Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages by Mycobacterium bovis BCG and the 19-Kilodalton Mycobacterial Lipoprotein

Mice.Specific pathogen-free, female C57BL/6 (H-2^b) mice were purchased from Charles River Laboratories (North Wilmington, Mass.) and used at between 8 and 12 weeks of age. TLR-2 gene knockout mice were generously provided by O. Takeuchi and S. Akira (Osaka University, Osaka, Japan) and bred onto the C57BL/6 background. Mice were housed under specific-pathogen-free conditions by using microisolator cages (Lab Products, Inc., Maywood, N.J.) and fed a standard rodent diet and water ad libitum. Studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University.

Bacteria. M. bovis BCG Connaught (ATCC 35745) was grown at 37°C for 18 to 21 days in Proskeur-Beck medium (pH 7.0) containing l-asparagine (5.0 g/liter), potassium dihydrogen phosphate (5 g/liter), sesquimagnesium citrate (1.5 g/liter), dipotassium sulfate (0.5 g/liter), d-glucose (30 g/liter), and Tween-80 (0.05%). During mid-log growth, the culture was supplemented with glycerol (6%, vol/vol), aliquoted, and stored at −70°C until further use. Bacterial titers were determined by plating serial 10-fold dilutions on 7H10 agar and counting CFU after incubation for 2 to 3 weeks. The viability of M. bovis BCG is retained for at least 2 years. At the time of use, bacteria were thawed, sonicated for 40 s by means of an ultrasonic Cup Horn water bath (90 W; 20 kHz; Heat Systems-Ultrasonics, Farmingdale, N.Y.) and added to cultured alveolar macrophages as indicated.

Fluorescein labeling of M. bovis BCG.A stock solution of FLUOS [5(6)-carboxyfluorescein-N-hydroxysuccinimide ester] (Boehringer Mannheim, Mannheim, Germany) at a concentration of 20 mg/ml was made in dimethyl sulfoxide and stored at −20°C. A 1.0-ml aliquot of M. bovis BCG (3 × 10⁷ to 5 × 10⁷ CFU) was thawed, centrifuged at 4°C (10,000 × g for 15 min), resuspended in 0.5 ml of phosphate-buffered saline (PBS; pH 9.12) and sonicated for 40 s by means of an ultrasonic Cup Horn water bath. A total of 7.5 μl of FLUOS solution was added and mixed with bacteria at 4°C for 30 min. Fluorescein-stained M. bovis BCG (fluos-BCG) was pelleted (10,000 × g for 15 min), washed with 1.0 ml of PBS (pH 9.12), resuspended in Dulbecco's modified Eagle's medium (DMEM), and sonicated for 40 s. Fluos-BCG was prepared fresh for each experiment, and the number of CFU was determined by plating serial 10-fold dilutions on 7H10 agar plates. FLUOS labeling did not affect the viability of mycobacteria. Alveolar macrophages were infected overnight (37°C) with fluos-BCG (multiplicity of infection [MOI], 3:1 to 5:1) and subsequently treated with (or without) IFN-γ for 24, 48, and 72 h. After infection, cells were stained for surface MHC-II and B7.1 as described below and analyzed by flow cytometry. Where indicated, infected cells were gated on FL1 and examined for coexpression of MHC-II or B7.1. Ungated cells were also analyzed to determine the degree of phagocytosis.

Antibodies and cytokines.The following fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated, and biotinylated antibodies (identified by catalogue number) were purchased from PharMingen (San Diego, Calif.): biotin-mouse immunoglobulin G2a(κ) [IgG2a(κ)] (no. 553455), biotin-mouse antimouse I-A^b (no. 06042D), PE-hamster IgG2(λ) (no. 553935), PE-hamster antimouse CD80 (B7.1) (no. 553769), biotin-hamster IgG2(λ) (no. 550084), biotin-hamster antimouse CD80 (B7.1) (no. 09602D), rat-antimouse CD16/CD32 Fc-Block (Fcγ III/II Receptor) (no. 553141), FITC-rat IgG2a(κ) (no. 553929), FITC-rat antimouse CD86 (B7.2) (no. 09274D), FITC-hamster IgG1(λ) antitrinitrophenol (no. 553953), FITC-hamster antimouse CD11c (no. 09704A), FITC-rat antimouse CD40 (no. 09664D), PE-rat IgG2b(κ) (no. 553989), PE-rat antimouse CD11b (Mac1) (no. 01715B), FITC-rat IgG1(κ) (no. 553923), and FITC-rat antimouse Mac3 (no. 01715D). PE-rat antimouse F4/80 (no. MCA497PE) and PE-rat IgG2b (no. MCA1125PE) were purchased from Serotec Inc. (Raleigh, N.C.). Streptavidin-red 670 (no. 19543-024) was purchased from GibcoBRL (Grand Island, N.Y.). Murine IFN-γ was purchased from R & D Systems (Minneapolis, Minn.).

