Type II alveolar epithelial cells (AEC) can produce various antimicrobial and proinflammatory effector molecules. This, together with their abundance and strategic location, suggests a role in host defense against pulmonary pathogens. We report that murine type II AEC, like their human counterparts, express class II major histocompatibility complex (MHC). Using a murine model of pulmonary tuberculosis, we find that type II AEC become activated and have increased cell surface expression of class II MHC, CD54, and CD95 following infection. Type II AEC use the class II MHC pathway to process and present mycobacterial antigens to immune CD4+ T cells isolated from mice infected with Mycobacterium tuberculosis. Therefore, not only can type II AEC contribute to the pulmonary immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can also serve as antigen-presenting cells. Although type II AEC are unlikely to prime naïve T cells, their ability to present antigens to T cells demonstrates that they can participate in the effector phase of the immune response. This represents a novel role for type II AEC in the immunological response to pulmonary pathogens.
- major histocompatibility complex
- antigen presentation
once inhaled, Mycobacterium tuberculosis infects alveolar macrophages in the alveoli of the lung. Although the early events that occur following in vivo infection are not entirely clear, it is thought that M. tuberculosis replicates inside the phagolysosome and ultimately kills the alveolar macrophage. Released soluble mediators, bacteria, or bacterial products can then interact with other cells. In particular, type II alveolar epithelial cells (AEC) are strategically located in pulmonary airways and alveoli where they can contribute to the innate cellular immune response against airborne pathogens (4, 5, 36). The activation of type II AEC, either by secreted macrophage products or by bacterial components, may play a role in the initiation of the early inflammatory response. Many studies have used the human tumor cell line A549 as a model of type II AEC. M. tuberculosis infects and grows intracellularly within A549 cells and also has a direct cytotoxic effect on A549 cells (3, 14, 28). Type II AEC have been proposed to have a role in host resistance to pulmonary mycobacterial infections because soluble factors derived from A549 cells enhance the ability of infected macrophages to control bacterial replication (36). In addition, M. tuberculosis infection of A549 cells induces chemokine secretion that recruits inflammatory cells to the lung (24, 39).
Although human and rat type II AEC express class II major histocompatibility complex (MHC) antigens on their cell surface, their role as antigen presenting cells has never been directly addressed (11, 12, 19, 21). We hypothesize that type II AEC may function as antigen-presenting cells (APC) during pulmonary infection. One limitation of human type II AEC or A549 cells is the difficulty in obtaining MHC-matched immune T cells. To explore this hypothesis we have used type II AEC from two different sources. In some of our experiments, we use the type II AEC clone T7, which was derived from transgenic H-2Kb-tsA58 mice that express a temperature-sensitive mutant of the simian virus 40 large tumor antigen (T antigen) under the control of the IFN-γ-inducible H-2Kb promoter. When cultured under permissive conditions (33°C; +IFN-γ), T7 cells express the large T antigen, which drives cell proliferation. Under nonpermissive conditions, T antigen expression is turned off, and T7 cells differentiate into type II AEC that contain lamellar bodies and produce surfactant (13). To further substantiate the physiological significance of our findings, we also purified primary murine type II AEC and investigated whether these cells can present class II MHC-restricted antigens to immune CD4+ T cells. These experiments provide new insight into the role of type II AEC in pulmonary infection.
MATERIALS AND METHODS
Female C57BL/6 and C3H/HeJ mice, 6–8 wk old, were obtained from Jackson Laboratories (Bar Harbor, ME) and housed under specific pathogen-free conditions.
Bacteria and aerosol infections.
Six-week-old female mice were infected with virulent M. tuberculosis (Erdman strain) using a nose-only aerosol exposure unit as previously described (Intox Products, Albuquerque, NM) and housed in a biosafety level 3 facility (6, 7).
Type II AEC line.
The mouse type II AEC line T7 was maintained at 30°C in presence of IFN-γ 100 U/ml (US Biological) and differentiated as described previously (13).
Purification of cells.
