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Long-Term Deposition of Inhaled Antigen in Lung Resident CD11b^–CD11c⁺ Cells

Malaghan Institute of Medical Research, Wellington, New Zealand; Department of Dermatology, DIAID, and Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria; and INSERM U503-IFR128, Lyon, France

Correspondence and requests for reprints should be addressed to Dr. Franca Ronchese, Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand. E-mail: fronchese{at}malaghan.org.nz

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
In this study we report the characterization of a population of lung resident CD11b^–CD11c⁺ cells that are able to take up inhaled antigen and retain it for extended periods of time. Ovalbumin conjugated to fluorescein-isothiocyanate (FITC-OVA) administered intranasally to mice was taken up by two main populations of cells in the lung, a migratory CD11c⁺CD11b⁺ population consisting of dendritic cells (DC), which rapidly transported antigen to the draining lymph node (LN), and a resident CD11b^–CD11c⁺ population that retained engulfed antigen without apparently degrading it for up to 8 wk after administration. The FITC⁺CD11b^–CD11c⁺ cells did not migrate to draining LN at a detectable rate, and did not up-regulate expression of costimulatory molecules in response to LPS treatment. FITC⁺CD11b^–CD11c⁺ cells were found in the lung and bronchoalveolar lavage fluid, and their distribution was compatible with macrophages. Although FITC⁺CD11b^–CD11c⁺ cells expressed the DC marker DEC205 and other molecules associated with antigen-presenting cell function, they did not induce proliferation of antigen-specific CD4⁺ T cells in vitro or acute cytokine production by activated CD4⁺ T cells in vivo. Thus, FITC⁺CD11b^–CD11c⁺ cells appear to represent an intermediate cell type sharing properties with DC and macrophages. These cells may have a role in modulating the responses of lung resident T cells to inhaled antigens.

Key Words: animal models • antigen presentation/processing • lung inflammation

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   This article reports the characterization of a population of lung cells that take up inhaled antigen and retain it for several weeks. Retention of antigen by these cells had not been reported before, and may have important implications for immune responses in the lung.

 
The immune response to inhaled allergen involves distinct and well-characterized phases. Pulmonary and airway dendritic cells (DC) are required to transport antigen from tissues to secondary lymphoid organs during the sensitization phase, to drive the activation of naïve CD4⁺ T cells (1, 2). Antigen-presenting cells (APC) are again required upon antigen re-encounter to present antigen to effector or memory CD4⁺ T cells in the lung and airways (3). They may include migratory DC populations, that present antigen to T cells in the lung before migrating to the draining lymph node (LN) (4), and other populations of lung-resident APC that migrate to the draining LN at a minimal rate (5). Such resident APC may have an important function in presenting antigen in lung tissue, and perhaps supporting the retention of antigen-specific memory T cells within the lung. In one study, DC isolated from the airway were shown to present inhaled antigen for up to 8 wk after antigen exposure (6). However, it was not established whether those DC are long-lived, or whether they acquire antigen from a separate, long-lived population that cannot directly present antigen to T cells.

Several APC populations are present in lung. Alveolar macrophages phagocytose large amounts of inhaled antigen (7), but also down-regulate the activity of pulmonary DC (8) and suppress T cell activation through the production of NO (9). Murine alveolar macrophages express major histocompatibility complex class II (MHC II) molecules (10) and can present antigen to T cell hybridomas, which have little need for costimulation (11). This suggests that alveolar macrophages could be able to stimulate antigen-specific effector or memory T cells that also have low costimulatory requirements. B cells are also found in the pulmonary tissue of mice, and may also contribute to antigen presentation in situ (12).