Isolation of murine alveolar macrophages.Age-matched mice were anesthetized with a lethal dose of tribromoethanol (240 mg/kg of body weight), and alveolar macrophages were isolated as described previously (13). The abdominal cavity was incised, mice were exsanguinated by transecting the left renal artery, and the chest was decompressed by a transverse incision at the level of the xiphoid. The trachea was exposed aseptically, and an 18-gauge Angiocath intravenous catheter (Becton Dickinson, Sandy, Utah) was inserted through a 1-mm incision at the first tracheal ring. A total of 3.6 ml of PBS (Sigma, St. Louis, Mo.) was instilled in nine separate 0.4-ml bronchoalveolar lavages (BAL). Alveolar macrophages were centrifuged at 2,000 × g for 10 min at 4°C and resuspended in DMEM (BioWhittaker, Walkersville, Md.) supplemented with 10% fetal calf serum, 100 IU of penicillin per ml, 100 μg of streptomycin per ml, 100 mM HEPES, 1× nonessential amino acids, 2.0 mM l-glutamine, and 0.05 mM 2-mercaptoethanol. Cell viability was routinely greater than 95% as determined by trypan blue (0.4%) exclusion. Cytospin slides of 2.5 × 10⁴ cells/BAL/mouse were prepared by using a Cytospin 3 centrifuge (600 rpm for 6 min) (Shandon, Pittsburgh, Pa.) and stained with Diff-Quik (Dade Diagnostics, Aguada, Puerto Rico) for differential cell counts. Resident BAL cells were comprised of alveolar macrophages (90 to 95%), lymphocytes (5 to 10%), and occasional granulocytes (<1 to 2%).

Purification of the 19-kDa lipoprotein of M. tuberculosis (H37Ra).Stock cultures of M. tuberculosis were grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) supplemented with albumin, dextrose, and catalase (Difco). After 2 to 3 weeks, cultures were supplemented with glycerol (final volume, 6% [vol/vol]), thoroughly mixed, aliquoted, and frozen at −70°C. The M. tuberculosis H37Ra 19-kDa lipoprotein was isolated as described previously (32, 34). M. tuberculosis H37Ra was cultured to mid-log phase in 4 to 6 liters of 7H9 medium supplemented with albumin, dextrose, and catalase. Bacteria were pelleted in 500-ml tubes spun for 20 min at 8,000 rpm (5,220 × g) by a Sorvall RC-5B rotor. Bacterial pellets were combined and resuspended in 20 to 30 ml of lipopolysaccharide-free deionized water containing EDTA (7.5 mM), leupeptin (0.7 μg/ml), phenylmethylsulfonyl fluoride (0.2 mM), pepstatin A (0.7 μg/ml), DNase (10 U/ml), and RNase A (25 U/ml) (Boehringer-Mannheim, Indianapolis, Ind.). The solution was passed three times through a French press and centrifuged for 2 h at 100,000 × g and 4°C. Next, mycobacterial-cell-wall-containing pellets were resuspended in cold extraction buffer (pH 7.5) containing 20% Triton X-114 (Sigma) in Tris-HCl (50 mM) and NaCl (150 mM). After mixing overnight at 4°C, the cell wall preparation was centrifuged (100,000 × g) for 2 h at 4°C, and the remaining insoluble pellet was discarded. The supernatant was warmed to 37°C and centrifuged (2,400 × g) for 10 to 15 min at 37°C to separate aqueous and TX-114 layers. The upper aqueous layer was discarded, and the TX-114 layer was remixed and washed three to five times with an equal volume of cold buffer containing Tris-HCl (50 mM) and NaCl (150 mM). After washing, TX-114 soluble proteins were precipitated overnight (at −20°C) with 10 volumes of acetone (Fisher Chemicals, Fair Lawn, N.J.). Precipitated proteins were centrifuged (2,400 × g) for 20 to 30 min at 4°C, washed with cold acetone (−20°C), pelleted, and air dried at room temperature. The pellet was solubilized in reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 0.7 M 2-mercaptoethanol, and 0.01 μg of bromophenol blue per ml) and electroeluted through an SDS-12% polyacrylamide gel by using a Bio-Rad Model 491 Prep Cell as previously described (32). Fractions enriched for the M. tuberculosis H37Ra 19-kDa lipoprotein were identified by Western blotting, pooled, and reextracted with TX-114. After reprecipitation with acetone, the pellet was solubilized in 5 mM HEPES buffer (pH 7.0) containing 90% dimethyl sulfoxide, aliquoted, and stored at −70°C. Protein content was determined by using a detergent-compatible protein assay (Bio-Rad). Working 19-kDa lipoprotein preparations (50 μg/ml) were 85 to 90% pure and primarily contaminated with lipomannan and lipoarabinomannan (data not shown). Electroelution fractions collected before and after elution of the 19-kDa lipoprotein were used as negative control fractions and did not significantly affect IFN-γ-induced MHC-II expression or antigen processing and presentation (data not shown). Electroelution fractions contained less than 20 pg of lipopolysaccharide per ml as measured by Limulus amoebocyte lysate assay (E-Toxate kit; Sigma).