Primary type II AEC were prepared from C57BL/6 mice using Corti's protocol with minor modifications (9). Dispase was instilled into the lung via a tracheal catheter; the lungs were removed and incubated in a dispase-containing solution for 45 min at room temperature. The parenchymal tissue was carefully teased apart, and the cell suspension treated with DNase I (Sigma). The cells were passed through 100- and 40-μM metal strainers. After red blood cell lysis, the leukocytes were depleted with anti-CD45 microbeads as per the manufacturer's protocol using an LS MACS separation column (see http://www.miltenyi.com for more details) (Miltenyi Biotec). Subsequently, the cells were stained with biotinylated anti-CD31 antibody (BD Pharmingen), and antibiotin magnetic beads (Miltenyi) were used to deplete contaminating endothelial cells. The remaining cells were 80% pure based on staining using the LBM180 antibody (Covance). In some cases, class II MHC microbeads (Miltenyi) were used to positively select the class II MHCHI type II AEC population: these cells were >95% pure. CD4+ T cells were purified from the spleens of uninfected or M. tuberculosis infected mice using anti-CD4 immunomagnetic microbeads (Miltenyi) by positive selection.
In vitro stimulation of T7 epithelial cells.
Cells were cultured in the presence of 1 μg/ml ultrapure LPS (from E. coli 0111:B4; Invivogen, San Diego, CA), 20 ng/ml TNF-α (BD Pharmingen), 10 μg/ml M. tuberculosis sonicate, or in complete medium. After culture, the cells were analyzed by flow cytometry.
T7 and primary type II AEC were stained with antibodies to CD1d-PE (clone 1B1, rat IgG2b), CD11b-PE (clone M1/70, rat IgG2b), CD11c-PE (clone HL3, hamster IgG1), CD40-PE (clone 3/23, rat IgG2a), CD45 biotin (clone 30-F11, rat IgG2b), CD54-PE (clone 3E2, hamster IgG1), CD80-PE (clone 16-10A1, hamster IgG2), CD86-PE (clone GL1, rat IgG2a), CD95-PE (clone Jo2, hamster IgG2), CD106 biotin (clone 429.MVCAM.A, rat IgG2a), MHCI-PE (clone AF6-88.5, mouse IgG2a), MHC II-PE (M5/114, rat IgG2b), and LBM180-Alexa 488 (clone 3C9, mouse IgG2a) or an appropriate isotype control (rat IgG2a-FITC, clone MPC-11; rat IgG2a-PE, clone G155-178; or rat IgG2b-PE, clone A95-1; hamster IgG1-PE, clone A19–3; or rat IgG2a-biotin, clone R35-95). Biotin-conjugated antibodies were detected with streptavidin-Cy-5. Purified T cells were stained with antibodies to CD4-PE (clone GK1.5, rat IgG2b), CD8-PE/Cy5 (clone 53-6.7, rat IgG2a), and CD19-FITC (clone 1D3, rat IgG2a) to check their purity. All reagents were obtained from Pharmingen (San Diego, CA), except the antibody to LBM180 (Covance, Princeton, NJ). LBM180 staining was performed after fixation and permeabilization of cells. Cells were analyzed using a FACSort (Becton Dickinson, San Jose, CA) and FlowJo software (Treestar, Palo Alto, CA).
In vitro antigen-presentation assays.
