lungs of mice following pulmonary infection with a fungal pathogen. Collectively,
our data imply that the accumulation of CD11b⫹ lung DC in mice
with cryptococcal infection results from the continuous CCR2mediated recruitment and differentiation of Ly-6Chigh monocytes.
The following important observations support this conclusion: 1)
CCR2 expression was strongly associated with an increase in the
Table I. Proliferation of bone marrow and lung leukocytes in CCR2⫹/⫹
micea
Leukocytes
Positive EdU (⫾SEM)
Bone marrow
Total cells
Ly-6Chigh monocytes
Ly-6Cmod PMN
16.6 ⫾ 0.5*
19.9 ⫾ 1.4*
11.0 ⫾ 1.1*
Lung
CD11b⫹ DC
Ly-6Chigh monocytes
Ly-6Cmod PMN
⫺0.1 ⫾ 0.3
⫺0.6 ⫾ 2.0
⫺0.1 ⫾ 0.3
a
Cells identified by flow cytometric analysis as per Materials and Methods. Data
represent percentage of cells staining for EdU (mean ⫾ SEM of three mice assayed
individually per time point).
ⴱ, p ⬍ 0.05 by an unpaired Student t test comparing EdU expression from EdUtreated mice vs untreated (but infected) controls; the experiment was repeated with
similar results.
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FIGURE 6. Ly-6Chigh monocytes differentiate into CD11b⫹ DC in
vitro. A, Ly-6Chigh monocytes were isolated from bone marrow of uninfected CCR2⫹/⫹ mice and cultured for 7 days in GM-CSF (20 ng/ml).
Nonadherent cells were assessed at day 0 (freshly isolated Ly-6Chigh monocytes) and days (D) 3, 5, and 7 of culture for expression of Ly-6C (top
panels: shaded histograms, isotype control; open histograms, specific Ab
staining) or coexpression of CD11c and CD11b (bottom panels: rectangular gate identifies CD11c⫹CD11b⫹ cells). Note that Ly-6C expression decreased whereas CD11c and CD11b expression increased over time in
culture. B, Representative histograms of MHC class II (I-Ad) and costimulatory molecule (CD40, CD80, and CD86) expression on nonadherent cells
after 7 days culture in medium containing either GM-CSF alone (medium,
top panels), GM-CSF with the addition of heat-killed C. neoformans (HKCneo) on days 6 –7 (middle panels), or GM-CSF with the addition of LPS
on days 6 –7 (bottom panels). Note the presence of cells strongly expressing MHC class II and costimulatory molecules that were further up-regulated in response to HC-Cneo or LPS consistent with a DC phenotype. The
experiment was performed three times with similar results.
FIGURE 7. CD11b⫹ DC and Ly-6Chigh monocytes are not proliferating
in the lungs of CCR2⫹/⫹ mice infected with C. neoformans. A and B,
CCR2⫹/⫹ mice were infected intratracheally with C. neoformans. At day
10 postinfection mice received an i.p. injection of the thymidine analog
EdU. Two hours later, bone marrow and lung leukocytes were removed
and specific cell populations were assayed (by flow cytometric analysis, see
Materials and Methods and gating strategies described in Figs. 3 and 4) for
evidence of EdU uptake (as an indicator of cell proliferation). Representative histograms of bone marrow (A) and lung (B) leukocyte populations.
Shaded histograms, EdU staining performed on leukocytes from infected
control mice not receiving EdU administration; open histograms, EdU
staining performed on leukocytes from infected mice receiving EdU administration. Gates indicate positive EdU staining. The experiment was
performed three times with similar results. Ly6Cmod, Ly6C moderate.
