Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG drive the primary immune response towards a type 1 cytokine production

Abstract

We showed in a previous study that the intranasal (i.n) delivery of bacille Calmette-Guérin (BCG) to BP2 mice (H-2q) inhibits eosinophilia and bronchial hyperreactivity in a mouse model of asthma. The present work has been performed to characterize the leucocyte lineages recruited to the lungs of mice after i.n. delivery of BCG and potentially involved in the polarization of T lymphocytes. The different antigen-presenting cells (APC) recruited to bronchoalveolar lavage (BAL) and to lung tissue of mice shortly after the delivery of BCG were analysed in parallel as well as their capacity to drive the immune response towards a T helper type 1 cytokine production. Alveolar macrophages (AM) from the BAL were CD11c+, F4/80+ and CD11b-, and in the lung tissue two major populations of potential APC were detected: one CD11c-, F4/80+, CD11b+ and I-Aq- was identified as interstitial macrophages (IM) and a second expressing CD11c+ and I-Aq+ antigens, negative for CD11b and F4/80 markers as leucocytic dendritic cells (DC). Freshly isolated DC up-regulated CD11b and CD40 antigens after overnight culture, but remained negative for CD8alpha antigen, suggesting a myeloid origin. Lung DC which produced high amount of interleukin (IL)-12 were potent inducers of naive CD4+ T lymphocyte priming, as assessed by interferon-gamma (IFN-gamma) production by these naive CD4+ T cells. Lung explants recovered long term after BCG delivery produced sustained levels of IFN-gamma. Our results suggest that AM and particularly DC by secreting IL-12 shortly after BCG delivery induce the long-term persistence of IFN-gamma-secreting T cells percolating in BCG-loaded lung tissue.

Figure 1

Recruitment of CD11c⁺ F4/80⁺ CD11b⁻ (AM), CD11c⁻ F4/80⁺ CD11b⁺ (IM) and CD11c⁺ F4/80⁻ CD11b⁻ (DC) to the lungs of mice shortly after i.n. delivery of BCG. Cells harvested from the BAL and from digested lung tissue 6–96 hr after i.n. delivery of BCG (10⁶ CFU) were stained with antibodies as follows: FITC- or PE-conjugated CD11c, F4/80 and CD11b. Cells expressing CD11c⁺ F4/80⁺ CD11b⁻ (AM) or CD11c⁻ F4/80⁺ CD11b⁺ (IM) or CD11c⁺ F4/80⁻ CD11b⁻ (DC) were analysed by FACScan flow cytometry. Rat IgG2a, rat IgG2b isotypes and hamster IgG were used as controls. The number of cells present in the three populations was calculated as: percentage of each subset as determined by FACS analysis × number of cells counted in the BAL or lung digest, and was expressed as the mean ± SEM. Values of P < 0·05 (*) were considered significant. The results shown are representative of three separate experiments.

Figure 2

Flow cytometric analysis of cell surface phenotype of purified lung DC freshly isolated or overnight cultured in RPMI medium. Lung DC were purified from 6 to 96 hr after i.n. delivery of BCG and stained for MHC class II I-A^q and costimulatory molecule (CD80, CD86, CD40) and DEC205, CD11b, CD8α expression. The thin open histograms represent isotype control mAb, grey histograms freshly isolated cells and bold open histograms overnight cultured cells. Data are representative of three separate experiments.

Figure 3

Laser scanning image cytometry on lung-derived DC freshly isolated or overnight cultured with RPMI medium supplemented with GM-CSF. Lung DC were purified 48 hr after i.n. delivery of CFDA-label BCG, cells were cultured for 1 h (a, b, c) or 24 hr (d, e, f) with GM-CSF. The laser scanning image cytometry allows visualization of the cells of same field with cells either unstained (a, d), monolabelled in red (I-A^q conjugated to PE) (b, e) and then double labelled in red and green (I-A^q and B7-2 conjugated, respectively, to PE and FITC) (c, f). CD11c DCs freshly isolated expressed moderate levels of I-A^q molecule (b), the expression of B7-2 was not observed (c). After overnight culture with GM-CSF the majority of the cells were double-labelled in red and green expressing I-A^q and B7-2 molecules (f). Figures are representative of two separate experiments.

