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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-03-0673.
IMMUNOBIOLOGY
From the Department of Pulmonary and Critical Care
Medicine and the Department of Immunology, Erasmus University Medical
Center, Rotterdam, The Netherlands, and the Department of
Physiology, Free University Brussels, Belgium.
Airway dendritic cells (DCs) are held responsible for inducing
sensitization to inhaled antigen, leading to eosinophilic airway inflammation, typical of asthma. However, less information is available
about the role of these cells in ongoing inflammation. In a mouse model
of asthma, sensitization to ovalbumin (OVA) was induced by
intratracheal injection of myeloid OVA-pulsed DCs. Upon OVA aerosol
challenge and induction of eosinophilic airway inflammation in
sensitized mice, there was a time-dependent and almost 100-fold
increase in the number of MHCII+ CD11b+
CD11c+ endogenous airway DCs as well as CD11b+
blood DCs. The mechanism of this increase was studied. Adoptive transfer experiments demonstrated that accumulation of airway DCs was
not due to reduced migration to the mediastinal lymph nodes. Rather,
the massive increase in airway and lymph node DCs was supported by an
almost 3-fold expansion of myeloid
CD31hiLy-6Cneg hematopoietic precursor cells in
the bone marrow (BM). There was no change in any of the other 5 populations revealed by CD31/Ly-6C staining. When these
CD31hiLy-6Cneg BM precursors were sorted and
grown in granulocyte macrophage-colony-stimulating factor, they
differentiated into MHCII+ CD11c+ DCs. The same
CD31hiLy-6Cneg precursors also expressed the
eotaxin receptor CCR3 and differentiated into eosinophils when grown in
interleukin 5. Serum levels of eotaxin were doubled in mice with
inflammation. These findings in an animal model of asthma suggest that
the BM increases its output of myeloid precursors to meet the enhanced
demand for DCs and eosinophils in inflamed airways.
(Blood. 2002;100:3663-3671) The role of the dendritic cell (DC) as a
professional antigen-presenting cell (APC) in the primary
immune response is now well established.1 Airway DCs form
a network in the epithelium, capture inhaled antigen (Ag), and migrate
to the mediastinal lymph nodes (MLN) where Ag is presented to
recirculating naive T cells.2-5 Not surprisingly, these
cells have been implicated to cause sensitization to inhaled allergens,
typical of Th2-mediated allergic asthma. In a mouse model of asthma,
intratracheal immunization with ovalbumin (OVA)-pulsed DCs generates
Th2 effector cells that control eosinophilic airway inflammation,
goblet cell hyperplasia, and bronchial hyperreactivity upon repeated
challenge with OVA aerosol.6,7 These observations indicate
that airway DCs are essential in the early steps of sensitization. However, less information is available on the role of DCs in
stimulating memory and/or effector Th2 cells upon repeated encounter
with allergens.8 In patients with stable allergic asthma,
the number of airway DCs is elevated compared with healthy
controls, and local allergen challenge leads to rapid accumulation of
CD1a+ HLA-DR+ DCs in the airway lamina propria,
suggesting that DCs also present allergens to T cells in the secondary
immune response leading to airway inflammation.9
The mechanisms by which DC numbers increase in asthmatic airways
include several not mutually exclusive possibilities. First, the
increase could be caused by enhanced recruitment of DCs from the
bloodstream into the site of airway inflammation. To support an
enhanced demand for DCs in the inflamed airways, the bone marrow (BM)
might enhance its output of DCs or DC progenitors. Such a mechanism
would be similar to the enhanced recruitment of eosinophils into sites
of allergic inflammation, supported by a release of eosinophilic
progenitors from the BM.10-14 Second, enhanced
differentiation of freshly recruited monocytes into DCs could also lead
to increased numbers of DCs being found at sites of airway
inflammation.15 In such a scenario, one would expect to
find enhanced production of DC differentiation and/or growth factors
within the lung. Finally, as there is continuous and high throughput
