Introduction

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Our understanding of the pathogenesis of asthma has evolved substantially over the past decade (1). Endobronchial biopsy and bronchoalveolar lavage (BAL) studies have revealed chronic eosinophilic mucosal inflammation, even in the airways of patients with mild persistent asthma (2, 3). These and additional studies performed in animal models of the disease have suggested that inflammation results from the induction and persistence of a T-helper (Th)-2 lymphocyte response to inhaled antigen (4, 5). Indeed, Th2 cytokines are implicated in the pathogenesis of numerous aspects of allergic airway inflammation; interleukin (IL)-4 (and IL-13 in humans) promotes B-cell immunoglobulin (Ig) class switching to IgE, and IL-5 is important for the growth, differentiation, and maturation of tissue eosinophils (6). Consequently, antigen-induced eosinophilic airway inflammation and bronchial hyperreactivity do not develop in IL-4 or IL-5 knockout mice or mice depleted of CD4⁺ lymphocytes (7). Despite this essential role for Th2 lymphocytes in the pathogenesis of asthma, few studies have addressed exactly how T cells are activated in the airways. In the two-signal hypothesis, naive T cells respond to antigen presented in the context of major histocompatibility (MHC) molecules (signal 1) together with costimulatory molecules (signal 2) on the surface of professional antigen-presenting cells (APC), such as dendritic cells (DC), macrophages, and B cells (10, 11). Recent studies have emphasized the importance of the DC as the most potent APC for the induction of a primary immune response to exogenous antigen (11). The ability of T cells to respond vigorously to DC is largely attributable to the high expression of costimulatory ligands B7-1, B7-2, and intercellular adhesion molecule-1 on the surface of DC (14). A highly developed network of DC is present in the airway mucosa, and this network is thought to be involved in the capture and presentation of inhaled antigen, leading to the sensitization of naive T cells in the lymph nodes draining the lung (15). In addition to the critical role for DC in sensitization to inhaled antigen, we have recently found that airway DC are essential for the acquisition of effector function in memory Th2 cells and the subsequent development of eosinophilic airway inflammation in a mouse model of asthma (19). These findings, together with the knowledge that alveolar macrophages downregulate responses to inhaled antigen (20), suggest that DC are the most relevant APC for the stimulation of Th2 cells in the pathogenesis of asthma.

It was recently shown that the airways of patients with atopic asthma contain increased numbers of CD1a⁺ airway DC and that the density of the network was decreased by treatment with inhaled corticosteroids, suggesting that modulation of the DC network is a mechanism of action of these drugs (21, 22). The mechanisms by which DC increase in the airways of these patients are largely unexplored, but may involve increased chemotaxis of DC into the inflammatory site due to expression of proinflammatory cytokines and chemokines (eotaxin, macrophage inflammatory protein [MIP]-3, monocyte chemotactic protein [MCP]-1, MCP-3, etc.) in asthmatic airways. As airway DC are derived from a myeloid-lineage precursor in the bone marrow (19, 23), another contributing yet unexplored factor could be an increased production of these bone-marrow progenitors, caused by an increase in DC growth-factor secretion or sensitivity. The bone marrow has been implicated in the pathogenesis of allergic inflammation; an increase in CD34⁺ bone-marrow progenitors for eosinophils and basophils has been observed in animal models of asthma (24, 25) and in atopic patients during the allergen exposure season (26).

We have previously described a rat model that mimics many of the features of human asthma, such as eosinophilic airway inflammation, bronchial hyperreactivity, and production of antigen-specific IgE (27), and that allows us to define clearly the role of a particular cell or mediator in various aspects of the disease. In this report we show that ovalbumin (OVA)-induced eosinophilic airway inflammation in sensitized Brown Norway (BN) rats is accompanied by upregulation of the airway DC network; and we relate the observed changes to those in other inflammatory cells, such as T cells, eosinophils, and monocytes. Further, we provide evidence that the upregulation of the local airway DC network is accompanied by marked effects on the DC progenitor in the bone marrow.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

All experiments were performed in inbred, pathogen-free, 12-wk-old male BN rats (200 to 250 g; Harlan, Zeist, The Netherlands). To minimize the baseline airway inflammation frequently observed in BN rats housed on wood shavings (17), animals were housed in sterile microisolator units on low-dust shredded-paper bedding (RS Systems, Finedon, UK) throughout the experiments. Fisher-344 rats served as donors for allogeneic T lymphocytes in mixed leukocyte reactions (MLRs).

