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The Journal of Immunology, 2006, 177: 2536-2542.
Copyright © 2006 by The American Association of Immunologists

Local CD11c+ MHC Class II– Precursors Generate Lung Dendritic Cells during Respiratory Viral Infection, but Are Depleted in the Process1

Hongwei Wang, Nina Peters, Vasile Laza-Stanca, Niga Nawroly, Sebastian L. Johnston and Jürgen Schwarze2

Department of Respiratory Medicine, National Heart and Lung Institute and Wright Fleming Institute of Infection and Immunity, Imperial College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Increases in numbers of lung dendritic cells (DC) observed during respiratory viral infections are assumed to be due to recruitment from bone marrow precursors. No local production has been demonstrated. In this study, we isolated defined populations of murine lung cells based on CD11c and MHC class II (MHC II) expression. After culture for 12 days with GM-CSF, we analyzed cell numbers, DC surface markers, and Ag-presenting capacity. Only CD11c+ MHC II– cells from naive mice proliferated, yielding myeloid DC, which induced Ag-specific proliferation of naive T cells. After respiratory syncytial virus (RSV) infection, numbers of pulmonary CD11c+ MHC II– precursor cells were significantly reduced and DC could not be generated. Moreover, RSV infection prevented subsequent in vivo expansion of pulmonary DC in response to influenza infection or LPS treatment. These results provide direct evidence of local generation of fully functional myeloid DC in the lung from CD11c+ MHC II– precursor cells that are depleted by RSV infection, leading to an inability to expand lung DC numbers in response to subsequent viral infection or exposure to bacterial products. This depletion of local DC precursors in respiratory viral infections may be important in explaining complex interactions between multiple and intercurrent pulmonary infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 are highly specialized APCs that have a unique ability to induce and direct Ag-specific responses in naive T cells. They are widely distributed in lymphoid and nonlymphoid tissues and are of particular importance as sentinels in mucosal surfaces, including the respiratory tract. In the lung, DC form a network within the epithelium and submucosa of conducting airways as well as in lung parenchyma, where they can be found both on alveolar surfaces and in the vascular compartment of the lung (1). In the absence of infection and inflammation, most pulmonary DC display an immature phenotype and induce T cell anergy or regulatory T cell responses to exogenous Ags and self Ags (2). Respiratory viral infections have been shown to prevent tolerance induction to harmless Ags in the lung (3, 4). Respiratory syncytial virus (RSV) is an important respiratory pathogen in infancy (5) and in the elderly (6), which can lead to bronchiolitis, a severe inflammation of the small airways, and to pneumonia. Severe RSV infection in infants has also been associated with the development of asthma symptoms throughout childhood (7) and early allergic sensitization (8, 9). Using a murine model, we have demonstrated recently that RSV infection results in sustained increases in numbers of pulmonary DC, which display a mature phenotype and increased Ag-presenting capacity (10). Numbers of these mature pulmonary DC remain elevated beyond the resolution of RSV disease and most likely influence T cell responses to both RSV and unrelated Ags during virus-induced inflammation and beyond. Similar increases in numbers of mature lung DC have been described following influenza infection (11, 12), but the origin of mature pulmonary DC following viral infections has not been determined.

Progenitors of conventional DC have been defined in the bone marrow both in humans and in mice (13). Culture with GM-CSF can differentiate these cells into conventional DC (14). In peripheral blood, CD14+ monocytes in humans and CD11c+ MHC class II (MHC II)– precursors in mice can give rise to DC in culture (15, 16). In addition, committed DC precursor cells have also been identified in lymphoid organs (17, 18). However, in nonlymphoid organs, such DC precursors have not been demonstrated. In this study, we show that precursor cells resident in the lung contribute to the expansion of mature pulmonary DC following RSV infection, and that their depletion in this process leads to sustained hyporesponsiveness to subsequent activating challenges.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female BALB/c mice aged 8–10 wk from Charles River Laboratories and OVA peptide 323–339-specific, TCR transgenic DO11.10 mice from The Jackson Laboratory were housed under specific pathogen-free conditions. Mice were allowed free access to food and water and were used under experimental protocols approved by the Home Office (London, U.K.).