migration of airway DCs from the epithelium to the draining MLNs, a
small decrease of DC efflux could lead to rapid and profound
accumulation of DCs within the epithelium.5,16 To study
which of these mechanisms might predominate, we have used a DC-driven
mouse model of asthma.6
Animals
Murine model of asthma
Detection of airway dendritic cells in whole mounts of the trachea At 24 hours after the last OVA challenge, animals were anesthetized and tracheal whole mounts were prepared as described earlier with a modification that the secondary antibody, used to detect rat anti-mouse major histocompatibility complex II (MHCII), was goat anti-rat F(ab')2 fragments conjugated to horseradish peroxidase (Serotec, Oxford, United Kingdom).18 The entire trachea was mounted in Entellan (Merck, Darmstadt, Germany) and viewed under a transmission light microscope equipped with Nomarski optics (Leica, Cambridge, United Kingdom).Collection of cells and tissues Bronchoalveolaire lavage fluid. At 24 hours after the last aerosol, groups of mice were killed by avertin overdose followed by exsanguination. Bronchoalveolaire lavage (BAL) was performed with 3 × 1 mL Ca2+- and Mg2+-free PBS supplemented with 0.1 mM ethylenediaminetetraacetic acid (EDTA). After red blood cells (RBCs) were lysed using ammoniumchloride lysis buffer, cytospin slides were prepared and remaining cells were used for flow cytometric analysis. Supernatants of BAL fluid (BALF) and serum were stored for enzyme-linked immunosorbent assay (ELISA) quantification of GM-CSF, interleukin 6 (IL-6) (OptEIA, PharMingen, Becton Dickinson, San Diego, CA; threshold 8 pg/mL), eotaxin, and fms-like tyrosine kinase 3 ligand (Flt-3L) (R&D Systems, Abingdon, United Kingdom; threshold 5 pg/mL). Lymph nodes. Lymph node (LN) cell suspensions were prepared by a 1-hour incubation at 37°C with 0.02 mg/mL DNAse I (Sigma Chemical) and 100 U/mL Collagenase IV (Life Technologies). RBCs were lysed and cells were passed through a 40-µm cell sieve (Becton Dickinson). Blood. Blood was collected in heparinized tubes from the iliac artery and lysed with 20 mL RBC lysis solution for 4 minutes at 4°C. Bone marrow. BM cells were prepared by flushing femurs and tibiae with 5 mL sterile PBS, followed by RBC lysis and passage through a 100-µm cell sieve. Staining for major basic protein-positive eosinophils Cytospin preparations of BALF and cultured BM were acetone fixed and blocked with 1% bovine serum albumin/PBS. Major basic protein (MBP) was detected using a rabbit anti-mouse MBP antibody (Ab) (J. J. Lee, Mayo Clinic, Scottsdale, AZ), followed by alkaline phosphatase-conjugated goat anti-rabbit Abs (Sigma) and development of signal with New Fuchsin in Tris-HCl. Slides were counterstained with Mayer haematoxilin (Merck). One investigator counted all cells.Flow cytometric analysis on BALF, LN, and blood cells Cells were washed in PBS containing 5% FCS and 5 mM sodium azide (FACS wash), and 1 × 106 cells were stained for 30 minutes on ice. To reduce nonspecific binding, cells were incubated with 2.4G2 blocking reagent for 15 minutes. Monoclonals used were as follows: MHCII-fluorescein isothiocyanate (MHCII-FITC) (2G9), allophycocyanin (APC)-labeled CD11c (HL3), phycoerythrin (PE)-labeled Abs against CD3 (145-2C11), B220 (RA3-6B2), and CCR3 (R&D Systems), biotin-labeled CD11b (M1/70), followed by streptavidin (SA)-PE-Cy5 (Quantum Red; Sigma). Propidium iodide (Sigma) was added for exclusion of dead cells before analysis on a FACScalibur flow cytometer using CellQuest (Becton Dickinson Immunocytometry Systems, San Jose, CA) and FlowJo software (Treestar, Costa Mesa, CA).Flow cytometric analysis and sorting of BM cells BM cells were stained with anti-CD31 (anti-platelet endothelial cell adhesion molecule PECAM-1; ER-MP12-bio), and anti-Ly-6C (ER-MP20-FITC; both produced in-house) followed by SA-PE or SA-PE-Cy5, allowing the discrimination of 6 distinct populations of cells.19-22 For phenotype description the following mAbs were used: CD11b-APC, CD127-PE (IL-7R ; SB/14), CD131-PE (JORO50),
CCR3-PE, CD3-PECy5 and CD4-APC (GK1.5), CD11c-APC and B220-PECy5, or
Gr1-PE (RB6-8C5). Antibodies were from PharMingen or R&D Systems.