T7 epithelial cells were plated (4 × 104/well) with IFN-γ at 30°C for 5–6 days and differentiated 2 days before the assay. Primary type II AEC (105/well) were used fresh or after culture on fibronectin-coated plates for 72 h (9). In some experiments, adherent peritoneal exudate cells were used for comparison. APC were pulsed with H37Rv M. tuberculosis sonicate, culture filtrate proteins (CFP), or Ag85 [CFP and Ag85 were obtained from Colorado State University through the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIAID) contract NO1-AI-75320 entitled “Tuberculosis Research Materials and Vaccine Testing”] at the indicated concentrations for 6 h (T7 epithelial cells) or overnight (type II AEC), and excess antigen was removed by washing (1). Fixation was performed using 0.0125% glutaraldehyde for 30 s and quenched with l-lysine. Splenic CD4+ T cells (2 × 105/well) or BB7 T cell hybridoma cells [mycobacterial Ag85B-specific class II MHC-restricted; kindly provided by Drs. Henry Boom and Cliff Harding, Case Western Reserve University, Ohio (30)] were added to the wells containing epithelial cells or macrophages. The amount of IL-2 (after 20 h) or IFN-γ and TNF-α (after 48 h) in culture supernatants was quantified by ELISA (BD Pharmingen) (6). Antibody blocking experiments were done using the following MAb: anti-CD4 (GK1.5), anti-class II MHC (M5/114.15.2), or an isotype control (Y13–238; all hybridomas obtained from American Tissue Culture Collection, Manassas, VA). All antibodies were added at a final concentration of 20 μg/ml and were purified from culture supernatant by standard methods.
One- or two-way ANOVA with Bonferroni posttest were performed using GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).
T7 epithelial cells express MHC and costimulatory molecules.
Differentiated T7 epithelial cells constitutively express high levels of MHC class I and class II molecules (Fig. 1). T7 epithelial cells express the H-2Kb and Db antigens, but not H-2Kk or Dk, as determined by flow cytometry using haplotype-specific MAb (data not shown). These cells also express CD106 (VCAM-1) and CD80 (Fig. 1), but not CD86 or CD40 (data not shown). Culture of T7 epithelial cells with TNF-α increases cell surface expression of MHC class II, CD54 (ICAM-1), and VCAM-1, whereas treatment with M. tuberculosis sonicate led to smaller increases in ICAM-1 and VCAM-1 only. Thus type II AEC express the necessary antigen presenting and accessory molecules required to present microbial antigens to T cells.
T7 epithelial cells present M. tuberculosis antigens to CD4+ T cells.
The constitutive class II MHC expression by T7 epithelial cells led us to consider whether they can function as APC. Differentiated T7 cells were pulsed with increasing concentrations of M. tuberculosis sonicate and excess antigen removed by washing. Presentation of antigen by T7 epithelial cells activated CD4+ T cells isolated from M. tuberculosis-infected mice to produce IFN-γ and IL-2 (immune T cells, Fig. 2A). Under these conditions, CD4+ T cells from uninfected mice did not secrete cytokines (nonimmune T cells, Fig. 2A). Similarly, T7 epithelial cells present M. tuberculosis CFP to immune CD4+ T cells in an antigen dose-dependent manner (Fig. 2B). In contrast, neither the nonimmune CD4+ T cells cultured with epithelial cells nor the purified T cells cultured with antigen alone produced significant amounts of IFN-γ. Antigen presentation by T7 epithelial cells elicited a similar antigen-induced response as peritoneal macrophages (data not shown).
Antibody blocking studies established that antigen presentation by T7 epithelial cells is class II MHC restricted. Activation of antigen-specific CD4+ T cells was inhibited 60–70% by anti-class II MHC antibody and completely blocked by anti-CD4 antibody (Fig. 2C). This is consistent with our finding that antigen presentation by T7 epithelial cells did not activate MHC mismatched immune T cells (data not shown). Thus presentation of M. tuberculosis antigens by T7 epithelial cells to CD4+ T cells is class II MHC restricted.
To exclude the possibility that free peptides in the crude M. tuberculosis sonicates bind directly to class II MHC and are presented without processing, T7 epithelial cells or peritoneal macrophages were fixed with glutaraldehyde before or after adding antigen. Fixation before adding antigen abrogated antigen presentation by both APC (Fig. 2D). T7 epithelial cells required 2–3 h of processing before antigen presentation could occur. Peritoneal macrophages were more efficient APC than T7 epithelial cells, based on their ability to elicit a maximal T cell response more rapidly (Fig. 2E). Thus intracellular processing of intact proteins by type II AEC is required for antigen presentation to CD4+ T cells.