Viral infection and LPS administration

Human RSV (A2 strain) from LGC Promochem free of chlamydia or mycoplasma contamination was plaque purified and grown in HEp-2 cells (LGC Promochem) cultured in complete medium (RPMI 1640, 2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, all from Invitrogen Life Technologies). Mice were infected by intranasal inoculation of 6 x 105 PFUs of RSV in 60 µl of PBS or sham infected with PBS or UV-inactivated virus under light anesthesia. To exclude GM-CSF contamination from HEp-2 cells, in one experiment RSV was purified by centrifugation using a 30% sucrose cushion, as described (19). For influenza A virus infection, mice were inoculated intranasally with five hemaglutinin units of strain X31. In LPS exposure experiments, 10 µg of LPS (from Escherichia coli 055:B5; Sigma-Aldrich) in 100 µl of PBS was administrated intranasally. Controls received 100 µl of PBS.

GM-CSF ELISA

Lungs harvested from naive mice or on days 1, 2, 3, 4, and 7 after RSV infection were homogenized, and GM-CSF concentrations were assessed in supernatants using a GM-CSF ELISA kit from R&D Systems Europe.

Isolation of lung cells

Lungs were harvested from naive mice or on days 4, 8, 12, 21, and 45 after RSV infection. Before harvest, lungs were gently perfused via the right ventricle with 5–10 ml of PBS containing 0.6 mM EDTA to remove blood cells from the pulmonary circulation. They were then cut into small pieces and incubated with collagenase type IA-S (0.7 mg/ml PBS; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 mg/ml PBS; Sigma-Aldrich) for 30 min at 37°C. After digestion, lung cells were dispersed by shearing through a 20-gauge needle, followed by filtration through a nylon screen cell strainer (70 µm). Single cell suspensions were washed, contaminating erythrocytes (always <10% of lung cells) were lysed using ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA), and viable cells were counted by trypan blue exclusion.

Generation of DC from lung or bone marrow cells

Both lung- and bone marrow-derived DC were generated, as described (20). Briefly, total lung cells or bone marrow cells (obtained by flushing the femoral bones) were cultured in 6-well plates (106 cells/well) at 37°C in complete medium with 10% FBS, supplemented with 20 ng/ml mouse rGM-CSF (BioSource International). During the culture period of 12 days, medium with GM-CSF was replaced every 3 days. Cells were harvested by vigorous pipetting and PBS washes with Versene 1:5000 (Invitrogen Life Technologies).

Flow cytometry

Following FcR blockade with anti-mouse CD16/CD32 (24G2; BD Biosciences), cells were stained with the following anti-mouse Abs (all from BD Biosciences, except for anti-DO11.10 TCR-TRIcolor and anti-CD11c CyChrome from Caltag Laboratories, and anti-TLR4 PE from eBiosciences): anti-CD3e FITC (145-2C11), anti-CD19 FITC (1D3), anti-CD49b/pan-NK FITC (DX5), anti-Ly-6C FITC (AL21), anti-I-A/I-E FITC (2G9), anti-Ki67 PE (B56), anti-CD11c PE (HL3), anti-CD11b PE (M1/70), anti-CD40 PE (3/23), anti-CD80 PE (16-10A1), anti-CD86 PE (GL1), anti-CD31 PE (MEC 13.3), anti-CD8 PE (53-6.7), anti-CD4 PE (GK1.5), and anti-CD45R/B220 CyChrome (RA3-6B2). Hamster IgG2 (500A2), rat IgG1 (A110-1), rat IgG2a (R35-95), rat IgG2b (A95-1), mouse IgG2a (G155-178), and hamster IgG1 (A19-3) were used as isotype controls. Samples were stained at 4°C in PBS with 1% FBS, acquired using an LSR flow cytometer and CellQuest software (both BD Biosciences), and analyzed using WinList software (Verity Software House).