We wished to track the cells that engulf and present antigen within the lung tissue following intranasal instillation of ovalbumin (OVA) protein conjugated to the fluorescent marker fluorescein isothiocyanate (FITC) (FITC-OVA). We found that several populations of cells take up FITC-OVA within the lung tissue; however, 1 wk after antigen exposure, FITC-OVA becomes restricted to a population of CD11b^–CD11c⁺ cells (hereafter referred to as CD11b^–FITC⁺). These CD11b^–FITC⁺ cells could be isolated from both the lung tissue and bronchoalveolar lavage (BAL) for longer than 8 wk after intranasal antigen administration. Despite expression of MHC II and T cell costimulatory molecules, CD11b^–FITC⁺ cells appeared unable to present their retained antigen to activated Th2 cells in vivo or naïve CD4⁺ T cells in vitro. It is possible that these previously unidentified long-lived cells may modulate the response of lung T cells to inhaled antigens, affecting the chronicity of the allergic response that is observed in some disease situations (6, 13).

	MATERIALS AND METHODS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Mice
C57Bl/6 mice were from breeding pairs originally obtained from The Jackson Laboratories (Bar Harbor, ME). OT-II mice (14) were a kind gift of Dr. Frank Carbone (Melbourne University, Australia). Experimental procedures were performed with the approval of the Wellington School of Medicine Animal Ethics Committee in accordance with the University of Otago guidelines. C57Bl/6 mice were bred and maintained in the VIRCC animal facility (Vienna, Austria), and used with the approval of the Medical University of Vienna Animal Ethics Committee.

In Vitro Culture Media and Reagents
All cultures were in complete IMDM (cIMDM), which consisted of IMDM with GlutaMAX and HEPES buffer supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen Corp., Auckland, NZ) and 50 µM 2-ME (Sigma-Aldrich, Castle Hill, NSW, Australia). Grade V chicken OVA was from Sigma-Aldrich; OVA_323–339 peptide was from Chiron Mimotopes, Clayton, Australia. Low-endotoxin FITC-OVA and Texas Red–conjugated OVA (TR-OVA) were from Molecular Probes (Eugene, OR).

Intranasal Instillations and Preparation CD11c⁺ Cells from Lung and LN Digests
Mice were anesthetized and 100 µg of OVA protein, FITC-OVA or TR-OVA in 50 µl of PBS were instilled into one nostril. At different times, lungs were collected, finely sliced, and digested by two consecutive incubations in IMDM containing 1 mg/ml DNase I (Sigma-Aldrich) and 2.4 mg/ml collagenase I (Invitrogen Corp.) at 37°C for 30 min. In some experiments airway cells were removed by BAL before lung collection; this did not affect the number or phenotype of the recovered FITC⁺ populations. Mononuclear cells were purified by centrifugation over 60% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ). Lung-draining mediastinal LN were harvested and digested for 1 h at 37°C in 2.4 mg/ml collagenase II (Invitrogen Corp.) and 1 mg/ml DNase I in IMDM (15).

Total lung cell preparations were enriched for CD11c⁺ cells using anti-CD11c MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and an Auto MACS sorter (Miltenyi Biotech). The enriched population was labeled with fluorescent anti-CD11c and anti-CD11b antibodies, and further purified by electronic sorting using a FACS-Vantage SE (Becton Dickinson, Mountain View, CA).

Antibodies and Flow Cytometric Analysis
Antibodies were from BD Pharmingen (San Diego, CA), with the exception of F4/80 which was from Serotec Inc. (Raleigh, NC). Anti-FcRII, anti-CD11c, anti–I-A^b, anti-CD86, anti-CD28, anti-CD3, and anti–DEC-205 were purified from hybridoma supernatants using HyTrap protein G columns (Pharmacia Biotech, Uppsala, Sweden) and conjugated to FITC (Sigma-Aldrich) or to allophycocyanin (Prozyme, San Leandro, CA). Cells were pre-incubated in anti-FcRII mAb and labeled in PBS containing 2% FCS, 2 mM EDTA and 0.01% sodium azide. Analysis was on a FACSort using the Cell Quest software (BD, Mountain View, CA). Live cells were identified by forward scatter/side scatter (FSC/SSC) properties and PI (BD Biosciences) exclusion.