Pulmonary infection leads to activation of primary type II AEC.
To determine whether primary type II AEC had a similar phenotype as the T7 epithelial cells, single cell suspensions from the lungs of C57BL/6 mice were depleted of leukocytes. Type II AEC were identified by flow cytometry based on intracellular staining with MAb specific for LBM180, a component of the limiting membrane of the lamellar body (29). By gating on the LBM180+ cells, we were able to determine that type II AEC constitutively express high levels class II MHC and CD54 (ICAM-1) and moderate levels of class I MHC and CD95 (Fig. 3). No expression of CD106, CD1d, CD11b, or CD11c was detected (data not shown). Type II AEC isolated from the lungs of M. tuberculosis-infected mice expressed higher levels of class I and II MHC, ICAM-1, and CD95 and slightly higher levels of CD80. These observations show that M. tuberculosis infection leads to generalized activation of primary type II AEC as indicated by a change in their cell surface phenotype. This is the first demonstration of the upregulation of antigen presenting molecules on type II AEC following infection.
Primary type II AEC can present antigen to CD4+ T cells.
Depletion of CD45+ cells using antibody-conjugated magnetic beads efficiently removed leukocytes from single cell suspensions prepared from dispase-digested lungs (Fig. 4A). The remaining cells were ∼60% type II AEC based on intracellular staining for the LBM180 antigen (Fig. 4A and data not shown). The resulting cells present mycobacterial antigen both to CD4+ T cells (Fig. 5A) and the BB7 T cell hybridoma (data not shown). However, careful analysis revealed a population of class II MHCLO cells that were CD45−CD31+ and likely to be endothelial cells (data not shown). Depletion of CD31+ cells and further positive selection of cells for high expression of class II MHC resulted in cell preparations that were >95% type II AEC. These cells were free of contamination from CD45+ leukocytes including any CD11b+ and CD11c+ cells (Fig. 4, A and B). Nearly 100% of the class II MHC+ cells were LMB180+ (Fig. 4C). These highly purified primary type II AEC were tested for their ability to present antigen using the class II MHC pathway. For this experiment we used the BB7 T cell hybridoma. BB7 is a well-characterized T cell hybridoma that recognizes M. tuberculosis antigen 85B presented by the class II MHC molecule H-2 I-Ab (31). Furthermore, BB7 T cells do not express class II MHC and therefore cannot present this antigen to itself. BB7 T cell hybridomas recognized mycobacterial antigen presented by highly purified type II AEC as indicated by their production of IL-2 in an antigen dose-dependent manner (Fig. 5B). These results highlight the possibility that primary type II AEC can present microbial antigens to class II MHC-restricted CD4+ T cells in vivo.
It is easy to imagine that type II AEC play a role in host defense against microbial pathogens. They are abundant throughout the lung and have many innate immune functions. Type II AEC express Toll-like receptors (TLR) that serve as microbial pattern recognition receptors, secrete chemokines critical for the recruitment of leukocytes to the lung, and produce proteins with antimicrobial properties including surfactant and β-defensins (10, 15, 16, 23). A549 cells produce granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF), IL-8, interferon-inducible protein-10 (IP-10), IFN-γ-inducible T cell α-chemoattractant (I-TAC), monokine induced by IFN-γ (MIG), monocyte chemoattractant protein-1 (MCP-1), and leukotriene B4 after stimulation with proinflammatory cytokines (IL-1β or TNF-α) or with microbial products (LPS or M. tuberculosis infection) (22, 24, 36, 37, 39). Interestingly, IL-1, which is secreted by infected human myeloid cells, is an even more potent inducer of IL-8 and regulated on activation, normal T cell expressed, and secreted (RANTES) production by A549 cells than M. tuberculosis infection (39, 40). Therefore, cross talk between type II AEC and macrophages may be important in the elaboration of chemokines and the subsequent recruitment of leukocytes to the lung.