Sensitization and Allergen Challenge of BN Rats

On the first day of the experiment (Day 0), groups of rats (n = 6-10) were immunized by intraperitoneal injection of 10 µg OVA (Grade V; Sigma, St. Louis, MO) emulsified in 0.5 ml heat-killed Bordetella pertussis (BP) in Al(OH)₃ adjuvant (Vaxicoq; Pasteur-Merieux, Lyon, France), as described (27, 28). Control animals were sham-sensitized to phosphate-buffered saline (PBS) (GIBCO, Merelbeke, Belgium) in BP adjuvant.

From Day 10 to Day 17 after the sensitization, animals were exposed daily (0, 1, 3, or 7 d) for 30 min to an aerosol of 1% OVA in PBS. Groups of awake rats were placed in an exposure chamber connected to the outlet of an ultrasonic nebulizer that delivers an aerosol of particles with a mean diameter of 3.5 µm (Microvernebler R80; Microvernebler, Zürich, Switzerland). Control animals were exposed to an aerosol of PBS.

BAL

Twenty-four hours after the last aerosol exposure, rats were killed by intraperitoneal injection of pentobarbital sodium (60 mg/kg body weight; Abbott Laboratories, Louvain la Neuve, Belgium) followed by exsanguination from the iliac vessels. The trachea was surgically exposed and cannulated, and BAL was performed with 6 × 6 ml of Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution supplemented with 0.05 mM sodium ethylenediaminetetraacetic acid, as described (27). The BAL fluid (BALF) was centrifuged (10 min, 4°C, 700 × g) and resuspended in Tris-ammoniumchloride lysis buffer solution (2.06 g/liter Tris and 7.47 g/liter ammoniumchloride, pH 7.2) for 3 min at room temperature. After washing, cells were counted in a hemocytometer (Coulter Counter ZF; Coulter, Hertfordshire, UK). Differential cell counts were performed on cytospin preparations (Cytospin 2; Shandon Ltd., Cheshire, UK) stained with May-Grünwald-Giemsa by classification of 300 cells on standard morphologic criteria. Flow cytometry labeling and analysis were performed on aliquots of 1 × 10⁶ cells.

Airway Histology

After BAL was performed, fixative (4% paraformaldehyde in PBS) was gently infused through the lavage catheter by a continuous-release pump under pressure- and volume-controlled conditions. The lungs were resected and fixed for an additional 4 h. After routine paraffin embedding, 4-µm sections were stained with May-Grünwald- Giemsa and hematoxylin-eosin and examined by light microscopy for histologic changes.

Flow Cytometry

The antirat monoclonal antibodies (mAbs) OX62 (mouse IgG₁, recognizing an _E7 integrin on DC and putative T cells [30]), OX19-fluorescein isothiocyanate (FITC) and OX-19-bi (mouse IgG₁, anti-CD5, pan-T-cell marker), OX39-FITC (mouse IgG₁, anti-IL2-receptor), W3/25-FITC (mouse IgG₁, anti-CD4), OX6-phycoerythrin (PE) and OX6-bi (mouse IgG₁, anti-MHC class II [Ia] molecules), OX8-PE (mouse IgG₁, anti-CD8), R73-PE (mouse IgG₁, anti--T-cell receptor [TCR]), and OX33-PE (mouse IgG₁, -CD45RA on B cells) were obtained from Serotec (Kidlington, UK). mAbs G4.18-PE (mouse IgG₃, anti-CD3, a molecule of the TCR) and V65-PE (mouse IgG₁, anti- -TCR) were from PharMingen (San Diego, CA). Isotypic controls conjugated to FITC or PE were from PharMingen (mouse IgG₁ and IgG₂) and Cedarlane (Hornby, ON, Canada) (IgG₃). Unconjugated antibodies (OX62 and unlabeled isotype IgG₁ control) were detected with FITC-conjugated F(ab')₂ fragment rat-antimouse IgG (RAM-FITC) (Jackson Immunoresearch Laboratories, Westgrove, PA), and biotin-linked antibodies (OX6 and OX19) were detected using streptavidin-PECy5 (Tricolor; Burlingame, Caltag, CA).