Cell sorting

Following density gradient centrifugation with underlaying Biocoll solution (density = 1.09 mg/ml; obtained from Autogen Bioclear) of lung cell suspensions, CD11c+ and CD11c– cells were separated using anti-CD11c-coated MACS beads and an AutoMACS (Miltenyi Biotec). To isolate CD11c+ MHC II+ and CD11c+ MHC II– cells, mononuclear lung cells were labeled with anti-I-A/I-E FITC (2G9) and anti-CD11c PE (HL3) Abs. Cells were separated using a FACSDiva cell sorter and software (BD Biosciences). Sorted cells were collected into complete medium supplemented with 10% FBS and quickly aliquoted into 6-well plates for culture.

Microscopic analysis

Lung mononuclear cells or bone marrow cells were cultured in the presence of GM-CSF on 8-well chamber slides (BD Biosciences). After 10 days of culture, nonadherent cells were washed off and adherent cells were fixed with 4% paraformaldehyde, blocked with PBS supplemented with 1% BSA and 1% FBS, and stained with anti I-A/I-E FITC and anti-CD11c PE Abs in PBS with 1% FBS. Slides were mounted with coverslips using a 4',6'-diamino-2-phenylindole dilactate containing mounting medium (Sigma-Aldrich). Cell images were captured using an LSM 510 META confocal microscope and analyzed with LSM 510 software (both from Zeiss).

Ag-specific T cell proliferation

DO11.10 T cells were sorted and labeled with CFSE (Molecular Probes), as described (21). Briefly, CD4+ DO11.10 T cells were isolated from spleen mononuclear cells by AutoMACS using anti-CD4-coated MACS beads (Miltenyi Biotec). Purified CD4+ DO11.10 T cells were washed and resuspended at 1 x 107 cells/ml in PBS, and CFSE (5 µM) was added. After 15 min of incubation at 37°C, unbound dye was quenched by addition of an equal volume of FBS and then washed three times with ice-cold medium containing 10% FBS. For proliferation assays, lung- or bone marrow-derived DC (0.5 x 104/well), incubated with OVA323–339 peptide or with PBS as a control, were cultured with CFSE-labeled CD4+ DO11.10 T cells (2.5 x 104/well) in 200 µl of complete medium. Cells were harvested after 4 days of culture, and reduced CFSE fluorescence intensity indicating proliferation was determined by flow cytometry. Assays were performed in triplicate.

Statistical analysis

Groups were compared by Student’s t test. Values of p for significance were set at <0.05. All values are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RSV infection results in increased numbers of pulmonary CD11b+CD11c+ MHC IIhigh DC

We have recently reported sustained increases in numbers of conventional CD11c+ MHC II+ DC following RSV infection (10). To determine whether these DC are of myeloid or lymphoid origin, we assessed their phenotype in more detail. Although expression of CD4 and CD8 on pulmonary DC was hardly detectable both in naive and RSV-infected mice, the percentage of lung DC expressing the myeloid marker CD11b increased from 23.9 ± 4.5% in naive mice to 79.8 ± 3% (n = 12; p < 0.05) on day 8 after RSV infection (Fig. 1A). In parallel, the percentage of DC expressing high levels of MHC II was also increased with a maximum on day 12 and remained elevated until day 45 (Fig. 1B).


Figure 1
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  		FIGURE 1. MHC II and CD11b expression on lung DC. Lung cells were isolated from RSV-infected and naive mice, stained with fluorescent Abs, and analyzed by flow cytometry. A, Dot plots represent typical samples of lung cells stained for CD11c and CD11b from naive mice and mice on day 8 postinfection. Figures indicate percentages of lung cells expressing both CD11c and CD11b (right upper quadrant) or CD11c only (right lower quadrant). B, Dot plots represent typical samples of lung cells stained for CD11c and MHC II from naive mice and mice on days 12 and 45 after RSV infection. Figures indicate percentages of lung cells expressing CD11c and MHC II at intermediate (R1) and at high levels (R2). All results were obtained in three independent experiments; n = 12 mice per group.