In separate sorting experiments, 80 × 106 cells were stained with CD31-bio followed by SA-PE and Ly-6C-FITC and were sorted into CD31hiLy-6Cneg and into CD31negLy-6Cmed populations under sterile conditions on a FACS Vantage flow cytometer (Becton Dickinson). Culture of CD31hiLy-6Cneg and CD31negLy-6Cmed populations After sorting, cells were washed twice in TCM and cultured for 7 days at 0.25 × 106/well in 24-well plates with 40 ng GM-CSF per milliliter to induce DC differentiation. In separate experiments, cells were grown at 8 × 105 cells/well in round-bottom 96-well plates in TCM supplemented with 30% FCS and 24 ng/mL murine recombinant IL-5 (rIL-5) (PharMingen) to induce eosinophil differentiation. As a control, unsorted BM was stained and cultured under identical conditions. After 7 days, cells were analyzed for expression of MHCII-FITC, CD11c-bi (N418) in combination with CD80-PE (16-10A1), CD86-PE (GL-1), or CD40-PE (3/23), followed by SA-PECy5. For eosinophil differentiation, cytospin preparations were stained with an anti-MBP Ab as described above.Detection of labeled DCs after adoptive transfer BM DCs were labeled using carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR), as previously described.6 CFSE+ DCs (2-5 × 106) were transferred intratracheally into mice with established eosinophilic airway inflammation (OVA-DC/3xOVA) or into control mice (PBS-DC/3xPBS). At 48 hours after the intratracheal cell transfer, mice were killed. The number of CD11c+, CFSE+ DCs was determined on BALF, MLN, and inguinal LN samples.Statistical analysis All experiments were performed using 3 to 10 mice per group, and per time point in kinetic experiments. Comparison of means between different groups was performed with a Kruskal-Wallis test for equality among the different groups and in the case of a significant difference, the Mann-Whitney test for unpaired data was used for comparing 2 groups (SPSS 10.0 for Windows) separately. Differences were considered significant if P < .05.
OVA exposure time-dependently induces eosinophilic airway inflammation in OVA-DC-immunized mice Sensitization was induced by intratracheal injection of 1 × 106 OVA-pulsed DCs. As a marker for inflammation in the lungs, the total number of BAL cells was measured 24 hours after 1, 3, or 7 OVA aerosol exposures in sensitized mice (Figure 1A). The total recovery of BAL cells was not different from control mice (PBS-DC/PBS) after 1 OVA exposure, but sequentially increased 10-fold (P = .008) after 3 OVA exposures and 46-fold (P = .008) after 7 OVA exposures.
Differential analysis of the BALF cells using flow cytometry (Figure
1B) showed a significant proportional increase in lymphocytes (CD3+ or B220+ cells) and granulocytes (based
on scatter characteristics) with a concomitant decrease in alveolar
macrophages/monocytes (highly autofluorescent cells) in OVA-DC/OVA mice
(Figure 2A). As the discrimination of
eosinophils from other polymorphonuclear granulocytes is impossible
based on scatter characteristics alone, eosinophils were further
characterized as nonautofluorescent highly granular (SSChi)
cells expressing intermediate levels of CD11c, and lacking expression of MHCII, B220, and CD3.23 These highly granular cells
also expressed the eotaxin receptor CCR3 (Figure 1D).24
This method of counting eosinophils was compared with counting BALF
cytospins stained with an anti-MBP Ab, yielding a highly statistically
significant Pearson correlation coefficient of 0.82 (P = .0001) (Figure 1C). Performing 3 OVA aerosols
induced an eosinophilia of 30.7 ± 6.0%, and 7 OVA aerosols
induced an eosinophilia of 49.0% ± 5.1 of all BALF cells. Thus, OVA
exposure in OVA-DC-immunized mice time-dependently induces
eosinophilic airway inflammation.
OVA exposure leads to a massive increase in endogenous airway DCs in OVA-sensitized mice To determine the number of DCs in the airways of PBS- and OVA-exposed mice, we have analyzed BALF cells 24 hours after the last OVA aerosol (Figure 2A). Dendritic cells were identified with multiparameter flow cytometry as nonautofluorescent CD11chi/MHCIIhi/B220 /CD3
cells, as described previously.3 Additional staining
revealed that these cells expressed CD11b, identifying them as myeloid DCs (Figure 2D). First we verified that injected DCs could no longer be
recovered from the BALF 5 days following intratracheal injection (data
not shown). This eliminates the possibility that nonendogenous DCs
still remaining in the BALF could confound the counting of DCs after
the aerosol challenge period (day 11 to day 17 after injection). The
absolute number of DCs was elevated about 10 times in OVA-DC-immunized
mice challenged with 3 OVA aerosols (P = .008) and
increased about 100 times after 7 OVA aerosols (P = .016)
compared with control PBS-DC/PBS mice (Figure 2B). In addition to an
absolute increase in cell number, the percentage of DCs found in BALF
cells was similarly increased following 3 aerosols, although not
significantly (P = .056), and doubled after 7 aerosols
(P = .016). In control mice the number of DCs remained at
low levels, comparable to the situation in unmanipulated mice.