For routine analysis, BAL cells were washed twice in staining buffer (PBS, 1% bovine serum albumin [BSA], and 0.02% sodium azide) before aliquotting 1 × 10⁶ cells in flexible 96-well plates (ICN, Costa Mesa, CA). All reactions were performed in staining buffer at  1 µg mAb/10⁶ cells for 30 min on ice. The first layer consisted of primary unconjugated or FITC-conjugated mAbs for 30 min on ice, followed by secondary labeling with RAM-FITC, if appropriate. After blocking the residual binding of RAM-FITC with normal mouse serum, a second layer of PE- or biotin-labeled mouse antibody was added. The final layer was to add streptavidin-PECy5. Three-color fluorescence intensity analysis was performed on 5 × 10⁴ cells on a FACS-Vantage flow cytometer using Cellquest software (Becton Dickinson, Mountain View, CA).

For some sorting experiments on BALF cells, DC were pre-enriched by panning adherent alveolar macrophages on plastic Petri dishes (Falcon no. 1029; Becton Dickinson, Erembodegem, Belgium) for 1 h, followed by centrifugation of the nonadherent population over a metrizamide gradient (Sigma; 14.5% wt/vol in RPMI 1640 at 600 × g for 20 min at room temperature). Sorted cells were collected in 30% fetal calf serum (FCS) in culture medium (CM).

Measurement of OVA-Specific IgE

Immediately before exsanguination, blood samples were taken from the sinus cavernosus for measurement of OVA-specific IgE by isotype-specific enzyme-linked immunosorbent assay (ELISA). OVA grade V (Sigma) was coated overnight onto 96-well flat-bottomed microtest plates (Nunc, Roskilde, Denmark) at a concentration of 50 µg/ml in PBS, followed by incubation with 1% BSA. Serial dilutions of rat serum were applied for 1 h at 37°C, followed by mouse-antirat IgE (MARE; H. Bazin, Experimental Immunology Unit, UCL Brussels, Belgium) for 2 h at 37°C. Bound MARE was detected by labeling with secondary horseradish peroxidase-conjugated goat antimouse Igs (1 µg/ml) (DAKO, Glostrup, Denmark). Diaminobenzidine tetrahydrochloride substrate was then applied and plates were developed for 30 min at room temperature. A serum pool of OVA-sensitized rats was used as an internal laboratory standard. Units were arbitrarily defined from optical density (405 nm) values from a one-one-hundredth dilution of this pool.

Isolation and Culture of Bone Marrow DC

On Day 13 of the experiment, bone-marrow cultures were initiated from various groups of rats according to a protocol for the generation of DC, described in detail by others (31). Briefly, bone-marrow cells were isolated by flushing the long bones with ice-cold CM (RPMI 1640 medium supplemented with 2 mM L-glutamine, 15 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid buffer, 10 µg/ml streptomycin, 100 U/ml penicillin, and 5% FCS; all from GIBCO), and were depleted of Fc-receptor-positive cells by panning on serum-coated Petri dishes (5% normal human serum and 10% normal goat serum in PBS) for 1 h. Nonadherent cells were removed by gentle pipetting and cultured for 6 d in CM containing 0.5 µg/ml N--monomethyl-L-arginine (Calbiochem, La Jolla, CA), 50 µM 2-mercaptoethanol, and 0.4 ng/ml r-murine granulocyte macrophage colony-stimulating factor (GM-CSF) (R&D Systems, Abingdon, UK) in culture flasks coated with porcine skin gelatin (Sigma). After 6 d of culture, nonadherent cells were collected and depleted of Fc-receptor-positive cells by serum panning. The Fc-receptor-negative, nonadherent DC were density-enriched by centrifugation over a metrizamide gradient. Aliquots of cells were stained with the mAb OX62, which stains the majority of bone marrow-derived DC (31), and with the anti-Ia mAb OX-6. The number of DC in the culture was calculated as the percentage of OX62⁺OX6⁺-positive cells multiplied by the yield of total cells in the culture.