 
Increased GM-CSF concentrations and up-regulation of Ki67 expression on CD11c+ lung cells during RSV infection

GM-CSF is a critical growth factor for the differentiation of myeloid DC. We therefore asked whether RSV infection induces increased levels of this cytokine in the lung, potentially enabling proliferation and differentiation of precursors into pulmonary DC. Analyzing supernatants of lung homogenates by ELISA at different time points after RSV infection, we observed that GM-CSF concentrations were elevated ~10-fold compared with noninfected controls 24 h after infection, and that they remained significantly increased on day 2 postinfection before returning to baseline on day 3 (Fig. 2A). This increase in lung GM-CSF levels after RSV infection was not due to contamination from HEp-2 cell cultures, because purified virus also induced marked increases in lung GM-CSF concentrations, while nonpurified UV-inactivated RSV did not (data not shown). To determine whether this early increase in GM-CSF is associated with proliferation of lung DC from precursors in addition to maturation, we determined the expression of Ki67, a nuclear proliferation marker (22), by flow cytometric analysis. Compared with naive animals, Ki67 expression was significantly increased in lung CD11c+ cells on day 1 after RSV infection (Fig. 2B), but not at later time points once numbers of lung DC had increased (data not shown). This suggests that proliferation of CD11c+ cells in the lung may contribute to the expansion of lung DC following RSV infection.


Figure 2
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  		FIGURE 2. Lung GM-CSF levels and Ki67 expression in CD11c+ lung cells. A, Lungs harvested from naive mice and on days 1, 2, 3, 4, and 7 after RSV infection were homogenized, and GM-CSF concentrations were determined by ELISA in cell-free supernatants. Data shown represent mean ± SEM from two independent experiments; n = 6 per group; significant difference **, p < 0.01 and *, p < 0.0.5. B, Lung cells isolated on day 1 after RSV infection and from naive mice were stained with Abs to CD11c and Ki67 and analyzed by flow cytometry. Dot plots represent typical results from two independent experiments (n = 6 per group). Figures indicate percentages of lung cells expressing both CD11c and Ki67.

 
Precursor cells resident in the naive lung can give rise to CD11b+ myeloid DC

To test the hypothesis that precursor cells resident in the lung may provide a local contribution to the expansion of pulmonary DC after RSV infection, we cultured isolated lung cells from noninfected mice in the presence of GM-CSF. During the first 3 days of culture, the majority of lung cells died, but the small population of surviving cells subsequently gave rise to substantial numbers of large cells. By the end of the 12-day culture period, total cell numbers were 2- to 3-fold higher than the numbers of lung cells seeded at the start of culture (2.1 ± 0.7 x 106 and 1.0 ± 0.1 x 106, respectively; n = 6; p < 0.05). Flow cytometric analysis of these resulting cells showed that they had a similar size and granularity compared with bone marrow-derived DC and that almost all cells expressed CD11c. A total of 22 ± 8% of these lung-derived cells also expressed high levels of MHC II (Fig. 3A). Coexpression of these markers on the surface of lung-derived cells was confirmed by confocal microscopy (Fig. 3B). Total numbers of CD11c+ MHC IIhigh cells increased 27-fold in GM-CSF culture from 1.6 ± 0.4 x 104 in seeded naive lung cells to 44 ± 16 x 104 in cultured cells (n = 12; p < 0.01). Further phenotypic analysis showed that while these lung-derived cells did not stain for CD4 or CD8, most of them expressed the myeloid marker CD11b (95 ± 5%). Stimulation of these cells with RSV resulted in increased expression of the costimulatory molecules CD40, CD80, and CD86, as has been observed in vivo in pulmonary DC after RSV infection and in vitro in RSV-infected bone marrow-derived DC (our unpublished data). To assess the functional capacity of lung-derived cells after GM-CSF culture, we next compared their Ag-presenting capacity to bone marrow-derived DC. Using Ag-specific proliferation assays, we found that CD4+ DO11.10 T cells proliferated equally after stimulation with chicken OVA-pulsed DC generated from lungs or bone marrow, indicating similar degrees of functional activity in the two different populations of APCs (Fig. 3C). Taken together, these results indicate that fully functional DC can be generated in vitro from precursor cells resident in naive murine lungs in response to GM-CSF stimulation. The finding that GM-CSF levels are significantly increased in lungs in response to RSV infection in vivo, and our previous observations that mature DC are expanded in the lung following RSV infection (10) suggest that the same may occur in vivo.