For detection of intracellular Langerin expression, Fc receptors were blocked by incubation in 5% mouse serum. Cells were fixed and permeabilized using a cytofix/cytoperm kit (BD Pharmingen) before incubation with anti-Langerin Ab (16) and anti-rat IgG-PE.

Immunofluorescence and Histologic Analysis
Seven days after intranasal instillation, mice were killed by injection of a lethal dose of anesthetic. The lungs were harvested and frozen in OCT on dry ice and maintained at –20°C. Frozen lung sections (4 µm) were fixed with acetone for 10 min and washed in PBS. Slides were then incubated with anti–CD11c-FITC or anti–CD11b-FITC (Pharmingen) for 60 min at 37°C, washed with PBS and distilled water, and incubated with hematoxylin for 15 s before embedding with Fluorosave (Molecular Probes). Immunofluorescence was visualized with a Nikon Optiphot 2-UD Fluorescence Microscope (Nikon, Tokyo, Japan) with a x40 objective and a Multiband Filtersystem triple filter (DAPI/FITC/Texas Red; Af-Analysentechnik, Tübingen, Germany).

In Vitro T Cell Proliferation Assays
Decreasing numbers of FACS-sorted CD11c⁺CD11b⁺ and CD11b^–FITC⁺ lung cells were distributed in triplicate in 96-well plates. OT-II thymocyte suspensions were depleted of APC by 2 h plastic adherence as described (17), and 2 x 10⁵ cells were added to each well. OVA_323–339 was added to some wells at 10 µg/ml. Bone marrow–derived DC (BM-DC) were generated as described (17) and used at 1.5 x 10³ cells/well; rhIL-2 was used at 100 U/ml. Plates were incubated for 3 d at 37°C and proliferation of T cells was evaluated by measuring [³H]-thymidine (Perkin Elmer Life Sciences Inc., Boston, MA) incorporation over the last 8 h of culture. Cells were harvested using a Tomtec automated cell harvester and a liquid scintillation Beta counter (Wallac, Turku, Finland).

Generation of Th2 Cells In Vitro and Adoptive Transfer
Th2 cells were generated as previously described (18). Cell suspensions from LN of OT-II mice were depleted of CD8⁺ T cells and B cells using Dynabeads (Dynal Biotech ASA, Oslo, Norway) and cultured on plates coated with 5 µg/ml anti-CD3 mAb in cIMDM containing 2 ng/ml rhIL-6, 20 U/ml rhIL-2, 1,000 U/ml rmIL-4 and 2 µg/ml anti-CD28 mAb. After 5 d cells were harvested, resuspended in cIMDM containing 100 U/ml rhIL-2 and cultured in fresh plates for a further 2 d. 1 x 10⁷ V2⁺V5.1, 5.2⁺ cells were injected into the lateral tail vein of recipient mice.

Quantitative and Qualitative Assessment of Leukocytes in BAL Fluid
Airway-infiltrating cells were collected by BAL. Cells were spun on a glass slide, stained using Diff-Quik (Dade International, Dudingen, Switzerland) and counted under a microscope as described (19). A minimum of 200 cells/sample were counted.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
DC Transport Antigen from the Lung to the Draining LN
To follow the migration of DC from the lung to the mediastinal LN, mice were given 100 µg of FITC-OVA by i.n. instillation, and the phenotype and number of FITC-OVA⁺ cells in the LN was determined by FACS. At 24 h after instillation, 81% of FITC⁺ cells were CD11c⁺ MHC II⁺, indicating that antigen was mostly transported by DC (Figures 1A and 1B). In addition, virtually all of the CD11c⁺ FITC⁺ cells expressed high levels of MHC II, a phenotype compatible with mature cells emigrating from tissue. Most of the FITC⁺ cells expressed intermediate or high levels of CD11b. The number of FITC⁺ cells in LN was 1,000 at 6 h after instillation and peaked at 20,000 at 24 h (Figure 1C). By 96 h, there were few FITC⁺ cells remaining in the LN, suggesting that lung deposits of FITC-OVA had been exhausted.