Staining and flow cytometry of alveolar macrophages.Alveolar macrophages (5 × 10⁴) were resuspended in 0.015 ml of Fc-block (at a concentration of 2 μg/ml in 0.1% bovine serum albumin [BSA] in PBS) for 15 min on ice. Cocktails of primary antibodies or isotype control antibodies were diluted (for biotinylated MHC-II [I-A^b], 1:100; for B7.1, 1:50; for B7.2, 1:25; for CD40, 1:25; for CD11c, 1:25; for Mac3, 1:25; and for biotinylated F4/80, 1:25), and 0.030 ml was added to cells for 20 min on ice. Cells were washed with 5% BSA in PBS (pH 7.4), stained with 0.025 ml of streptavidin-red 670 (1:100 in 0.1% BSA) for 15 min on ice, washed with 5% BSA in PBS, and fixed with 1% paraformaldehyde in PBS (pH 7.4). Samples were stored in the dark at 4°C for subsequent analysis by flow cytometry.

Flow cytometry was performed with a Becton Dickinson FACScan flow cytometer (San Jose, Calif.). Forward light scatter, side light scatter, FL1 (FITC), FL2 (PE), and FL3 (streptavidin-red 670) settings were determined empirically to maximize the difference between isotype and specific staining. A total of 5,000 to 10,000 ungated events were collected. Histogram, dot plot, and mean fluorescence intensity (MFI) analyses were performed by using Cellquest (Becton Dickinson) software. Differences in mean specific MFI (MFI − isotype MFI ± standard error of the means [SEM]) values were statistically analyzed where indicated. In some cases (as indicated) where cells were not uniformly positive for surface staining, differences in mean specific geometric MFI (geoMFI) were analyzed.

Antigen processing and presentation assay.Alveolar macrophages were harvested as described above, plated (65,000 cells/well) into flat bottom 96-well plates (Falcon 35-3072) in complete medium and allowed to adhere overnight at 37°C. Alveolar macrophages were washed five times and pretreated with complete medium with or without the 19-kDa lipoprotein (250 ng/ml). After 24 h fresh medium containing murine IFN-γ (2 ng/ml) was added with or without fresh 19-kDa lipoprotein (250 ng/ml). After 48 h of incubation, alveolar macrophages were washed extensively with warm DMEM to remove residual IFN-γ and 19-kDa lipoprotein. Next, alveolar macrophages were incubated for 2 h with 300 to 5,000 μg of ovalbumin (OVA; Sigma) per ml, washed five times with warm DMEM, and allowed to process OVA overnight. After fixation with 1% paraformaldehyde (15 min) and extensive washing, OVA peptide (OVA_323-339)-specific (18), MHC-II-restricted T-hybridoma cells (DOBW) were added (100,000/0.2 ml/well) and allowed to incubate overnight as described previously (31). Culture supernatants were removed after 18 to 24 h and stored at −70°C until they were assayed for interleukin 2 (IL-2). IL-2 was measured by using the standard colorimetric CTLL bioassay with Alamar Blue (Alamar Biosciences, Sacramento, Calif.) and a Bio-Rad 550 microplate reader as previously described (18). The CTLL assay was capable of detecting 20 to 100 pg of IL-2 per ml, which typically corresponds to 0.05 to 0.500 relative absorbance units (the optical density at 550 nm [OD₅₅₀]). At each antigen concentration, the inhibition of antigen processing and presentation was determined by calculating the percent decrease in the OD₅₅₀ between control cells and cells treated with the 19-kDa lipoprotein [percent inhibition = (control OD₅₅₀ − experimental OD₅₅₀)/control OD₅₅₀ × 100].