Figure 3
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  		FIGURE 3. Morphology, phenotype, and function of lung-derived DC. Isolated lung cells from naive mice were cultured for 12 days in 20 ng/ml GM-CSF. As comparison, bone marrow-derived DC were generated in the same manner. A, Lung- and bone marrow-derived DC were stained with Abs to CD11c and MHC II and analyzed by flow cytometry. Typical results are shown as dot plots. Figures indicate percentages of lung cells coexpressing CD11c and high levels of MHC II. B, Typical confocal microphotographs of cultured lung cells stained with Abs to CD11c and MHC II, counterstained with 4',6'-diamino-2-phenylindole dilactate, magnification: x400. C, Flow cytometric analysis of CFSE-stained OVA peptide323–339-specific CD4 T cells (DO11.10) after coculture without DC, bone marrow-derived DC (BM-DC), or lung-derived DC pulsed with OVA. Reduction in CFSE staining intensity represents T cell proliferation. Dot plots show typical results from three independent experiments (n = 6 per group).

 
CD11c+ MHC II– lung cells comprise a population of DC precursors

To define the cell population constituting lung-resident DC precursors, we separated CD11c+ from CD11c– lung cells before culture with GM-CSF for 12 days. Using positive selection by AutoMACS of cells labeled with anti-CD11c-coated MACS beads, purity of CD11c+ cells was >85% and CD11c– cells were >98% pure. Hardly any CD11c– lung cells survived in culture except for few cells of heterogeneous morphology. In contrast, culture of CD11c+ cells yielded large numbers of CD11c+ MHC II+ cells (Fig. 4, A and B).


Figure 4
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  		FIGURE 4. CD11c+ MHC II– lung cells proliferate and differentiate into DC in culture with GM-CSF. A, CD11c+ cells, CD11c– cells, CD11c+ MHC II+, and CD11c+ MHC II– lung cells isolated by MACS and FACS sorting were cultured with GM-CSF for 12 days. The phenotype of cultured cells was assessed by flow cytometry. A, Dot plots show the frequency of cells expressing CD11c and MHC II after culture of CD11c–, CD11c+, and CD11c+ MHC II– lung cells compared with an isotype control stain. B, Total cell numbers were enumerated after culture in the same experiments and are indicated in the graph. At the start of culture, 1 x 106 cells/well were seeded in 6-well plates. Data represent mean ± SEM from three individual experiments; n = 6 per group. C, Typical microphotograph of lung CD11c+ MHC II– DC precursor cells after FACS sorting, cytospin centrifugation, and staining with Quickdiff (Reagena); magnification, x200.

 
Based on these findings and using FACS sorting, we separated CD11c+ lung cells further into MHC II+ and MHC II– populations achieving a purity of 95 and 98%, respectively. Although culture of CD11c+ MHC II+ cells, which consist of immature and mature lung DC, hardly yielded any viable cells after 12 days, large numbers of CD11c+ MHC II+ DC were generated from cultures of CD11c+ MHC II– cells (Fig. 4, A and B). Interestingly, in addition to the newly generated DC, CD11c+ MHC II– cells were also still present after 12 days of culture, suggesting either that lung CD11c+ MHC II– cells are a heterogeneous population of cells including both DC precursors and cells that do not differentiate into DC in response to GM-CSF, or that CD11c+ MHC II– cells have stem cell-like properties, being capable of self regeneration as well as providing the population of differentiated cells. Flow cytometric analysis of lung CD11c+ MHC II– DC precursor cells revealed that only ~10% of these cells expressed CD11b, while the costimulatory molecules CD40, CD80, and CD86 as well as CD4 and CD8a were absent. In addition, lineage markers for T cells (CD3), B cells (CD19), and NK cells (DX5), the pre-B cell marker CD45R (B220) and CD31 (PECAM-1), a molecule expressed on platelets, monocytes, granulocytes, and B cells, were undetectable. We also directly assessed the morphology of freshly sorted CD11c+ MHC II– cells from naive lungs. These DC precursors are mononuclear cells, larger than resting lymphocytes with little cytoplasm and large nuclei, as often seen in nonproliferating cells (Fig. 4C).