To determine if the increase in the number of BALF DCs was also
supported by an increase in airway mucosal DCs, we visualized the DC
network in tracheal whole mounts, as previously
described.18 The pattern of the MHCII staining revealed a
dendritic network in the control PBS-DC/PBS mice (Figure
3A), whereas in the OVA-DC-immunized mice exposed to 3 OVA aerosols, numerous dense areas of rounded MHCII+ cells lacking the typical dendritic morphology were
seen (Figure 3B). Due to the fact that MHCII+ cells were
rounded and could also represent MHCII+ B cells
or eosinophils, we could not directly compare DC numbers at the
tissue level, although clearly, overall MHCII staining was enhanced.
OVA exposure leads to an increase in peripheral blood DCs in OVA-sensitized mice To investigate if the accumulation of lung CD11c+ CD11b+ DCs was supported by recruitment from the bloodstream, the number of DCs (CD11c+/MHCII+/B220/CD3
cells) was determined in the blood 24 hours after the last of 3 aerosols. The percentage of MHCII+ CD11c+ blood
DCs was significantly raised (OVA 0.81 ± 0.09% vs PBS
0.37 ± 0.03 vs naive 0.39 ± 0.03, P < .0001) in
response to OVA challenges in OVA-DC mice (Figure
4A,C).
In the blood of the control PBS-DC/PBS-immunized mice, levels were
comparable with those found in untreated animals. Additional experiments were carried out to define the CD11b+ myeloid
and CD11b OVA exposure leads to an increase in CD31hiLy-6Cneg BM cells in OVA-sensitized mice The increased presence of CD11c+ DCs in the bloodstream during OVA challenge, despite the massive influx of DCs into the airways, suggested that DC output from the BM might be enhanced.Staining of whole BM cells with the monoclonal Abs CD31 (ER-MP12) and
Ly-6C (ER-MP20), gives rise to 6 distinct populations of BM cells, each
with varying degrees of lineage commitment and progenitor potential
(see Figure 5A).19 This
staining was used to define the cellular composition of BM in mice with
or without eosinophilic airway inflammation. Performing 1, 3, or 7 OVA aerosol exposures to OVA-DC mice sequentially induced an
increase in the CD31hiLy-6Cneg population, from
4.61 ± 0.75% of total BM at baseline up to 7.84 ± 1.25%
after 3 aerosols and 11.1 ± 1.48% after 7 aerosols (Figure 5A-B). There was no increase in this population after exposure to PBS
aerosols. None of the 5 other distinct populations was altered
significantly by OVA aerosol in OVA-DC mice or by PBS aerosol in PBS-DC
mice.
To explore whether the increase of the
CD31hiLy-6Cneg population was due to an
increase of cells with myeloid or lymphoid commitment, additional
staining was performed using the myeloid differentiation marker CD11b
in combination with a CD127 Ab against the IL-7R The CD31hiLy-6Cneg BM subset gives rise to DCs and eosinophils under different culture conditions As the CD31hiLy-6Cneg subset of cells was the only population that was increased in the BM of mice with eosinophilic airway inflammation, we examined if this subset could give rise to DCs. The CD31hiLy-6Cneg population was purified by flow cytometric sorting to 85% to 95% purity, and cultured in GM-CSF (40 ng/mL) (Figure 6).
After 7 days culture in the presence of GM-CSF, many colonies of proliferating cells were seen. About 77% of cells were CD11c+ and more than half of these expressed MHCII, indicating maturation in culture (Figure 6C). In contrast, the CD31negLy-6Cmed population was sorted and cultured under the same conditions and yielded only 2.2% MHCII+/CD11c+cells. Airway eosinophilia is a prominent feature of allergic airway
inflammation and was also observed in our model. Therefore, we
investigated whether the same CD31hiLy-6Cneg
population contained eosinophil precursors. First, to support the
concept that cells with eosinophil potential were contained within the
CD31hiLy-6Cneg population, bone marrow subsets
were stained with a monoclonal antibody against the eotaxin
receptor CCR3.24 Cells within this subset expressed CCR3
at intermediate levels (see Figure 7A). Mature granulocytes contained within the
CD31negLy-6Cmed also contained
CCR3hi mature eosinophils. Next, cells in the
allergen-induced enlarged CD31hiLy-6Cneg
population were sorted (about 85% to 95% pure
CD31hiLy-6Cneg cells) and cultured in the
presence of IL-5 (24 ng/mL) for 6 days (Figure 7B-C). Eosinophils were
detected after 6 days by MBP staining and morphology on cytospins. The
CD31hiLy-6Cneg subset yielded a 4-fold higher
number of cells (P = .029) and a higher percentage of
eosinophils compared with whole BM cultured under the same conditions
(CD31hiLy-6Cneg: 51.2 ± 1.1% vs whole BM:
25.6 ± 2.2%, P = .029).