View larger version (14K): [in this window] [in a new window]   Figure 1. FITC+ cells in the draining LN express CD11c, MHCII, and CD11b. C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, and 24 h later the draining LN were harvested, digested with collagenase, and analyzed by FACS. Dead cells were identified by FSC/SSC and PI staining and were excluded from analysis. (A) FITC staining in total LN suspensions from representative untreated and FITC-OVA treated mice. Gating of FITC+ cells is shown. (B) Dot plot analysis of MHCII, CD11b, and CD11c expression on total or FITC+ LN cells. (C) Number of FITC+ cells in the draining mediastinal LN at different times after FITC-OVA administration. FITC+ cells were gated as in A. Bars show the mean ± SEM number of FITC+ cells in the draining LN of four mice.

View larger version (29K): [in this window] [in a new window]   Figure 2. Phenotype of FITC+ cells in the lungs and BAL of FITC-OVA treated mice. C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, and at the indicated time points lungs were harvested, digested with collagenase, and analyzed by FACS. Dead cells were identified by FSC/SSC and PI staining and were excluded from analysis. (A) Number of FITC+ cells in the lung at different times after FITC-OVA administration. FITC+ cells were gated as in B. Mean ± SEM for groups of at least four mice are shown. (B) FITC staining in total lung suspensions from representative untreated and FITC-OVA–treated mice. Gating of FITC+ cells is shown. (C) Dot plot analysis of CD11b and CD11c expression on representative total or FITC+ lung cells at the indicated times after FITC-OVA administration. The CD11b– CD11c+ population is highlighted. (D) Six days after FITC-OVA treatment, airway cells were collected by BAL and analyzed by FACS. The top panel shows gating for FITC+ cells on total live BAL cells; the bottom panel shows expression of CD11b and CD11c on FITC+ cells. Representative dot plots are shown.

View larger version (43K): [in this window] [in a new window]   Figure 3. Phenotype of FITC+ cells that remain in the lung after i.n. administration of FITC-OVA. C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, and 5 d later their lungs were harvested, digested with collagenase, and analyzed by FACS. All samples were incubated with anti-CD11c plus the marker indicated in each panel; dead cells were identified by FSC/SSC and PI staining and were excluded from analysis. For the detection of intracellular markers (e.g., Langerin), cells were fixed after surface staining and permeabilized. Gating of FITC+ cells is shown in each panel; FITC+ cells also expressed CD11c (data not shown).

View larger version (91K): [in this window] [in a new window]   Figure 4. Morphology and localization of CD11b–FITC+ cells in lung tissue. (A) C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, and 7 d later their lungs were harvested and made into single-cell suspensions. The CD11b–CD11c+FITC+ cells were then sorted by FACS, cytospun onto glass slides, stained with Diff-Quik, and examined under a light microscope. Panel illustrates typical morphology of CD11b–CD11c+FITC+ cells. (B) As in A, with the exception that mice were treated with 100 µg TR-OVA, and sorted cells were observed under a fluorescent microscope. A representative TR+ cell is shown. "n" indicates the nucleus and "c" indicates the cytoplasm of the cells. (C–E) C57BL/6 mice were treated with 100 µg TR-OVA by intranasal instillation. Sixteen days later the lungs were harvested, frozen, and stained with Haematoxylin and anti–CD11b-FITC or anti–CD11c-FITC. (C) Photomicrograph of frozen section demonstrates an area of lung parenchyma and airway. (D) Overlay of hematoxylin and fluorescent image in E showing that the majority of the cells are present near the airway. (E) Frozen lung section visualized with triple filter (DAPI/FITC/Texas Red) illustrates a cluster of single positive TR-red cells and CD11b-FITC single positive green cells. Although cells are often in close proximity, no double labeling is observed. (F) Anti–CD11c-FITC staining of frozen sections. (G) Overlay of fluorescent images in F and H to show double labeling of TR-red+ CD11c-FITC+ cells, which appear yellow-orange. TR-red–CD11c+ cells appear green. (H) Unstained frozen section to reveal TR+ cells after intranasal administration of TR-OVA.