Prolonged depletion of lung DC precursors after RSV infection

Having established that lung-resident DC precursor cells can give rise to myeloid DC similar to those found in the lung following RSV infection, we next asked whether DC precursors in the lung are depleted during RSV infection and whether DC can still be generated from infected lungs. Importantly, we found that the percentage of CD11c+ MHC II– precursor cells in lung cells was reduced 8-fold on days 12 (data not shown) and 21 after RSV infection compared with naive controls (Fig. 5A). Absolute numbers of these DC precursors were reduced 5-fold from 6.50 ± 0.54 x 105 in naive lungs to 1.22 ± 0.16 x 105 (n = 12; p < 0.01) after RSV infection. Corresponding to this finding, newly generated DC were absent or significantly reduced in numbers if lung cells from RSV-infected mice were cultured in the presence of GM-CSF (Fig. 5B). Comparing different time points after RSV infection, we found that on days 12 and 21 postinfection DC could not be generated, while on days 4 and 45 postinfection numbers of DC generated in culture were significantly reduced compared with naive lungs. These data indicate that respiratory virus infection leads to depletion of the lung DC precursor cell population, and inability to expand mature DC from lung cells ex vivo in response to GM-CSF.


Figure 5
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  		FIGURE 5. Generation of lung DC following RSV infection. A, Lung cells from mice 21 days after RSV infection or naive controls were stained with Abs to CD11c and MHC II. R1 gates in representative dot plots from three independent experiments (n = 6) highlight CD11c+ MHC II– cells. Percentages of CD11c+ MHC II– cells of total lung cells are indicated. B, Lung cells harvested from mice on days 4, 12, 21, and 45 after RSV infection and from naive mice, as well as bone marrow cells were cultured with GM-CSF for 12 days. Means ± SEM of total cell numbers after culture are depicted in the graph. Significant differences vs naive lung: **, p < 0.01; *, p < 0.05; n = 6, from three independent experiments.

 
RSV infection prevents a secondary expansion of pulmonary DC in response to influenza infection and LPS exposure

In addition to the in vitro studies outlined above, we finally asked whether RSV infection also reduces or prevents expansion of lung DC in response to a secondary viral infection or bacterial stimulation in vivo. To this end, mice were sham infected or infected with RSV, and on day 21 postinfection they were treated with LPS intranasally or infected with influenza A virus. In sham-infected mice, both treatments resulted in increases in numbers of pulmonary DC on days 8 and 15 after bacterial product treatment/secondary infection, respectively. If RSV infection preceded the bacterial product treatment/secondary infection, no significant increases in pulmonary DC numbers beyond those initially induced by the RSV infection were noted (Fig. 6).