Isolation of Spleen Cells

Spleens were collected from unimmunized rats and mechanically dispersed by pressing through an 80-mesh metal sieve using a syringe plunger. Red blood cells were lysed by resuspending in Tris-ammoniumchloride lysis buffer solution. For the purification of spleen T cells, the cells were further depleted of adherent APC and B cells by adherence to nylon wool columns, as described elsewhere (32). The enriched fraction contained > 90% CD3⁺ T cells.

MLR

For MLR experiments, putative lavage DC and unfractionated fresh spleen cells from BN rats were treated with 50 µg/ml of Mitomycin C (Sigma). Increasing numbers of these cells were added as stimulators to 2 × 10⁵ allogeneic nylon wool-enriched Fisher-344 spleen T cells in round-bottom 96-well microtest plates (Falcon no. 3077; Becton Dickinson, Erembodegem, Belgium) in 200 µl CM supplemented with 50 µM 2-mercaptoethanol. Stimulator-to- responder ratios ranged from 2.5/1 to 1/820 for unfractionated spleen-cell stimulators and from 1/7 to 1/14,000 for OX62-enriched stimulators. Each well was labeled with 0.5 µCi/ml final concentration of ³H-thymidine (Amersham, Buckinghamshire, UK) for a 12-h period, 5 d after starting the MLR. Cells were automatically harvested (Inotech, Dottikon, Switzerland) and thymidine incorporation was determined using an automated liquid scintillation counter (1450 Microbeta Plus; Wallach, Turku, Finland). Results are expressed as mean counts per minute (c.p.m.) from triplicate wells.

Statistical Analysis

All results are expressed as means ± standard error of the mean (SEM). Comparison of means of different groups was conducted with a Mann-Whitney U test (33) using the Systat statistical package (Spreadware Statistics, Palm Desert, CA). Differences were considered significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of OVA Exposure on Cellular Composition of BALF in Sensitized BN Rats

OVA-sensitized and sham-sensitized rats were exposed to three daily aerosols of OVA aerosol from Days 10 through 13 after sensitization. The cellular composition of BALF was measured as an indicator of airway inflammation (Figure 1a). The total number of BAL cells was increased significantly in OVA-exposed compared with saline-exposed sensitized rats (P = 0.01) and compared with OVA- exposed sham-sensitized rats (P = 0.008). Differential cell counting on cytospin preparations revealed that the majority of cells in the OVA-sensitized/PBS-exposed group and the sham-sensitized/OVA-exposed group consisted of alveolar macrophages and some lymphocytes. In the sensitized/OVA-exposed group, significantly higher numbers of neutrophils, eosinophils, and T lymphocytes were found compared with both control groups (respectively, P = 0.005, 0.003, and 0.003 for OVA/PBS control group; and P = 0.005, 0.003, and 0.005 for PBS/OVA control group).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Effect of OVA exposure on the cellular composition of BALF. On Day 0, BN rats (n = 6-10) were immunized with OVA in BP adjuvant or sham sensitized (PBS) as described in MATERIALS AND METHODS. On Days 10 to 12, rats were exposed to 30 min daily OVA aerosol or PBS. At 24 h after the last exposure (Day 13), BAL was performed. (a) Differential cell counts on BALF cells based on Giemsa staining. (b) Total T lymphocytes (CD5+) and subsets (CD4+ and CD8+) in BALF as determined by flow cytometry. Results are expressed as means ± SEM from six to 10 rats.

Five-parameter flow cytometric analysis was performed to reveal the phenotype of T lymphocytes (gated on scatter characteristics and the pan-T cell marker CD5) present in BALF and for comparison with spleen cells. The majority of lavage CD5⁺ T cells were CD4-positive (Figure 1b). These T lymphocytes had an antigen-experienced phenotype as revealed by positive staining for the IL-2 receptor CD25 (54.3 ± 2.9%), not shown. Further, 94.9 ± 1.7% of lavage T cells (as detected with the pan-T cell marker CD5) expressed -TCR, 2.1 ± 0.7% expressed -TCR, and almost all (98.4 ± 0.9%) expressed CD3 in the OVA-immunized/OVA-exposed group at Day 13 of the response (not shown). We consistently found expression of the _E7-integrin on 32.9 ± 4.8% of CD3⁺CD5⁺ T cells in BALF at Day 13 of the response, as compared with 4.9 ± 0.3% of spleen T cells (P = 0.02) (Figure 2). Of the CD5⁺ T cells expressing the -TCR chain, 25.6 ± 4.8% expressed the _E7-integrin in BALF as compared with 5.2 ± 0.3% in the spleen (P = 0.02). Of those CD5⁺ T cells expressing the -TCR, 36.6 ± 4.7% expressed the _E7-integrin in BALF as compared with 9.4 ± 1.5% in the spleen (P = 0.03).