Quantitative real-time reverse transcription-PCR for CIITA and MHC-II mRNA.Alveolar macrophages were isolated and first cultured overnight (18 to 24 h) without stimulation. After 24 h, cells were incubated with or without 19-kDa lipoprotein (500 ng/ml) for 24 h. Next, IFN-γ (2 ng/ml) with or without fresh 19-kDa lipoprotein (500 ng/ml) was added, and the cells were incubated for an additional 24 h. Afterwards, cells were pelleted (2,000 × g for 10 min), resuspended in lysis buffer, and passed through a QiaShredder according to the manufacturer's instructions (QIAGEN, Valencia, Calif.). Cellular RNA was purified by using the RNeasy kit (QIAGEN) according to the manufacturer's instructions. RNA (1 μg) was reverse transcribed by the SuperScript preamplification system (Life Technologies) as previously described (34). The cDNA mixture was diluted 1:5 and 10 μl was used for each gene-specific amplification reaction using a high-speed thermal cycler (I-Cycler; Bio-Rad, Hercules, Calif.). PCR products were detected by iQ-SYBR Green Supermix (Bio-Rad). The amplification cycle was 95^°C for 10 s, 52^°C for 15 s, and 72°C for 20 s (35 cycles). Primers were designed by using OLIGO version 6.4 (Molecular Biology Insights, Cascade, Colo.) and are shown as follows: for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 5′-AACGACCCCTTCATTGAC-3′ (sense) and 5′-TCCACGACATACTCAGCAC-3′(antisense) (predicted size, 191 bp); for total class II transactivator (CIITA), 5′-ACGCTTTCTGGCTGGATTAGT-3′ (sense) and 5′-TCAACGCCAGTCTGACGAAGG-3′ (antisense) (predicted size, 342 bp); and for MHC-II (I-A^b α-chain), 5′-GCTCCTCAAGCGACTGTG-3′ (sense) and 5′-AAGCTGGTCTCATAAACACCG-3′ (antisense) (predicted size, 155 bp). cDNA copy numbers for each gene were quantified by using standard curves of known quantities of gel-purified PCR products prepared with a QiaQuick gel extraction kit. Standard curves of the PCR threshold cycle number versus cDNA copy number were generated and used to quantify the corresponding CIITA and MHC-II mRNA copy numbers in alveolar macrophages treated as described. The copy numbers of CIITA and MHC-II in both control and treated alveolar macrophages were normalized to GAPDH expression.

Statistical analysis.The Wilcoxon rank sum test or the Student's t test was used where indicated. Significant differences are indicated (P ≤ 0.05).

Murine alveolar macrophages constitutively express surface MHC-II, CD80 (B7.1), and CD11c.Alveolar macrophages comprise more than 90 to 95% of alveolar cells and are the first immune effector cells to encounter aerosolized mycobacteria transmitted to alveolar spaces. By using flow cytometry, resident alveolar macrophages from healthy normal mice were analyzed for the expression of MHC-II and the costimulatory molecules CD80 (B7.1) and CD86 (B7.2) essential for the activation of CD4⁺ T cells. In Fig. 1A to F, representative histograms show the degree of surface MHC-II, B7.1, B7.2, CD11b (Mac-1), CD11c, and CD40 expression on alveolar macrophages isolated and pooled from age-matched mice. Mean specific MFIs (± SEM) from five different experiments also are shown on the figure. Resident alveolar macrophages expressed B7.1 (panel A), MHC-II (panel D), CD11c (panel E), and FcRIII/II (data not shown) constitutively but did not express B7.2, CD11b, and CD40 (panels B, C, and F, respectively). In addition, expression of Mac-3 or F4/80 was not detected (data not shown). However, CD11b was expressed on alveolar macrophages cultured overnight in vitro (data not shown). Thus, resident alveolar macrophages were characterized by the expression of MHC-II, B7.1, CD11c, and FcRIII/II. By means of two-color analysis, resident alveolar macrophages were also analyzed for coexpression of B7.1 and MHC-II (panel H) and CD11c (panel J) molecules. We determined that the majority (85.2% ± 1.3%) of resident alveolar macrophages coexpressed B7.1 and CD11c. In addition, 20.3% ± 3.2% also coexpressed MHC-II (panel H), thereby characterizing the two major subsets of resident alveolar macrophages as MHC-II⁺/CD11c⁺/B7.1⁺ and MHC-II⁻/CD11c⁺/B7.1⁺. In addition, a small proportion of bronchoalveolar cells expressed MHC-II only (7.6% ± 1.0%), and 7.26% ± 0.45% of cells did not stain for either surface B7.1 or MHC-II. Thus, a subset of resident alveolar macrophages expresses the molecules necessary for CD4⁺-T-cell recognition and activation.