What remains inexplicable is why type II AEC express class II MHC that is usually restricted to professional APC. Although a variety of other cell types can express class II MHC following activation, type II AEC are one of the few non-bone marrow-derived cell types that constitutively express class II MHC (11, 12, 19, 21). Engagement of the T cell receptor by the MHC/peptide complex is not sufficient to activate T cells; costimulatory and accessory signals are required. Memory T cells are less dependent upon costimulatory signal for activation but still require certain accessory signals. ICAM-1 is induced on A549 as well as other human alveolar epithelial cell lines by the pulmonary pathogens Haemophilus influenzae, Pseudomonas aeruginosa, and respiratory syncytial virus (17, 38). Primary murine type II AEC express high levels of ICAM-1 but little or no VCAM-1 and CD80. This pattern is similar to type II AEC isolated from humans and rats (8, 11, 12, 18). Thus type II AEC express accessory molecules required for the stimulation of antigen-specific T cells.
Our data show that type II AEC not only express class II MHC proteins and accessory molecules but also present microbial antigens to CD4+ T cells. Both T7 epithelial cells and primary type II AEC present mycobacterial proteins to class II MHC-restricted CD4+ T cells, including both the BB7 T cell hybridoma and immune CD4+ T cells isolated from M. tuberculosis-infected mice. This is the first demonstration that type II AEC use the class II MHC pathway to present microbial proteins to CD4+ T cells. Although we have not yet characterized this pathway, the molecular details of antigen uptake and processing have been examined in intestinal epithelial cells and type II AEC lines. Intestinal epithelial cells take up soluble protein antigens by fluid phase endocytosis into an endolysosomal pathway (2). These endocytic compartments contain class II MHC and cathepsins, which are required for antigen processing by professional APC (20). The uptake of antigen in the A549 and BEAS-2B cell lines also follows an endocytic pathway, and exogenously administered proteins can be colocalized with intracellular class II MHC (35). Thus much of the machinery required for antigen processing exists within epithelial cells.
Type II AEC are particularly interesting as they have a specialized endocytic pathway for the storage, secretion, and recycling of surfactant proteins. Lamellar bodies are intracellular organelles unique to type II AEC that contain surfactant proteins. Surfactant is secreted into the alveolar air space when lamellar bodies fuse to the cell membrane and the contents of the vesicle is extruded. Lamellar bodies contain CD208 (DC-LAMP), a member of the lysosomal-associated membrane protein (LAMP) family, which is found in the MHC class II-containing intracellular compartment (MIIC) after dendritic cell (DC) activation (33, 34). Mouse, sheep, and human type II AEC express DC-LAMP constitutively, and it colocalizes with LBM180 and class II MHC to the lamellar body (34). Our data showing that type II AEC use the class II MHC pathway to present antigen to T cells makes the parallels between the MIIC compartment of DC and lamellar bodies of type II AEC even more intriguing.
Several professional APC in the lung have the capacity to present antigen to class II MHC-restricted T cells (32). Why should type II AEC also express MHC class II and present antigen to T cells? Type II AEC are unlikely to play a role in the activation of naïve T cells; this function is more likely to be mediated by DC that acquire microbial antigens in the lung and migrate to the draining lymph node. However, type II AEC, which outnumber other MHC class II+ cells, could have a role in stimulation of the memory immune response, particularly for memory T cells that reside in peripheral tissues such as the lung (26). Although it is unknown whether type II AEC are a significant reservoir for M. tuberculosis, class II MHC-restricted CD4+ T cells could be important for eliminating infected type II AEC. Equally likely is the possibility that inadvertent presentation of mycobacterial antigens by type II AEC has untoward effects for the host. Type II AEC are sensitive to killing by TNF-α and Fas ligation, which could both be mediated by immune CD4+ T cells (25, 27). If this were to occur during M. tuberculosis infection, antigen presentation by type II AEC could give rise to some of the immunopathology observed in tuberculosis, manifested by epithelial cell death and lung parenchymal damage.
This work was supported by NIAID Grant R01-AI-47171 (S. M. Behar).
↵* H. Debbabi and S. Ghosh contributed equally to this work.
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