Microscopic examination of fluorescent frozen lung sections and sections stained with hematoxylin revealed that the fluorescent signal of OVA-TR⁺ cells was located almost exclusively in the lung periphery. The majority of OVA-TR⁺ cells were located in the alveoli (Figure 4D), and no large airways were observed in the vicinity of the OVA-TR⁺ cells. Although the alveolar walls were often compressed, by following the nuclear alignment alveolar lumina could often be appreciated, revealing that the OVA-TR⁺ cells were located on the luminal side of the basement membrane. OVA-TR⁺ cells were morphologically and functionally consistent with macrophages, as they were large, were found in large numbers within alveoli, and had phagocytosed OVA-TR. As shown in Figure 4E, the CD11b⁺ staining did not co-localize with the OVA-TR⁺ fluorescent signal. CD11b⁺ cells also appeared large and morphologically consistent with macrophages; however, they were found within the alveoli, as well as between epithelial cells.

CD11b^–FITC⁺ Cells Do Not Up-Regulate Expression of Co-Stimulatory Molecules in Response to LPS Stimulation In Vivo
To establish whether CD11b^–FITC⁺ cells might have a role in antigen presentation, the expression of T cell costimulatory molecules was investigated. Mice were treated with FITC-OVA and 10–28 d later the expression of CD80, CD86, and CD40 on FITC⁺ cells was examined by flow cytometry. At each time point, > 90% of total FITC⁺ cells were CD11b^–CD11c⁺ (data not shown). These FITC⁺ cells expressed moderate levels of CD80, low levels of CD86 and undetectable CD40 (Figure 5A).

View larger version (32K): [in this window] [in a new window]   Figure 5. CD11b–FITC+ cells express some T cell costimulatory molecules but do not up-regulate CD86 in response to LPS. C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, and were killed at the times indicated below. Lung suspensions were prepared and examined by FACS. Dead cells were identified by FSC/SSC and PI staining and were excluded from analysis. (A) Representative FACS stainings of lung cell suspensions prepared on Day 14 or 28 after intranasal challenge. Histograms show expression of the indicated markers on FITC+ cells, of which > 90% were CD11b–CD11c+. Open histograms, cells stained with the indicated antibodies; solid histograms, cells stained with an isotype-matched control antibody. (B) Representative FACS stainings of lung cell suspensions prepared on Day 6 after intranasal challenge. Some of the mice were injected with 50 µg of LPS given intraperitoneally 7 h before killing. Histograms show expression of CD86 in the indicated populations gated as indicated in the contour plot at the top of the figure. Solid histograms, cell populations from untreated mice; open histograms, populations from LPS-treated mice.

CD11b^–FITC⁺ Cells Do Not Induce Airway Eosinophilia In Vivo
To assess if CD11b^–FITC⁺ cells were able to induce T cells to secrete cytokines in vivo, mice were treated intranasally with FITC-OVA or saline. On Day 7, when the FITC⁺ population in the lung consisted almost exclusively of CD11b^–FITC⁺ cells, mice received an adoptive transfer of activated, OVA-specific Th2 cells; intranasal challenge with OVA protein or saline was on Day 8. Figure 6A shows that the BAL fluid of mice challenged with OVA contained large numbers of eosinophils. In contrast, no eosinophilia could be demonstrated in mice that had been treated with FITC-OVA 1 wk before the transfer of Th2 cells. Thus, CD11b^–FITC⁺ cells were unable to induce IL-5 secretion by activated Th2 cells in vivo. In addition, similar numbers of eosinophils were detected in mice pretreated with FITC-OVA or saline, suggesting that CD11b^–FITC⁺ cells did not suppress the activation of Th2 cells in vivo.