OVA exposure does not increase the number of plasmacytoid DCs in BM Despite the fact that none of the other populations identified by CD31 and Ly-6C staining were percentually changed, we studied these subsets in greater detail by 4-color analysis. More specifically, the percentage of CD31hiLy-6Chi cells, known to contain precursors for DCs as well as plasmacytoid DCs, was not altered by exposure to OVA.22,27,28 The percentage of plasmacytoid CD11c+ B220+ DCs within the CD31hiLy-6Chi subset was 12.2 ± 1.4% in OVA-exposed mice compared with 13.0 ± 0.8% in PBS-exposed mice after 3 aerosols (Figure 5C). However, in response to the OVA challenges, the CD31hiLy-6Chi subset, expressed more CD131 compared with the control mice (data not shown).The percentage of CD3+CD4+ T cells (falling within the CD31medLy-6Cneg fraction) in total BM was significantly lower (0.16 ± 0.03%) in the OVA-DC/OVA group compared with the PBS-DC/PBS group (0.37 ± 0.07%; P = .001). OVA exposure modifies the BALF and serum level of cytokines in OVA-sensitized mice An increase in DCs could also be caused by increased local differentiation of DCs from monocytic precursors within inflamed tissues. To determine the presence of early growth and differentiation factors for DCs, we measured the content of the cytokines IL-6, GM-CSF, and Flt-3L in BALF 24 hours after 3 OVA aerosol exposures, when the number of DCs was significantly increased. IL-6 was significantly raised after 3 aerosols in OVA-DC/OVA mice compared with PBS-DC/PBS mice (OVA: 418.2 ± 74.6 pg/mL, PBS: 12.5 ± 1.9 pg/mL; P = .016). Ag challenge did not effect the GM-CSF level (OVA: 3.90 ± 1.50 pg/mL vs PBS: 9.45 ± 2.3 pg/mL; P = .111). The Flt-3L levels in BALF were just above the detection level of our assay, showing no detectable difference in the various groups.In serum, levels of IL-6 (13.67 ± 3.39 pg/mL vs 2.55 ± 0.79 pg/mL; P = .016) and eotaxin (564 ± 62 pg/mL vs 282 ± 25 pg/mL; P = .008) were higher in the OVA-DC/OVA group compared with the PBS-DC/PBS group, whereas that of GM-CSF was below the detection limit. OVA exposure increases DC migration toward the draining lymph nodes in OVA-sensitized mice In addition to the mechanisms studied above, a decreased efflux to the draining MLN could contribute to an accumulation of DCs in inflamed airways. We observed that the draining MLNs of OVA-DC/OVA mice were grossly swollen compared with nondraining nodes or MLNs of PBS-DC/PBS mice. After 3 aerosol exposures, the total number (both relative and absolute) of DCs was increased in the OVA-DC/OVA group compared with the control PBS-DC/PBS group (7.00 ± 0.53% vs 2.37 ± 0.27%; P = .002). This was due primarily to an increase in the CD11bmed/hi myeloid DCs (3.5-fold increase), although the lymphoid CD11b subset was also increased following OVA
challenge (Figure 8A-B). To provide
further proof that migration of airway DCs was influenced by the
eosinophilic airway inflammation, we injected CFSE-labeled, BM-derived,
in vitro-cultured DCs intratracheally in mice with (OVA-DC/OVA group)
or without (PBS-DC/PBS) eosinophilic airway inflammation. At 48 hours
after injection, mice with inflamed lungs had a small, but
significantly higher number of CFSE-labeled DCs in the MLNs, compared
with control mice with uninflamed lungs (9.74 ± 1.81 × 102 vs
2.41 ± 0.84 × 102 CFSE-labeled CD11c+
cells; P = .029; Figure 8C), demonstrating that
DC efflux to the MLNs was actually enhanced in mice with eosinophilic
airway inflammation. After intratracheal injection, CFSE-labeled DCs could not be detected in peripheral LN.