View larger version (30K): [in this window] [in a new window]   Figure 2.   Expression pattern of OX-62 on lavage and spleen T cells. T cells were gated on forward- and sideward-scatter characteristics and for staining with CD5-PECy5 antibodies. Lavage cells (top row) or spleen cells (lower row) were first labeled with isotype control mouse IgG1 (left) or OX62 (middle and right), followed by secondary antimouse FITC. Cells were also stained with CD5-bi and CD3-PE (left and middle) or -TCR-PE (right) followed by SA-PECy5. Dot plots are representative of all animals in the OVA-immunized group exposed to three aerosols of OVA.

Effect of OVA Exposure on Airway Histology in Sensitized BN Rats

Histologic analysis of the lungs of sensitized rats revealed that a 3-d OVA-aerosol exposure led to the development of peribronchial and perivascular inflammatory lesions characterized by a predominance of eosinophils, mononuclear cells, and occasional giant cells, as previously reported (27). These changes were absent from sham-sensitized/ OVA-exposed and from OVA-sensitized/PBS-exposed animals (data not shown).

Effect of OVA Exposure on OVA-Specific Serum IgE Levels in Sensitized BN Rats

In view of the association of allergic disorders with the presence of detectable levels of IgE, we measured serum OVA-specific IgE by ELISA (Table 1). Ten days after immunization of BN rats with 10 µg of OVA in BP/AlOH₃ adjuvant the level of OVA IgE was 496 ± 162 U/ml, and this level increased to 1,750 ± 384 U/ml 13 d after immunization (P = 0.03). Aerosol exposure boosted the production of IgE considerably, as the level of IgE was 2.5 times higher in animals exposed to three repeated OVA exposures compared with PBS exposures (P = 0.003). Sham-sensitized animals had very low levels of OVA IgE, even after three repeated OVA exposures (2.5 ± 0.9 U/ml).

Identification of DC in BALF

In early experiments we used the marker OX-62 to detect DC in BALF, because this marker is specific for veiled DC in the afferent lymph of the rat and is expressed on DC of the airways (30, 34). However, it soon became clear that OX-62 was also expressed on other cells in BALF, such as T cells. Therefore, five-parameter flow cytometry was performed to determine more clearly the presence of DC. Cells were first gated based on forward-scatter and side angle-scatter profile to exclude eosinophils and dead and clustering cells. The FL2 channel, detecting emission in the 570 ± 20-nm spectral region, was used as a "dump" channel to gate out autofluorescent alveolar macrophages and remaining eosinophils, T lymphocytes (labeled with anti-CD3-PE), and B lymphocytes (labeled with CD45RA-PE). By gating on cells with an intermediate forward light scatter and low FL-2 signal, a cluster positive for both the DC marker OX-62-FITC and the MHC class II marker OX-6-PECy5 was readily observed (Figure 3). This population of cells was recovered in the nonadherent fraction after panning on serum-coated plates and in the low-density fraction after submitting fresh BALF to metrizamide density centrifugation (not shown). Cytospin preparations of freshly isolated, low-autofluorescent CD3CD45RA OX62⁺OX6⁺ cells sorted by fluorescence-activated cell sorting revealed numerous long surface extensions or veils by phase-contrast microscopy and a typical indented or cloverleaf-shaped nucleus (Figure 4). Functionally, these putative DC stimulated allogeneic T cells at least 1,000 times as efficiently as control unfractionated spleen-cell stimulators in a primary MLR using Fisher rat T lymphocytes as responders (Figure 5). On the basis of marker studies, morphology, and functional assays, we assume that the OX62⁺OX6⁺ population are in fact DC and we will refer to this population as such.

View larger version (31K): [in this window] [in a new window]   Figure 3.   Characterization of DC in BALF. High-autofluorescent macrophages, eosinophils, and T and B lymphocytes (stained with CD3-PE and CD45R-PE) were gated out of the FL-2 "dump" channel (a). The low-autofluorescent lineage population contained a population of OX-62+OX-6+ putative DC. (b) BALF of actively sensitized and exposed (OVA/OVA) and (c) PBS-exposed (OVA/PBS) group.