View larger version (20K): [in this window] [in a new window]   Figure 6. CD11b–FITC+ cells do not induce or suppress airway eosinophilia in vivo. Solid bars: eosinophils; shaded bars: macrophages; open bars: lymphocytes. (A) C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation, or saline as a control, and 1 wk later were injected intravenously with 107 in vitro activated, OVA-specific Th2 cells. One day later, some of the mice received a second intranasal instillation of 100 µg OVA protein. BAL cells were collected 3 d after the second intranasal challenge, counted and spun onto a glass slide. Differential cell counts were performed after Diff-Quik staining. Each bar shows the mean ± SEM number of eosinophils, macrophages, and lymphocytes per milliliter of BAL fluid from groups of four mice. (B) As in A, but Th2 cells were also given intranasally (106/mouse), or intranasal challenge was also with 100 ng LPS, as indicated.

CD11b^–FITC⁺ Suppress Naïve CD4⁺ T Cell Proliferation In Vitro
The ability of CD11b^–FITC⁺ cells to present antigen was also examined in vitro. Mice were treated with FITC-OVA and 1 wk later the CD11c⁺CD11b⁺ and the CD11b^–FITC⁺ cells were FACS sorted from lung digests, and their ability to present retained FITC-OVA antigen or exogenous OVA_323–339 peptide to OT-II T cells was tested in a proliferation assay. Figure 7A shows that addition of OVA_323–339 to cultures containing CD11c⁺CD11b⁺ lung DC resulted in robust T cell proliferation. In contrast, CD11b^–FITC⁺ cells were completely unable to present their retained antigen or exogenous peptide to specific CD4⁺ T cells.

View larger version (12K): [in this window] [in a new window]   Figure 7. CD11b–FITC+ cells do not induce the proliferation of OVA-specific OT-II T cells in vitro. C57BL/6 mice were treated with 100 µg FITC-OVA by intranasal instillation. One week later, lungs were harvested, made into single-cell suspensions, and CD11b–FITC+ and CD11c+CD11b+FITC– cells were purified by FACS sorting. (A) The indicated numbers of sorted lung cells were incubated with OT-II cells, ± 10 µg/ml OVA323–339, for 3 d at 37°C, and thymidine incorporation was measured over the final 8 h of culture. Mean ± SEM cpm for triplicate wells are shown. Circles, CD11b–FITC+ cells with no OVA added, or CD11c+CD11b+ cells with no OVA added; solid triangles, CD11b–FITC+ cells and OVA323–339; open triangles, CD11c+CD11b+ cells and OVA323–339. (B) Different numbers of sorted lung cells were mixed with 1.5 x 103 BM-DC to obtain the indicated cell ratios. OT-II cells, 10 µg/ml OVA323–339, and 100 U/ml IL-2 were added as indicated. Plates were incubated for 3 d at 37°C, and thymidine incorporation was measured over the final 8 h of culture. Mean ± SEM cpm for triplicate wells are shown. Line at bottom, OT-II cells only; circles, OT-II and OVA323–339; open triangles, OT-II, BM-DC, CD11b–FITC+ cells, and OVA323–339; solid triangles, OT-II, BM-DC, CD11b–FITC+ cells, OVA323–339, and IL-2.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Phenotype of CD11b^–FITC⁺ Cells
In this report, we describe the identification and characterization of a population of lung CD11b^–CD11c⁺ cells that retain inhaled FITC-OVA for longer than 8 wk after administration. While it is known that a population of CD11b^–CD11c⁺ cells able to take up antigen resides in the lung (5), the ability of these cells to store antigen for many weeks had not, to our knowledge, been reported.