Figure 6
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  		FIGURE 6. Expansion of lung DC numbers in vivo in response to secondary stimuli after RSV infection. Mice were infected with RSV or sham infected. On day 21 of RSV infection, mice were infected with influenza A virus (A) or LPS was administered (B). On days 15 or 8, respectively, after administration of these secondary stimuli, lung cells were harvested and expression of CD11c and MHC II was analyzed by flow cytometry. The graphs illustrate absolute numbers of lung DC in each group. Data shown are representative of three independent experiments; n = 12 per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DC as professional APCs are present throughout the respiratory tract and play an important role in the initiation of immune responses to environmental Ags and pathogens encountered in the lung (1). In healthy lungs, the majority of DC are immature and mediate immune tolerance to innocuous Ags (2). In contrast, marked increases in numbers of mature pulmonary DC have been observed following allergen challenge in allergen-sensitized mice (23) and after viral (4, 10, 11, 12) and bacterial infections (24) of the lung. Following RSV infection, conventional CD11c+ lung DC display increased expression of MHC class II and costimulatory molecules, associated with an increased capacity to present Ag to naive T cells (10). Using a higher inoculum of RSV, we have observed more sustained increases in MHC II expression in the present study than previously published (10), lasting at least until day 45 postinfection. In addition, we show that the vast majority of lung DC express the myeloid marker CD11b after RSV infection, but neither CD4 nor CD8. In naive lungs, most DC do not express CD11b and are thus thought to be of lymphoid origin (25, 26). According to current understanding, CD11b– and CD11b+ DC are individual populations most likely originating from different precursors. This suggests that the mature CD11b+ DC after RSV infection cannot originate from immature CD11b– DC found predominantly in naive lungs. The source of this RSV-induced population has not been defined.

It is generally assumed that conventional DC are recruited from the bone marrow and migrate to the lung as monocytic cells. van Rijt et al. (23) have detected increases in CD31high Ly-6c – bone marrow progenitors of DC following allergen challenge in sensitized mice. In addition, DC precursors have been described in lymphoid organs such as spleen and thymus (17, 18). In the lung, as a nonlymphoid organ, MHC II– cells bearing the DC marker OX 62 have been observed in neonatal rats and were thought to be precursors developing into mature lung DC (27). Furthermore, overexpression of GM-CSF, a potent DC growth factor, locally in the lung results in marked expansion of pulmonary DC (28, 29, 30). This phenomenon may not be sufficiently explained by DC recruitment from the bone marrow, especially because GM-CSF is not thought to be chemotactic for monocytes or DC. Importantly, RSV infection also results in increases in GM-CSF production in infected epithelial cells in vitro (31) and in lungs the first 2 days after RSV infection in vivo, as we demonstrate in this study. An initial increase in GM-CSF levels in the lung most likely stimulates proliferation of DC precursors and induces their differentiation into mature DC. This notion is supported by increased detection of the nuclear proliferation marker Ki67 in CD11c+ lung cells early after RSV infection, indicating proliferation within this population. Once differentiated, bone marrow-derived myeloid DC can survive in culture for prolonged periods in the presence of low levels of GM-CSF, as we have observed (data not shown). This may also apply to DC in the lung, where additional factors may be involved in long-term DC survival.

Considering these observations, we hypothesized that DC precursors resident in the naive lung contribute to the expansion of pulmonary DC after RSV infection. To test this hypothesis, we cultured isolated lung cells in the presence of GM-CSF, according to protocols previously used to derive DC from bone marrow precursors (20). Circulating blood DC precursors were removed from lungs before harvest by gentle perfusion of the pulmonary vasculature until lungs blanched. Using lung cells from naive or sham-infected mice, substantial numbers of DC were generated. Expressing CD11c, MHC class II, and CD11b, these cells were phenotypically similar to pulmonary DC after RSV infection, and they were capable of inducing Ag-specific T cell proliferation as efficiently as bone marrow-derived DC. Interestingly, no DC expressing the lymphoid markers CD4 or CD8, nor plasmacytoid DC were detected in these cultures, suggesting that lung-resident DC precursors are committed to develop exclusively into DC with a myeloid phenotype. Increases in total cell numbers and in numbers of DC after culture clearly demonstrate that the precursors proliferated to generate DC and did not only differentiate. In contrast to our findings, Swanson et al. (32) recently reported that they were not able to generate DC from mononuclear lung cells using GM-CSF/IL-4 culture. This is probably due to pretreatment of their mice with FMS-like tyrosine kinase 3 ligand, a DC growth factor, which induces expansion of pulmonary myeloid DC (26) and may have resulted in depletion of DC precursors in the lung.