View larger version (68K): [in this window] [in a new window]   Figure 4.   Morphology of low-autofluorescent, CD3, CD45RA, OX-62+, and OX-6+ cells. (a) Phase-contrast microscopy of freshly sorted cells (×400). Numerous cell extensions or veils can be seen. (b) Giemsa staining.

View larger version (23K): [in this window] [in a new window]   Figure 5.   Function of sorted low-autofluorescent, CD3, CD45RA, OX-62+, and OX-6+ cells in MLR. These cells were sorted from the BALF of a group of rats that were actively sensitized to OVA and exposed on Days 10 through 12 to OVA aerosol. Increasing numbers of freshly sorted BN-stimulators were added to 2 × 105 allogeneic T-cell responders. As a control, unfractionated spleen cells were used as stimulators. Results are expressed as mean cpm from triplicate wells, representative of three experiments.

Effect of OVA Exposure on the Number of DC in BALF of Sensitized Rats

In actively sensitized rats, the number of DC recovered in BALF was significantly higher after a 3-d OVA exposure compared with a PBS exposure (P = 0.003) (Figure 6). The increase was caused by both a percentage increase (0.53 ± 0.09% versus 0.056 ± 0.02%, P = 0.001) and an increase in the total amount of recovered BAL cells. Sham-sensitized animals did not show an increase in DC numbers in BALF after OVA exposure. The kinetics of increase in DC numbers were followed in the actively sensitized and OVA-exposed group (Figure 7a). Twenty-four hours after a single challenge with OVA aerosol (Day 11), an increase in the number of DC was observed compared with animals that were not challenged (Day 10) (P = 0.04). A maximum was reached after 3 d of OVA aerosol. This increase in DC numbers reflected a general increase in other inflammatory cells (T cells and eosinophils) recovered from lavage (Figure 7b).

View larger version (13K): [in this window] [in a new window]   Figure 6.   Effect of OVA aerosol on the number of BALF DC. Rats were immunized at Day 0 with OVA in BP adjuvant or sham-sensitized (PBS) and exposed to OVA or PBS aerosol from Days 10 to 12. BAL was performed 24 h after the last aerosol exposure. DC were identified as low-density, low-autofluorescent, CD3, CD45RA, OX-6+, OX-62+ cells. Groups are coded as immunization/exposure. Results are expressed as means ± SEM from eight to 10 rats.

View larger version (19K): [in this window] [in a new window]   Figure 7.   Kinetics of increase in DC (a) and T cells (b) after exposure to OVA aerosol in sensitized rats. Rats were immunized at Day 0 with OVA in BP adjuvant and exposed to 0 (Day 10), 1 (Day 11), 3 (Day 13), or 7 (Day 17) 30-min aerosols of OVA. BAL was performed 24 h after the last aerosol exposure. Results are expressed as means ± SEM from six to 10 rats.

Effect of OVA Exposure on the Number of DC Grown from Bone-Marrow Precursors in Sensitized Rats

On Day 13 of the experiment, when the influx of DC in BALF of the active group was maximal, bone marrow was isolated from various groups of rats. Equal amounts of cells were grown in recombinant-murine GM-CSF for 6 d, followed by an enrichment procedure for DC. DC were identified in bone marrow based on low density in a metrizamide gradient and strong expression of both OX62 and MHCII molecules. These cells had long surface projections and stimulated a primary MLR at least 2 logs more potently than did control spleen stimulators (not shown). In the OVA-sensitized group the outgrowth of DC at the end of the 6-d culture period was significantly higher in OVA-exposed compared with PBS-exposed animals (P = 0.01) (Figure 8). This increase was due to both a percentage increase and an increase in the total amount of cells recovered. When exposed to PBS, the OVA-sensitized group had equal numbers of DC in the culture compared with the sham-sensitized group (P = 0.28).