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FIG. 1.

Resident alveolar macrophages express CD80 (B7.1), CD11c, and MHC-II. (A to F) Alveolar macrophages were isolated from 8- to 10-week-old female C57BL/6 mice and stained as described in Materials and Methods. A total of 5,000 events per sample was analyzed by flow cytometry. Histograms show both specific staining (shaded) and isotype control staining (unshaded) for CD80 (B7.1), CD86 (B7.2), CD11b (Mac-1), CD11c, CD40, and MHC-II. Data shown on the panels are the mean specific MFIs (± SEM) from five independent experiments demonstrating the expression of B7.1, MHC-II, and CD11c (for FcRIII/II, MFI = 16.9 ± 7.1; data not shown). B7.2, Mac-1, and CD40 were not detected. (G to J) Dot blots show coexpression of MHC-II and CD11c on B7.1-expressing alveolar macrophages. The mean percentages (± SEM) of total cells expressing B7.1 and coexpressing MHC-II and CD11c from five independent experiments are shown on the panels inserts. Staining with isotype control antibodies was less than 2 to 3% of the total events detected in any single quadrant.

IFN-γ enhances surface MHC-II molecule expression on alveolar macrophages.IFN-γ regulates the microbicidal and antigen-presenting functions of macrophages and, thus, has a critical role in protective immunity to mycobacteria (11, 12). Alveolar macrophages were cultured either with or without IFN-γ and analyzed for the expression of MHC-II and B7.1. In Fig. 2, representative histograms demonstrate IFN-γ-enhanced MHC-II expression (panels A to C). Expression of B7.1 (panels D to F), B7.2 (data not shown), and CD11b (data not shown) was not effected by IFN-γ. Since the staining of untreated (control) cells was not uniform, mean specific geoMFI was used to quantify the increased expression of MHC-II. In panel G, the values of the mean specific geoMFI (± SEM) for MHC-II expression from six independent experiments are summarized. Equivalent levels of MHC-II were detected on freshly isolated alveolar macrophages (15.3 ± 1.2) (Fig. 1D) and alveolar macrophages cultured for 24 (17.8 ± 2.0), 48 (16.7 ± 2.5), and 72 h (14.9 ± 1.5) in medium alone (Fig. 2A to C). After 24 h, IFN-γ treatment significantly enhanced MHC-II expression by 1.5-fold (17.8 ± 4.9 versus 26.7 ± 6.9; P < 0.05). In addition, after 48 or 72 h of culture, IFN-γ treatment resulted in a significant increase in MHC-II expression levels of threefold (16.7 ± 2.5 versus 46.4 ± 5.5) and sixfold (14.9 ± 1.4 versus 83.7 ± 10.2), respectively, compared to controls (n = 6, P ≤ 0.05).

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FIG. 2.

IFN-γ enhances expression of MHC-II on alveolar macrophages. (A to F) Alveolar macrophages were isolated from mice and cultured at the indicated times in the presence (shaded) or absence (dark black line; unshaded) of murine IFN-γ (2 ng/ml). Cells were stained for MHC-II and B7.1 as described, and histograms from a representative experiment (n = 5) are shown. Specific staining in the absence and presence of IFN-γ are shown and compared to isotype control staining. In panel G, the mean specific geoMFI (± SEM) for MHC-II from five experiments is shown. The Wilcoxon rank sum test was used to compare MHC-II expression between control and IFN-γ-stimulated alveolar macrophages. Statistically significant differences in specific geoMFI are designated by an asterisk (P ≤ 0.05).