The identity of CD11b^–FITC⁺ cells was not firmly established. Their morphology was similar to macrophages, and immunohistologic analysis also revealed that the predominant location of CD11b^–FITC⁺ cells near alveoli and on the luminal side of the basement membrane was typical of a subpopulation of macrophages. Intracytoplasmic punctate deposits of fluorescent material were prominent in CD11b^–FITC⁺ cells. Thus, the accumulation of presumably undigested material in these cells would suggest that at least some of their properties differ from those of typical macrophages, which rapidly degrade internalized proteins (20), and are more reminiscent of immature DC (21).

Previous studies have also identified lung CD11c⁺CD11b^– as macrophages, including alveolar macrophages and a resident population of lung macrophages (12, 22, 23). Alveolar macrophages have previously been described to have a slow turnover (24, 25) and sequester particulate antigen (7), but their ability to retain antigen has not been reported and is not consistent with their proposed function of removing debris. Phenotypic analysis revealed that our CD11b^–FITC⁺ cells also expressed the DEC205 marker, but were CD40^–B220^–Gr-1^–, which is again consistent with the phenotype assigned to alveolar macrophages in two (22, 23), but not one (12) of those earlier studies. The phenotype of CD11b^–FITC⁺ cells was not consistent with conventional DC, which express the CD11b marker, or plasmacytoid DC, which express Gr-1 and B220 (26). However, it is interesting to note that initial studies where the expression of DEC205 by alveolar macrophages was first reported (27) proposed that alveolar macrophages might, in some conditions, give rise to DC. In keeping with this proposal, other authors have used a model of lung infection with Yersinia pestis to show that a population of CD11c⁺CD11b^–DEC205⁺ cells was able to take up bacteria, acquire expression of CCR7, and migrate from the lung tissue to the draining lymph node (28), all properties that are normally associated with DC. Thus, our CD11b^–FITC⁺ cells resemble a population of lung cells whose surface markers and reported properties are intermediate between macrophages and DC.

Antigen-Presenting Function of CD11b^–FITC⁺ Cells
Despite the expression of moderate levels of MHC II and costimulatory molecules, CD11b^–FITC⁺ cells appeared unable to present peptide or protein antigen to naïve CD4⁺ T cells in vitro. We obtained no evidence that CD11b^–FITC⁺ cells present their retained OVA to activated Th2 cells in vivo, and attempts to enhance presentation and migration to the airways using LPS were also unsuccessful. This lack of activity might not be surprising given the apparently slow degradation of ingested materials by these cells. Failure to activate Th2 cells in vivo in the lung could also reflect the use of limiting amounts of FITC-OVA for the intranasal instillation of antigen. While the dose used (100 µg/mouse) is usually sufficient to elicit a strong response (18), this is in conditions in which the CD11c⁺CD11b^– and CD11c⁺CD11b⁺ populations are both loaded with antigen. It is possible that higher amounts of antigen might be necessary to reveal presentation by CD11c⁺CD11b^– cells alone.

One previous report by Julia and coworkers documented the ability of a BAL CD11c⁺CD11b⁺ DC population to stimulate antigen-specific cells for several weeks after antigen exposure (6). The cell population described in that study differs from the population described here in both the expression of CD11b and in the ability to support cytokine secretion by T cells. In addition, Julia and colleagues identified their cells of interest only on the basis of stimulatory function, and their results are equally consistent with DC being long-lived, or DC taking up antigen from another long-lived population of cells. In our study, cells were directly identified on the basis of retention of fluorescent antigen, allowing us to conclude that the CD11b^–CD11c⁺ cells were loaded with antigen and long-lived. Together, the two studies suggest that a long-lived population of CD11b^–CD11c⁺ lung cells might act as a reservoir of inhaled antigen, which can become stimulatory for T cells if taken up and presented by neighboring lung DC (29, 8, 30), or under conditions that support the differentiation of CD11b^–CD11c⁺ cells into DC.