View larger version (14K): [in this window] [in a new window]   Figure 8.   Effect of OVA-aerosol exposure on the number of DC (low density, OX62+, OX6+) recovered from a 6-d culture of bone-marrow cells. Animals were immunized with OVA in BP or with PBS on Day 0 and exposed to three daily 30-min OVA or PBS aerosols (Days 10 to 12). At 24 h after the last exposure, bone marrow was collected and equal amounts of cells were grown in r-murine GM-CSF. Groups are coded as immunization/ exposure. Results are expressed as means ± SEM from four rats per group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have investigated the involvement of the DC network as part of an inflammatory response to inhaled antigen in sensitized BN rats. The problems that must be faced when studying DC are the relative paucity of these cells in peripheral tissues and the lack of specific markers. Recently, the monoclonal antibody OX-62, recognizing the mucosal integrin subunit _E2, has been described (30, 35). This integrin subunit forms a heterodimer with the ₇ chain and has been known to be expressed on intraepithelial -TCR T cells, veiled cells of the afferent lymph and intramucosal DC of the airways and gut (23, 30, 36). We have studied the expression of OX-62 on BALF cells and spleen cells. To our surprise, we found strong expression of this marker on CD3⁺CD5⁺ T lymphocytes in BALF but not in spleen. The expression of the _E7-integrin was observed on CD5-positive BAL T cells expressing -TCR (25% positive for OX-62) or -TCR (36% positive for OX-62). These findings are in line with recent studies on the phenotype of human lymphocytes that have similarly demonstrated strong expression of the _E17-integrin (cloned in humans as CD103 and recognized by the mAb HML-1) on lymphocytes obtained by BAL but not on peripheral blood lymphocytes (37, 38). It is unclear at present whether the _E7-integrin is necessary for extravasation of T cells into the lung or whether it merely acts as a retention signal for BAL T cells once these T cells have extravasated. It is possible that the _E7-integrin is specifically involved in the residence of T cells in the airway luminal compartment, as studies performed by Nelson and colleagues have demonstrated that CD5⁺ T cells that reside in the airway mucosa of the rat are negative for OX-62 (34).

The fact that OX-62 was also expressed on T cells led us to devise a more complex strategy to detect trace amounts of DC in BALF by multiparameter flow cytometry. Rare cell populations (< 1%) can best be identified using a cocktail of reagents. Ideally, the cells should be positive for at least two markers and negative for another (39). The use of a negative channel eliminates cells that bind both positively discriminative markers (e.g., both T cells and DC can express both OX-62 and MHC class II) as well as eliminating particularly autofluorescent cells. Alveolar macrophages have a high degree of spontaneous autofluorescence that impedes detection of fluorescently labeled antibodies, and DC are contained in the low-autofluorescent fraction of BALF (40). The use of a negative channel also eliminates two-cell events ("doublets") containing different cells individually expressing the different positive markers (39). Hence, the FL-2 channel of the cytometer, detecting signals in the 570 ± 20-nm spectral region, was used as a "dump" channel to gate out autofluorescent macrophages and T and B cells stained with PE-conjugated antibodies. Using this technique, we found a clear cluster of cells staining for the OX-62 marker and for MHC class II molecules. Morphologic analysis of freshly sorted cells revealed cells with long surface extensions and a typical nuclear structure, and the absence of cells with eosinophilic or lymphocytic characteristics. These cells were of low density when centrifuged over 14.5% metrizamide, a defining feature of DC (43, 44). When freshly sorted cells were used as stimulators in a primary mixed MLR, they stimulated T cells at least 1,000 times more potently than did control unfractionated spleen-cell stimulators. This is striking, as alveolar macrophages in BALF normally suppress the function of DC and the subsequent activation of T cells in an allogeneic MLR (15, 45). It was previously shown that lung DC become fully immunogenic only after complete removal of alveolar macrophages and after an overnight maturation step in the presence of GM-CSF, the main cytokine inducing maturation of DC (46, 47). It is possible that the alveolar DC, isolated from the airways of animals with eosinophilic inflammation, were induced to mature in situ, possibly by the presence of proinflammatory mediators such as tumor necrosis factor- and GM-CSF, strongly expressed in eosinophilic inflammation of the airways. Due to the low cell yield of alveolar DC in control rats, however, we were unable to compare the immunostimulatory capacity of DC from naive rats directly with those from allergic rats.