Two-color analysis determined the extent of MHC-II coexpression on B7.1⁺ and B7.1⁻ alveolar cells. In Fig. 3, representative dot blots for alveolar macrophages cultured with (panels A to C) or without (panels D to F) IFN-γ show the effect of IFN-γ on MHC-II expression, with data summarizing six independent experiments (mean percentage ± SEM). In the absence of IFN-γ, the proportion of B7.1- and MHC-II-coexpressing cells increased somewhat after 24 h (35.2% ± 1.7 in panel A versus 20.3% ± 3.2 in Fig. 1, panel H) but did not change further after 48 to 72 h (range, 27.9 to 35.2%; panels A to C). This result suggested that overnight culture conditions increased the proportion of cells expressing MHC-II by 10 to 15%, which was not discernible by single-color analysis (Fig. 1D and 2G). In the presence of IFN-γ (Fig. 3, panels D to F), the proportion of B7.1⁺/MHC-II⁺ cells increased markedly at each time point studied (P < 0.05 compared to untreated cells in panels A to C). After 72 h of IFN-γ treatment, the proportion of B7.1⁺/MHC-II⁺ cells increased from 52.7% ± 3.8% (panel D) to 67.8% ± 4.6% (panel F), corresponding to a concomitant decrease in the proportion of B7.1⁺/MHC-II⁻ cells (32.2% ± 4.1%, as seen in panel D, versus 7.7% ±1.7%, as seen in panel F; P < 0.05). After 72 h, nearly 90% of all B7.1⁺ cells coexpressed MHC-II. Although enhanced MHC-II expression occurred primarily on B7.1⁺ alveolar macrophages, 10 to 15% of total MHC-II was also detected on B7.1-negative cells. Thus, localized expression of IFN-γ in alveolar spaces may enhance MHC-II expression and the ability of alveolar macrophages to present antigen and activate CD4⁺ T cells.

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FIG. 3.

Enhanced coexpression of MHC-II on IFN-γ-treated B7.1⁺ alveolar macrophages. (A to F) Alveolar macrophages were isolated, cultured at the indicated times with IFN-γ (2 ng/ml) or without IFN-γ and then stained for B7.1 and MHC-II. A total of 5,000 to 7,500 events were collected for two-color analysis and representative dot blots are shown. Quadrant settings were used to calculate the distribution of cell types within the entire (ungated) alveolar macrophages population. The levels of expression of MHC-II and B7.1 from six different experiments are summarized and shown on panels (mean % total ± SEM) for upper- and lower-right quadrants. Isotype control staining was less than 2 to 3% in each quadrant.

M. bovis infection and the 19-kDa lipoprotein inhibit IFN-γ-mediated upregulation of MHC-II on alveolar macrophages.Since alveolar macrophages are phagocytic for M. bovis BCG and IFN-γ enhances expression of MHC-II on alveolar macrophages, we next studied IFN-γ-inducible MHC-II expression in alveolar macrophages infected with BCG. First, phagocytosis of M. bovis BCG by alveolar macrophages was analyzed by infecting alveolar macrophages overnight at a low MOI (3 to 5) with fluos-BCG. Figure 4 (panel A) demonstrates that at 24 h after infection, the change in MFI between uninfected and fluos-BCG-infected alveolar macrophages was 80.8 ± 7.2 and indicated that 47.8% ± 7.2% of alveolar macrophages had phagocytosed fluos-BCG (n = 6). Additional analysis of MHC-II expression on fluos-BCG-infected B7.1⁺ cells (R1 gated, panel B) indicated that there was a similar uptake of fluos-BCG by both MHC-II⁻/B7.1⁺ and MHC-II⁺/B7.1⁺ alveolar macrophages (45.8% ± 3.5% and 49.7% ± 3.1%, respectively). Uptake and viability of BCG were unaffected by fluorescein labeling (data not shown).

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FIG. 4.

Phagocytosis of M. bovis BCG by alveolar macrophages. Alveolar macrophages were incubated overnight with fluos-BCG at a low MOI (3 to 5) and analyzed by flow cytometry. (A) A representative histogram shows phagocytosis of fluos-BCG. (B) MHC-II and B7.1 expression on fluos-BCG-positive cells (region R1, panel A) from a representative experiment reveals similar uptake of bacteria by both MHC-II⁺ and MHC-II⁻ B7.1⁺ alveolar macrophages.