Airway DCs have been implicated in causing the sensitization to inhaled allergens, by taking up Ag in the lung mucosa, transporting it into the draining LN and finally by inducing differentiation of Th2 effector cells that can orchestrate eosinophilic airway inflammation.3,4,6,7 Despite these observations that airway DCs might be essential in the early steps of sensitization, less information is available on the role of DCs in stimulating memory and/or effector Th2 cells at times of repeated exposure to inhaled allergens. In this paper, we have demonstrated that the number of airway CD11c+ CD11b+ myeloid DCs is strongly increased within the airway epithelium and BALF following allergen challenge in sensitized animals. Within 3 days, an almost 10-fold expansion in the number of DCs was found following repeated OVA challenge, in parallel with an increase in CD4+ lymphocytes and eosinophils in the airways, reaching a 100-fold expansion at day 7. At the same time, the number of CD11c+ CD11b+ myeloid DCs in the bloodstream was increased 3-fold. The massive increase in airway DCs and the accompanying increase in
circulating blood DCs led us to investigate whether the production of
DCs from the BM ("dendropoiesis") might be enhanced in mice with
eosinophilic airway inflammation, to meet the enhanced demand for DCs
in the inflamed lung. Numerous studies have demonstrated that the BM
reacts to airway allergen challenge by increasing its output of
eosinophilic precursors.10-14 Previously, repopulation experiments in irradiated and BM-reconstituted rats have shown that
airway DCs stem from a rapidly dividing precursor cell in the
BM.16 It is possible to discriminate distinct populations of BM cells using multiparameter flow
cytometry.19,22,29,30 Here, we have used the expression of
Ly-6C in combination with CD31, platelet endothelial cell adhesion
molecule PECAM-1, to delineate 6 discrete populations of BM cells, each
with differential lineage commitment and differentiation
potential.19,22 When we stained BM cells from mice with or
without eosinophilic airway inflammation, there was a striking and
time-dependent increase in the population of
CD31hiLy-6Cneg cells, whereas none of the other
populations were affected by allergen challenge. In previous
experiments, this population of BM cells was shown to contain cells
with colony-forming unit (CFU) potential for granulocytes/monocytes,
erythrocytes, megakaryocytes, and mast cells as well as cells
with thymus repopulating capacity. More primitive precursors with
long-term repopulating ability were found in the
CD31medLy-6Cneg population.29,31
In mice with eosinophilic airway inflammation, CD31hiLy-6Cneg cells predominantly expressed
the myeloid marker CD11b, and the expression of the lymphoid
differentiation marker IL-7R The fact that a time-dependent increase in
CD31hiLy-6Cneg cells with DC potential was
observed in mice with massive accumulation of CD11b+
CD11c+ airway and blood DCs strongly suggests that the BM
increased its production of DCs to meet the enhanced demand in the
airways. Moreover, in mice with airway inflammation, there was no
increase in the CD31hiLy-6Chi population, known
to contain the plasmacytoid B220+ CD11c+ DCs
and some precursors of myeloid DCs.22 The fact that we did
not see an increase in any of these more mature DC populations following allergen challenge suggests that DC precursors leave the BM
at an early stage of differentiation. One possibility that needs
further investigation is that they were attracted to the airways
through the action of particular chemokines. Allergic inflammation is accompanied by enhanced production of eotaxin in the
airways.32 The subset of
CD31hiLy-6C The observed increase in CD31hiLy-6Cneg cells in BM, without any other increase in BM populations is unique for eosinophilic airway inflammation. Bacterial infection with Listeria monocytogenes leads to profound changes in BM composition with a predominant time-dependent increase in CD31hiLy-6Chi monocyte precursors, CD31negLy-6Chi mature monocytes, and CD31negLy-6Cmed granulocytes. At the same time, there was a depletion of CD31medLy-6Cneg lymphoid cells and more importantly, the CD31hiLy-6Cneg population described in this study.19 The changes in the BM of Listeria-infected mice reflect an increased need for granulocytes and monocytes, which are attracted to lesions consisting of mononuclear- and neutrophil-rich cell infiltrates. However, in our model of OVA-induced airway inflammation, eosinophils are strongly recruited to the airways of challenged mice together with DCs. Not surprisingly, we were able to demonstrate that the CD31hiLy-6Cneg population of BM cells could also differentiate into MBP-positive eosinophils after culture in IL-5, suggesting that enhanced production of eosinophils from the BM