Having established that the low-autofluorescent, low-density, CD3CD45RAOX62⁺OX6⁺ population had the morphology and functional capacity of DC, we determined the presence of these cells in animals with eosinophilic airway inflammation. We found that repeated exposure to antigen in previously sensitized rats was accompanied by a 60-fold increase in the number of DC recovered by BAL compared with PBS-exposed animals. Previous studies in patients with atopic rhinitis have revealed an increase in the number of CD1a⁺ human leukocyte-associated antigen-DR-positive Langerhans cells of the nasal mucosa during an out-of-season 2-wk allergen challenge and during the natural pollen season (48). The airways of patients with atopic asthma contain increased numbers of CD1a⁺ DC, but the effect of allergen exposition on the number of cells is unexplored (21, 22). Our results suggest that the increase in numbers is antigen-driven, as part of a general inflammatory response to inhaled antigen in sensitized individuals.

The kinetics of increase of DC after repeated exposure to antigen closely resembled the kinetics of increase of eosinophils and T cells in BALF, and a maximum number of all cells could be recovered 24 h after repeated exposure for 3 d. The coordinated increase in DC, T cells, and eosinophils suggests that these cells were attracted into the airways by similar mechanisms. The majority of chemokines that attract eosinophils (MCP-1; MCP-3; MIP-1; eotaxin; and regulated on activation, normal T cells expressed and secreted) have potent chemotactic activity on DC and Th2 cells (49, 50). In support of this theory, Schon-Hegrad and coworkers have previously reported that irritative chronic eosinophilic airway inflammation in rats, induced by inhalation of dust from Pinus radiata shavings is accompanied by an increase of the intramucosal DC network at areas of eosinophilic infiltration (17).

Airway DC originate from bone-marrow precursors that arrive in the airway mucosa via the circulation. They reside in the airway mucosa for a few days and then move in a fully mature form to the regional lymph nodes (23, 51). An increase of DC in the airways can be the result of an increased influx, a local proliferation, or enhanced survival, or a decreased efflux of DC via the lymph vessels. We found that a 3-d exposure to antigen in sensitized rats induced a 60% increase in the yield of DC grown from bone-marrow precursors in the DC growth factor GM-CSF (52). This is, to our knowledge, the first description of an effect of inflammatory conditions on bone-marrow precursors for DC. This could signify either increased sensitivity of precursors to the growth-stimulating activity of GM-CSF (e.g., mediated by receptor or postreceptor events) or an actual increase in the number of precursors for DC. It is possible that allergic airway inflammation is accompanied by the systemic release of growth factors or cytokines with DC growth-promoting activity such as GM-CSF, IL-4, stem cell factor, and transforming growth factor-, all of which are produced locally in the airways of patients with atopic asthma. These mediators could effect newly incoming precursors to differentiate into DC (53). The current study suggests that these factors (or other unknown mediators) could effect the bone-marrow output of precursors. These mediators could also influence newly arrived precursors to differentiate locally into DC (53). It will be interesting to study the effects of functionally antagonizing antibodies to chemokines or cytokines on the upregulation of the bone-marrow precursor activity induced by allergen challenge.

Whatever the mechanism involved, our results suggest that systemic and localized upregulation of the DC network is an integral part of the inflammatory response to inhaled antigen in sensitized animals. We have previously demonstrated that airway DC are essential for the presentation of inhaled antigen to memory T cells in the lungs and are indispensable for the development of allergen- induced eosinophilic airway inflammation in sensitized mice (19). Combined with the present data, we suggest that the increase in DC numbers found in asthmatic airways could be an important factor in the establishment of chronic T cell-mediated allergic inflammation. A relative increase in DC numbers over suppressive alveolar macrophages would switch the balance of immune regulation in the lung in favor of activation of T cells versus tolerance (47, 54). Increased numbers of DC, some of which carry the high-affinity receptor for IgE, would also lower the threshold for allergen recognition (22, 55). It is possible that the DC that are accessible by BAL migrate back to the central lymphoid organs to stimulate recirculating T cells (18). Another, more likely, possibility is that the airway DC present antigen locally to recirculating memory Th2 cells, leading to their activation and effector function in the airways. We are currently studying these various modalities of T-cell stimulation.

In conclusion, we found that exposure to inhaled antigen in sensitized rats leads to a profound increase in the DC network, not only at the site of antigen exposure but also at the bone-marrow precursor stage. This model will allow us to study more precisely the function of DC in allergic airway inflammation.