An alternative possibility is that CD11b^–CD11c⁺ cells may not be stimulatory, but promote tolerance and downregulate local immune responses to protect the fragile lung microenvironment (9). In vivo, pre-loading CD11b^–FITC⁺ cells with OVA did not appear to inhibit cytokine production by activated Th2 cells after intranasal OVA challenge. CD11b^–FITC⁺ cells did exhibit some suppressive activity in vitro; however, their inhibitory capacity was not particularly potent. Alveolar macrophages can suppress the activity of APC and the proliferation of T cells by producing NO (9, 31), and this inhibition can be reversed by stimulation with PMA or by addition of IL-2 (32, 9). Neither addition of IL-2, nor stimulation with PMA-ionomycin (data not shown) could reverse the suppression induced by CD11b^–FITC⁺ cells, suggesting that a different mechanism was involved. Cytokines such as TGF-1 and IL-10 have also been reported to inhibit immune responses in the lung (33, 34); however, secretion of these cytokines by CD11b^–FITC⁺ cells was not investigated.

We show in this paper that CD11b^–CD11c⁺ cells remain fluorescent for extended periods after FITC-OVA administration; this was also observed after administration of OVA conjugated to other fluorochromes such as TR or Alexa-Fluor488 (data not shown). Each of these OVA conjugates was rapidly and completely degraded by other cell types, such as for example the CD11c⁺CD11b⁺ DC. This suggests that the presence of fluorescence was a marker of incompletely degraded OVA within these cells, as complete protein degradation would be expected to lead to a loss of fluorescent signal.

CD11b^–FITC⁺ Cells in Pulmonary Disease
The apparent limited antigen degradation and processing in CD11b^–FITC⁺ cells could make them attractive targets for intracellular bacteria that reside in endocytic vacuoles, such as mycobacteria. Alveolar macrophages and DC are the favored host cells for mycobacteria, which are able to survive in phagosomes by preventing their acidification and fusion with lysosomes. Interestingly, in this paper we show that CD11b^–FITC⁺ cells do not readily process and present engulfed antigen, suggesting that they could potentially be exploited by infectious agents that do not possess specific immune evasion mechanisms. This may provide an additional explanation for ability of some bacteria to persist inside cells of the immune system.

A second possibility is that CD11b^–FITC⁺ cells might be specialized cells that gradually process stored antigen and present it, directly or indirectly, to resident T cells, modulating their function without inducing full activation. Such low-level stimulation could favor the retention of effector T cells in tissue and the maintenance of protective immunity (35), or possibly prevent T cell activation in conditions of limiting antigen. Memory Th2 cells and lymphocytic infiltrates containing CD4⁺ T cells have been demonstrated in the lungs of mice more than 400 d after the induction of acute allergic airway disease (13). Experiments are currently in progress to assess whether the lymphocytic infiltrates are in close proximity to CD11b^–FITC⁺ cells.

In summary, we report a cell population in the airways of mice that is able to retain fluorescent material for extended periods of time. This long-term depot of antigen could be important in modulating immune responses to inhaled antigens. Further work is required to determine the normal function of these cells in different inflammatory or infectious situations.

	Acknowledgments

 
The authors thank all staff of the Malaghan Institute of Medical Research for constructive suggestions and advice, and the Staff at the Biomedical Research Unit of the Wellington School of Medicine for animal husbandry and care. They also thank Sandra Jost for technical assistance, Oskar Hoffmann for assistance with fluorescent and confocal laser microscopy, and Hermine and Erich Berger (Vienna) for animal husbandry and care.

	Footnotes

 
This work was supported by research grants from the Cancer Society and Health Research Council of NZ to F.R. K.E.M. was supported by a Ph.D. Scholarship from the NZ Cancer Institute, and travel Scholarships from the Wellington Medical Research Foundation and Wellington Division of the Cancer Society.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0330OC on November 22, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 3, 2006

Accepted in final form November 13, 2006

	References

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