To define the phenotype of DC precursors in the lung, we sorted lung cells according to their expression of CD11c and MHC II and cultured the resulting cell populations separately. Only CD11c+ cells, which did not express MHC II, were capable of generating DC in GM-CSF culture, providing direct evidence that there are DC precursors among MHC II– lung cells, as previously postulated (27). Lacking expression of MHC II and costimulatory molecules, these precursor cells do not have the characteristics of APCs, and in the absence of lineage marker expression, they are unlikely to derive from T cells, B cells, NK cells, monocytes, or granulocytes. Despite a low frequency of CD11b expression only, these cells could be of myeloid origin. It seems likely that DC precursors from the blood, which have the same phenotype, are recruited into the lung. This notion is supported by the observation that leukocytes isolated from the lung vasculature contain comparatively high numbers of precursor cells from which DC can be generated (33). The DC precursors described in this work seem to reside in the lung because DC numbers generated from lungs with and without perfusion of the pulmonary vasculature did not differ significantly (data not shown). Functionally, CD11c+ MHC II– cells are a heterogeneous population, as some cells retained their phenotype during GM-CSF culture and did not differentiate into DC.

A distinct population of CD11c+ MHC II– cells is found in the lungs of naive animals. After RSV infection, this population is significantly diminished, suggesting a reduction in numbers of DC precursors. Culture of lung cells with GM-CSF after RSV infection supports this notion. From days 12 to 21 after RSV infection, it was not possible to generate DC from lung cells in GM-CSF culture, indicating that lung-resident DC precursors had been depleted. On days 4 and 45 after RSV infection, DC could be generated from lung cells, but at significantly lower numbers than from naive lungs. This is most likely due to the fact that the expansion of pulmonary DC is only beginning on day 4 of infection; thus, considerable numbers of unused DC precursors still remain in the lung; on day 45, lung DC precursors start to recover, and responses return. These results suggest that DC precursors in the lung are depleted during virus-induced expansion of lung DC. Inhibition of DC precursors, rather than their depletion, cannot be excluded entirely by our studies, but this seems less likely. To test the concept of RSV-induced DC precursor depletion in vivo, we challenged mice with influenza A virus or LPS on day 21 after RSV infection. In sham-infected mice, these stimuli resulted in marked expansion of pulmonary DC. In contrast, no increases in numbers of lung DC were detected in response to influenza A virus infection or LPS application in mice previously infected with RSV, suggesting that DC precursors were not available in the lung for a secondary expansion of lung DC. If pulmonary DC precursors are recruited from the pulmonary circulation, these findings indicate that recruitment does not happen acutely after an insult, but rather that DC precursors are recruited slowly over a prolonged period. These findings may have important implications for immune responses in the lung following viral infections. Respiratory viral infections have been associated with an increased risk for secondary bacterial infection of the respiratory tract, including pneumonia (27) and otitis media (34). We speculate that large increases in numbers of pulmonary DC are required for effective antibacterial immune responses, exceeding the numbers of DC still present in the lung after RSV infection. A role for lung DC in antibacterial responses is supported by the observation that increased numbers of pulmonary DC, induced by infection with a transgenic RSV strain expressing GM-CSF, resulted in increased titers of specific Abs to this virus (29). Specific Abs are of critical importance in antibacterial immune responses, and their production may well be impaired if numbers of lung DC presenting bacterial Ags are inadequate.

In conclusion, we demonstrate for the first time that naive lungs contain CD11c+ MHC II– precursor cells committed to proliferation and differentiation into fully functional myeloid DC. This is the first demonstration of precursors of DC in a peripheral organ. After RSV infection, these precursors give rise to increased numbers of pulmonary DC and are themselves depleted in the process. Thus, secondary expansions of pulmonary DC in response to viral or bacterial stimuli are inhibited for a prolonged period of time after RSV infection.


   Acknowledgments
 
We thank P. Openshaw for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 senior fellowship (Grant 067454) from the Wellcome Trust (to J.S.). Back

2 Address correspondence and reprint requests to Dr. Jürgen Schwarze, Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, Norfolk Place, London, W2 1PG, U.K. E-mail address: j.schwarze{at}ic.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; RSV, respiratory syncytial virus; MHC II, MHC class II. Back

Received for publication October 20, 2005. Accepted for publication May 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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