Second, to determine whether IFN-γ-induced MHC-II expression was affected by M. bovis BCG infection, cells were infected overnight at a low MOI and then stimulated with IFN-γ for 24, 48 or 72 h. As shown in a representative experiment (Fig. 5), IFN-γ treatment (panel A) enhanced MHC-II expression (two- to threefold) in both infected and uninfected control cells. However, after 72 h (panel B), M. bovis BCG infection inhibited upregulation of MHC-II by IFN-γ. In four independent experiments, a 47.8% ± 11.9% reduction in MHC-II was measured (P < 0.05, by paired Student's t test). In contrast, infection of alveolar macrophages in the absence of IFN-γ did not affect surface MHC-II expressed constitutively on the 20 to 30% of resident alveolar macrophages described in Fig. 1 (panel H) and Fig. 3 (panels A to C) (data not shown).

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FIG. 5.

M. bovis BCG infection and a 19-kDa mycobacterial lipoprotein inhibit IFN-γ-inducible MHC-II expression on alveolar macrophages. (A to D) Alveolar macrophages were cultured with or without BCG at an MOI of 3 to 5 and with or without the 19-kDa lipoprotein (250 ng/ml) for 24 h. IFN-γ (2 ng/ml) was added after infection or pretreatment with the 19-kDa lipoprotein, and alveolar macrophages were cultured for an additional 24, 48, and 72 h in the presence of IFN-γ. The expression of MHC-II was measured by flow cytometry, and histograms representative of four independent experiments are shown. IFN-γ-induced MHC-II expression on BCG-infected or 19-kDa-lipoprotein-treated macrophages (black lines) is compared to control treated cells (shaded) after 48 and 72 h of IFN-γ exposure. Isotype control staining for both treatment groups is shown with overlapping thin lines. (E to F) Alveolar macrophages were isolated, cultured overnight, and then treated without (medium) or with the 19-kDa lipoprotein (500 ng/ml) for 24 h. After 24 h, fresh 19-kDa lipoprotein was added along with IFN-γ (2 ng/ml), and cells were incubated for an additional 48 h. CIITA and MHC-II mRNA expression was measured by real-time PCR and normalized to GAPDH expression. Data from a representative experiment (n = 3) are shown. Decreases in CIITA and MHC-II mRNA expression were analyzed by using a paired Student's t test, and significant differences are designated with asterisks (P ≤ 0.05).

Recent studies have identified the 19-kDa lipoprotein of M. bovis BCG and M. tuberculosis as a TLR-2 ligand that modulates APC function (2, 32). As shown in Fig. 5C and D, the 19-kDa lipoprotein decreased the MFI for MHC-II by 69.8 ± 10.4 units (panel D) compared to the MFI of control cells at 72 h (P ≤ 0.05 by paired Student's t test). In five independent experiments, after 72 h a 35.5% ± 4.6% reduction in MHC-II was observed compared to MHC-II in cells at 24 h (P ≤ 0.05 by Wilcoxon rank sum). Thus, both whole bacilli and the 19-kDa lipoprotein, a major cell wall component of M. bovis BCG and M. tuberculosis, inhibited IFN-γ-inducible MHC-II expression on alveolar macrophages.

To determine the potential mechanisms for the 19-kDa lipoprotein-mediated inhibition of surface MHC-II expression, we also analyzed transcription of CIITA mRNA and MHC-II mRNA. RNA was isolated from alveolar macrophages pretreated with the 19-kDa lipoprotein (500 ng/ml) 24 h before IFN-γ treatment. After 24 h of IFN-γ treatment, mRNA for CIITA and MHC-II (I-A^b, beta chain) was measured by real-time reverse transcription-PCR. As shown in a representative experiment (Fig. 5E and F), the 19-kDa lipoprotein significantly decreased (P ≤ 0.05) the copy numbers of CIITA (panel E) and MHC-II (panel F) mRNA by 35.1 and 68.3%, respectively. In three independent experiments, a 41.9% ± 8.5% (mean ± SEM) reduction in CIITA mRNA and a 51.5% ± 19.7% (mean ± SEM) reduction in MHC-II mRNA were detected. Depletion of MHC-II mRNA correlated with the delayed loss of surface MHC-II detected after 72 h (Fig. 5B and D) when sufficient decay of surface MHC-II had occurred. Thus, the 19-kDa lipoprotein inhibited IFN-γ-induced transcription of both CIITA mRNA and MHC-II mRNA, resulting in decreased surface MHC-II.