The Journal of Immunology
fers from reports suggesting that CD11b⫹ lung DC are derived
from Ly-6Clow/⫺ monocytes (59, 60). Disparity in experimental
approaches may explain this difference and provide additional insight into factors that influence monocyte differentiation. The studies concluding that CD11b⫹ DC result from differentiated Ly6Clow/⫺ monocytes evaluated monocytes differentiation either in
the resting state (59), following the ablation of resident cells (60),
or in response to a single challenge with LPS (59, 60). Results of
those studies indicate that the replacement of resident DC or the
accumulation of DC in response to brief inflammatory stimuli can
be achieved via the differentiation of Ly-6Clow/⫺ monocytes. In
contrast, we evaluated the response to pulmonary challenge with a
live fungal pathogen. The resultant immune response is massive,
with upward of 150 million leukocytes recruited to the lung. Defense against the organism occurs over weeks and may be influenced by multiple pathogen-associated molecular patterns and virulence factors (4, 61– 64). We cannot exclude the possibility that
some CD11b⫹ lung DC in our study may have differentiated from
Ly-6Clow/⫺ monocytes. Yet, our observation that ⬎95% of F4/80⫹
monocytes identified in the lungs of Cryptococcus-infected
(CCR2⫹/⫹) mice expressed high amounts of Ly-6C suggests that
few Ly-6C⫺/low monocytes were present (relative to the large
number of Ly-6Chigh monocytes). Collectively, these results imply
that Ly-6Chigh monocytes are the dominant monocyte subset contributing to lung CD11b⫹ DC in response to the intense and prolonged inflammation induced by a replicating pathogen.
The findings that neither CD11b⫹ DC nor Ly-6Chigh monocytes
were proliferating in the lungs but that Ly-6Chigh monocytes
clearly did proliferate in the bone marrow is evidence that proliferation and differentiation of Ly-6Chigh monocytes is compartmentalized. Collectively, our data emphasize the important relationship
that develops between the lung and bone marrow in response to
pulmonary infection. The CCL2/CCL7/CCR2 axis is actively involved in the early phases of the response and mediates the egress
of additional Ly-6Chigh monocytes into the circulation. The role of
CCR2 or other chemokine receptors in facilitating monocyte recruitment directly into the lung continues to be investigated (10,
65, 66). Mechanisms involved in resolution of pulmonary inflammation remain poorly understood; however, our observation that
CD11b⫹ lung DC decrease after day 14 postinfection coincides
with a decrease in Ly-6Chigh monocytes in the peripheral blood.
This finding suggests that resolution may be closely linked to the
signals that mediate the release of Ly-6chigh monocytes.
Comparison of our findings with those obtained using other
model systems demonstrates that the infecting microbe influences
the magnitude, cellular phenotype, and microanatomic composition of the CCR2-mediated immune response in the lung. First, our
data show that upward of 10 million monocytes and 15 million DC
accumulate in the lungs of CCR2-expressing mice infected with C.
neoformans. This robust accumulation of these cells is 5- to 100fold greater than that in studies defining a role for CCR2 in mediating monocyte or DC recruitment to the lung in response to
bacterial (6, 9, 67), viral (8, 20, 68), or other fungal (69) pathogens.
Second, DC in the lungs of Cryptococcus-infected (CCR2-expressing) mice expressed B7-H1 and B7-DC. Expression of these molecules has not been previously reported on DC recovered from
mice infected with a fungal pathogen. These molecules may have
immunomodulatory properties and it will be important to determine whether they prevent immune-mediated lung injury, as can
occur in cryptococcal and influenza infection (8, 20). Third, use of
our model reveals an important link between CCR2-mediated pulmonary DC accumulation and the formation of neolymphoid structures in the lungs. We previously reported that recruited DC colocalize with CD4⫹ T cells specifically within neolymphoid
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percentage of Ly-6Chigh monocytes, but not pre-DC, in the peripheral blood of infected mice; 2) this observed increase in Ly-6Chigh
monocytes within peripheral blood was paralleled by their substantial accumulation in the lungs of CCR2⫹/⫹ mice (but not
CCR2⫺/⫺ mice); 3) CD11b⫹ DC were also strikingly more abundant in the lungs of infected CCR2⫹/⫹ mice than in those of
CCR2⫺/⫺ mice, and a comparative phenotypic analysis suggests
that Ly-6Chigh monocytes differentiate into CD11b⫹ DC in vivo;
4) Ly-6Chigh monocytes differentiate into CD11b⫹ DC in vitro;
and 5) neither CD11b⫹ DC nor Ly-6Chigh monocytes proliferated
within the lung.