was induced to meet the increased demand for eosinophils. This is in line with previous experiments in which mouse BM cells were cultured in the presence of IL-5 in semisolid media to asses eosinophil-colony-forming unit (CFU-Eo) potential and showing increased CFU-Eo in mice with eosinophilic airway inflammation.11-14 The increase in CFU-Eo in challenged mice has been attributed either to the presence of a serum factor distinct from IL-5, such as eotaxin, or to migration of IL-5-producing T cells to the BM.13,14,33 In support of a serum factor in our system, we measured a slightly enhanced level of the DC growth factor IL-6.34 One likely candidate that could be involved in the up-regulation of GM-CSF-responsive BM cells is eotaxin,33 of which the serum levels were indeed doubled in mice with inflammation. Further, the CD31hiLy-6Cneg cells expressed the eotaxin receptor CCR3, with identical levels in allergic and nonallergic mice (data not shown). The migration of "dendropoiesis"-promoting T cells to the BM is less likely as we measured a decrease in BM CD4+ T cells following allergen challenge, probably due to their migration to the lung. Although the direct recruitment of immature blood DCs is the most
likely explanation for the observed accumulation of lung DCs, one
contributing mechanism could also be enhanced local proliferation of
DCs from monocytic precursors in allergic lung. Although it was shown
in irradiation experiments that there is little if any local
self-renewal capacity for DCs in rat airways, this situation could be
different under inflammatory conditions.16 Indeed, in our
experiments, there was an increase in circulating
CD11c One final mechanism that could be responsible for the observed change
in airway DCs in mice with airway eosinophilia would be reduced
emigration of DCs. There is continuous and high throughput migration of
airway DCs from the epithelium to the draining MLN and a small decrease
of DC efflux could lead to rapid and profound accumulation within the
epithelium.3,5,16 To our surprise, we found that the
numbers of CD11c+CD11b+ myeloid and
CD11c+CD11b In summary, our data show that CD11b+CD11c+ DCs are massively attracted into the airways and draining LN upon OVA challenge in sensitized mice, a process supported by increased dendropoiesis in the BM. We have previously shown that systemic abolition of DCs in sensitized thymidine kinase transgenic mice immediately prior to secondary challenge completely suppresses eosinophilic airway inflammation, goblet cell hyperplasia, and IgE synthesis.18 Together, these data imply an important functional role for airway DCs not only in the induction of Th2 cells from naive precursors, but also in the maintenance of eosinophilic airway inflammation. Inhibiting the influx of DCs could prove to be a strategy for reducing airway inflammation that is typical of asthma.8
We thank C. Snoys (Cell Biology, Erasmus University) for sorting and J. Lee for antibodies.
Submitted March 1, 2002; accepted June 18, 2002.
Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-03-0673.
Supported by a grant from the Dutch Asthma Foundation (NAF3.2.99.37).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Leonie van Rijt, Erasmus University Rotterdam (Room Ee2263), Department of Pulmonary Medicine, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands; e-mail: vanrijt{at}longz.fgg.eur.nl.
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  M. Kool, M. van Nimwegen, M. A. M. Willart, F. Muskens, L. Boon, J. J. Smit, A. Coyle, B. E. Clausen, H. C. Hoogsteden, B. N. Lambrecht, et al. An Anti-Inflammatory Role for Plasmacytoid Dendritic Cells in Allergic Airway Inflammation J. Immunol., July 15, 2009; 183(2): 1074 - 1082. [Abstract] [Full Text] [PDF]
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N. Miyahara, H. Ohnishi, H. Matsuda, S. Miyahara, K. Takeda, T. Koya, S. Matsubara, M. Okamoto, A. Dakhama, B. Haribabu, et al. Leukotriene B4 Receptor 1 Expression on Dendritic Cells Is Required for the Development of Th2 Responses and Allergen-Induced Airway Hyperresponsiveness J. Immunol., July 15, 2008; 181(2): 1170 - 1178. [Abstract] [Full Text] [PDF]
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J. J. Osterholzer, J. L. Curtis, T. Polak, T. Ames, G.-H. Chen, R. McDonald, G. B. Huffnagle, and G. B. Toews CCR2 Mediates Conventional Dendritic Cell Recruitment and the Formation of Bronchovascular Mononuclear Cell Infiltrates in the Lungs of Mice Infected with Cryptococcus neoformans J. Immunol., July 1, 2008; 181(1): 610 - 620. [Abstract] [Full Text] [PDF]
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F. Blank, B. Rothen-Rutishauser, and P. Gehr Dendritic Cells and Macrophages Form a Transepithelial Network against Foreign Particulate Antigens Am. J. Respir. Cell Mol. Biol., June 1, 2007; 36(6): 669 - 677. [Abstract] [Full Text] [PDF]