Inhibition of MHC-II expression and antigen processing and presentation by the 19-kDa lipoprotein are dependent on TLR-2.In preliminary experiments we had determined that MHC-II on alveolar macrophages was capable of binding and presenting peptide to MHC-II-restricted DOBW (data not shown). To determine if changes in MHC-II expression (Fig. 5D) affected antigen processing, the effect of the 19-kDa lipoprotein on processing and presentation of ovalbumin was tested with an MHC-II-restricted T-cell DOBW. In Fig. 6, a representative experiment demonstrates that recognition of MHC-II-peptide complexes in normal alveolar macrophages pretreated with the 19-kDa lipoprotein and pulsed with ovalbumin at concentrations of 316, 1,000, or 3,160 μg/ml was significantly (P ≤ 0.05) inhibited by 77.2, 47.6, and 23.2%, respectively. In three different experiments, the average (± SEM) degree of inhibition was 85.8% ± 7.2%, 57.5% ± 8.7%, and 29.6% ± 7.0%.

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FIG. 6.

The 19-kDa mycobacterial lipoprotein inhibits antigen processing and presentation in alveolar macrophages. Alveolar macrophages were harvested, plated in duplicate in 96-well flat bottom plates (65,000 cells/well), and incubated overnight. Adherent cells were washed and incubated for 24 h with or without 19-kDa lipoprotein. Next, cells received IFN-γ (2 ng/ml) in fresh medium (controls) or medium supplemented with fresh 19-kDa lipoprotein. After 48 h, cells were washed extensively and pulsed for 2 h with ovalbumin, and antigen presentation was measured 24 h later as described. Ovalbumin-specific DOBW responses were measured, and mean (± standard deviation) DOBW responses from a representative experiment (n = 3) are shown. Percent inhibition was calculated as the reduction in the OD₅₅₀ measured by the CTLL assay for IL-2 (see Materials and Methods). Statistically significant inhibition (P ≤ 0.05) of ovalbumin processing was measured at each antigen dose by using a paired Student's t test.

Next we determined whether TLR-2 was necessary for the 19-kDa lipoprotein to inhibit MHC-II expression and antigen processing. Thus, we analyzed IFN-γ-inducible expression of MHC-II in the presence and absence of the 19-kDa lipoprotein in alveolar macrophages isolated from TLR-2 ^−/− and wild-type mice. In Fig. 7, representative histograms of three independent experiments show that the specific MFI for MHC-II expression was reduced 41.6% ± 4.9% (P ≤ 0.05) in wild-type alveolar macrophages after 72 h (panel A). In contrast, expression of IFN-γ-inducible MHC-II in alveolar macrophages isolated from TLR-2^−/− mice (panel B) was unaffected by the 19-kDa lipoprotein, suggesting that intact TLR-2 was necessary for the inhibition of MHC-II expression mediated by the 19-kDa lipoprotein. In addition, as shown in Fig. 7C, the 19-kDa lipoprotein did not inhibit processing of ovalbumin by alveolar macrophages obtained from TLR-2^−/− mice. Although it is not known why TLR-2 ^−/− alveolar macrophages appeared to process and present ovalbumin more efficiently than wild-type cells, the 19-kDa lipoprotein-mediated inhibition of MHC-II expression and antigen processing by wild-type alveolar macrophages was dependent upon TLR-2.

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FIG. 7.

Inhibition of MHC-II expression and antigen processing by the 19-kDa lipoprotein is dependent on TLR-2. (A to B) Alveolar macrophages were isolated from age-matched C57BL/6 wild-type and C57BL/6 TLR-2^−/− mice. After overnight incubation with (dark lines) or without (shaded) the 19-kDa lipoprotein (250 ng/ml), alveolar macrophages were treated for 48 h with IFN-γ (2 ng/ml) and then analyzed for surface MHC-II expression. Histograms representative of three different experiments are shown. (C) Alveolar macrophages were isolated from wild-type C57BL/6 and TLR-2 ^−/− mice, pretreated with the 19-kDa lipoprotein or medium, and then incubated for 48 h with IFN-γ with or without fresh 19-kDa lipoprotein or medium. Next, cells were pulsed with ovalbumin, and DOBW responses were analyzed as described in the legend of Fig. 6. Mean (± standard deviation) DOBW responses for wild-type alveolar macrophages and TLR-2 ^−/− alveolar macrophages pretreated with medium or 19-kDa lipoprotein from a representative experiment (n = 3) are shown. By a paired Student's t test, statistically significant inhibition (P ≤ 0.05) was detected at each ovalbumin dose in wild-type cells pretreated with the 19-kDa lipoprotein. Antigen processing and presentation were not inhibited in TLR-2 ^−/− cells pretreated with the 19-kDa lipoprotein.