The clear demonstration that Ly-6Chigh monocytes increase in
the blood of CCR2⫹/⫹ mice during cryptococcal lung infection
implies that the lung communicates with the bone marrow, likely
via a systemic signal. The onset of accumulation of Ly-6Chigh
monocytes in both blood and lungs on day 7 in the current study
coincides with the timing we previously found for pulmonary expression of two CCR2 ligands, CCL2 (MCP-1) and CCL7
(MCP-3) (7). The marked sequestration of Ly-6Chigh monocytes in
the bone marrow of infected CCR2⫺/⫺ mice suggests that CCR2
facilitates monocyte egress from the bone marrow, as proposed by
other investigators (36, 56, 57). Local production of CCR2 ligands
within the bone marrow could contribute to this process (58). This
collective evidence indicates that the peripheral pulmonary microenvironment facilitates the release of additional monocytes from
the bone marrow and that the CCL2/CCL7/CCR2 axis is a critical
mediator of this important process.
In contrast to our data for Ly-6Chigh monocytes, the percentage
of pre-DC (MHC class II⫹ CD11c⫹) in the peripheral blood did
not differ between CCR2⫹/⫹ and CCR2⫺/⫺ mice infected with C.
neoformans. This is the first study to demonstrate that their presence in the peripheral blood (in the resting state or in response to
infection) is CCR2 independent. The frequency of pre-DC in
PBMC did increase, albeit slightly, in both strains at day 14 postinfection. This finding was comparable to the small increase in circulating pre-DC we reported in wild-type C57BL/6 mice challenged with inhaled particulate Ag (13). The contribution of these
cells to total DC accumulation in the lungs in either the former or
current study is unclear. Importantly, however, they were 10-fold
less prevalent than Ly-6Chigh monocytes in both the blood and
bone marrow of CCR2⫹/⫹ mice (in this study). These findings lead
us to conclude that pre-DC are not major contributors to the total
population of CD11b⫹ lung DC following C. neoformans
infection.
Results of our study show that lung infection with a fungal
pathogen is a potent stimulus for the differentiation of Ly-6Chigh
monocytes into CD11b⫹ DC. Data from several of our experiments support this conclusion. First, our kinetic analysis in
CCR2⫹/⫹ mice reveals a strong association between the recruitment of Ly-6Chigh monocytes and the accumulation of lung
CD11b⫹ DC. Second, our comparative phenotypic analysis of
these cells recovered from the lung suggests that Ly-6Chigh monocytes do the following; 1) enlarge; 2) up-regulate the expression of
CD11c, MHC class II, and costimulatory molecules; and 3) decrease the expression of Ly-6C as they differentiate into CD11b⫹
DC. These findings are in agreement with others documenting the
loss of Ly-6C expression over time and as monocytes differentiate
(8, 52, 53). Third, results from our culture of Ly-6Chigh monocytes
verify that these cells differentiate into CD11c⫹CD11b⫹ cells in
vitro. Their resultant expression of costimulatory molecules was
similar to that observed for CD11b⫹ lung DC; expression of these
molecules was further enhanced by exposure to Cryptococcus.
Our conclusion that most lung CD11b⫹ DC in murine cryptococcal lung infection differentiated from Ly-6Chigh monocytes dif-
8051
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Ly-6Chigh MONOCYTES DIFFERENTIATE INTO LUNG DENDRITIC CELLS
Disclosures
The authors have no financial conflict of interest.
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