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H. Wang, N. Peters, V. Laza-Stanca, N. Nawroly, S. L. Johnston, and J. Schwarze Local CD11c+ MHC Class II- Precursors Generate Lung Dendritic Cells during Respiratory Viral Infection, but Are Depleted in the Process J. Immunol., August 15, 2006; 177(4): 2536 - 2542. [Abstract] [Full Text] [PDF]
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J. J. Osterholzer, T. Ames, T. Polak, J. Sonstein, B. B. Moore, S. W. Chensue, G. B. Toews, and J. L. Curtis CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen J. Immunol., July 15, 2005; 175(2): 874 - 883. [Abstract] [Full Text] [PDF]
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J. P. J. J. Hegmans, A. Hemmes, J. G. Aerts, H. C. Hoogsteden, and B. N. Lambrecht Immunotherapy of Murine Malignant Mesothelioma Using Tumor Lysate-pulsed Dendritic Cells Am. J. Respir. Crit. Care Med., May 15, 2005; 171(10): 1168 - 1177. [Abstract] [Full Text] [PDF]
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L. S. van Rijt, S. Jung, A. KleinJan, N. Vos, M. Willart, C. Duez, H. C. Hoogsteden, and B. N. Lambrecht In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma J. Exp. Med., March 21, 2005; 201(6): 981 - 991. [Abstract] [Full Text] [PDF]
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 H. Kuipers, C. Heirman, D. Hijdra, F. Muskens, M. Willart, S. van Meirvenne, K. Thielemans, H. C. Hoogsteden, and B. N. Lambrecht Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization J. Leukoc. Biol., November 1, 2004; 76(5): 1028 - 1038. [Abstract] [Full Text] [PDF]
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H. J. de Heer, H. Hammad, T. Soullie, D. Hijdra, N. Vos, M. A.M. Willart, H. C. Hoogsteden, and B. N. Lambrecht Essential Role of Lung Plasmacytoid Dendritic Cells in Preventing Asthmatic Reactions to Harmless Inhaled Antigen J. Exp. Med., July 6, 2004; 200(1): 89 - 98. [Abstract] [Full Text] [PDF]
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A. E. Kelly-Welch, M. E. F. Melo, E. Smith, A. Q. Ford, C. Haudenschild, N. Noben-Trauth, and A. D. Keegan Complex Role of the IL-4 Receptor {alpha} in a Murine Model of Airway Inflammation: Expression of the IL-4 Receptor {alpha} on Nonlymphoid Cells of Bone Marrow Origin Contributes to Severity of Inflammation J. Immunol., April 1, 2004; 172(7): 4545 - 4555. [Abstract] [Full Text] [PDF]
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 H. Hammad, H. J. de Heer, T. Soullie, V. Angeli, F. Trottein, H. C. Hoogsteden, and B. N. Lambrecht Activation of Peroxisome Proliferator-Activated Receptor-{gamma} in Dendritic Cells Inhibits the Development of Eosinophilic Airway Inflammation in a Mouse Model of Asthma Am. J. Pathol., January 1, 2004; 164(1): 263 - 271. [Abstract] [Full Text] [PDF]
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H. Hammad, H. Jan de Heer, T. Soullie, H. C. Hoogsteden, F. Trottein, and B. N. Lambrecht Prostaglandin D2 Inhibits Airway Dendritic Cell Migration and Function in Steady State Conditions by Selective Activation of the D Prostanoid Receptor 1 J. Immunol., October 15, 2003; 171(8): 3936 - 3940. [Abstract] [Full Text] [PDF]
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L. S. van Rijt, N. Vos, D. Hijdra, V. C. de Vries, H. C. Hoogsteden, and B. N. Lambrecht Airway Eosinophils Accumulate in the Mediastinal Lymph Nodes but Lack Antigen-Presenting Potential for Naive T Cells J. Immunol., October 1, 2003; 171(7): 3372 - 3378. [Abstract] [Full Text] [PDF]
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H. Kuipers, D. Hijdra, V. C. de Vries, H. Hammad, J.-B. Prins, A. J. Coyle, H. C. Hoogsteden, and B. N. Lambrecht Lipopolysaccharide-Induced Suppression of Airway Th2 Responses Does Not Require IL-12 Production by Dendritic Cells J. Immunol., October 1, 2003; 171(7): 3645 - 3654. [Abstract] [Full Text] [PDF]
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K. Vermaelen and R. Pauwels Accelerated Airway Dendritic Cell Maturation, Trafficking, and Elimination in a Mouse Model of Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 405 - 409. [Abstract] [Full Text] [PDF]
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K. Y. Vermaelen, D. Cataldo, K. Tournoy, T. Maes, A. Dhulst, R. Louis, J.-M. Foidart, A. Noel, and R. Pauwels Matrix Metalloproteinase-9-Mediated Dendritic Cell Recruitment into the Airways Is a Critical Step in a Mouse Model of Asthma J. Immunol., July 15, 2003; 171(2): 1016 - 1022. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-mercaptoethanol, all from Gibco BRL,
Paisley, Scotland) supplemented with 20 ng/mL recombinant murine
(rm) granulocyte macrophage-colony-stimulating factor
(GM-CSF).
correlates with enhanced numbers of CD11c+
DCs being found in the BALF.