Figure 4

Identification and morphology of lung DC after the i.n. delivery of BCG.Frozen lung tissue sections were stained with anti-I-A^q(a) or anti-CD11c mAb(b).Positive cells in red were easily detected in the peribronchiolar area and at a lower extent in the lung tissue. Original magnification ×20. The tissue sections shown are representative of 10 individual sections. All the CD11c enriched leucocytes purified from the lung tissue after i.n. delivery of BCG presented large nuclei and abundant cytoplasm with small cytoplasmic projections, the typical morphology of lung DC(c). A few cells had very long cytoplasmic processes, as shown in this higher magnification(d). Lung DCs purified after i.n delivery of unlabelled BCG (e) or CFDA-labelled BCG (f) were analysed by fluorescence microscopy after cytospin and Diff-Quick staining. Labelled bacteria are strongly fluorescent as compared to non labelled bacteria.

Figure 5

Phagocytic capacity and IL-12 p40 production of AM, IM and DC isolated from BAL and lungs of mice shortly after i.n. delivery of BCG.(a) BCG bacilli were labelled with CFDA, and 1, 6, 24 and 48 hr after their i.n. delivery, AM were obtained by bronchoalveolar lavage and IM and DC were isolated from digested lung tissue, followed by incubation with MACS CD11b or CD11c microbeads and passage through a column of an AutoMACS separator. Cytospins of purified AM, IM and DC were stained with Diff-Quick and the bacilli taken up by phagocytosis were visualized by fluorescence microscopy. Two hundred to 400 cells were counted and the number of cells that were phagocytic (more than two bacilli) is expressed as a percentage of the whole population studied. The experiment is representative of three separate experiments. (b) AM, IM and DC purified from naive mice and 6, 24, 48 and 96 hr after BCG delivery, were cultured for 24 hr and supernatants were assayed by ELISA for of IL-12 p40 production. IM were not able to produce IL-12 p40 and DC produced significantly higher amounts of IL-12 than AM (P < 0·05). The experiment shown is representative of three separate experiments. Results are expressed as mean ± SEM.

Figure 6

IFN-γ production by CD4⁺ T cells stimulated with APC (IM, AM or DC) isolated from the lungs of mice, shortly after i.n. delivery of BCG. Purified CD4⁺ T cells from lymph nodes of naive or BCG-immunized mice were cocultured for 3 days with AM, IM or DC isolated 24, 48 and 96 hr after i.n. delivery of BCG from BAL and lungs, respectively. A 1 : 5 ratio of APC/CD4⁺ T lymphocytes was used. Supernatants were assayed by ELISA for IFN-γ content. When APC were cocultured with CD4⁺ T cells from naive mice, IMs induced no IFN-γ production, AM very low levels, whereas DC allowed the production of high amounts of IFN-γ by these CD4⁺ T cells (a). When APC were cocultured with immune CD4⁺ T cells isolated from BCG-immunized mice, the IFN-γ production was found only if CD4⁺ T cells were stimulated with AM or DC. AM were less efficient than DC for initiating the production of IFN-γ by CD4⁺ T cells (P < 0·05), whereas IM failed to stimulate the production of IFN-γ(b). The addition of anti-IL-12 p40 mAb completely abrogated the production of IFN-γ(c). The experiment shown is representative of two separate experiments. Results are expressed as mean ± SEM.

Figure 7

Long-term production of IFN-γ and IL-5 by lung explants exposed to anti-CD3 mAb. Lung explants were prepared from 14 to 116 days after i.n. delivery of BCG. After stimulation with soluble anti-CD3 mAb, the secretion of IL-5 and IFN-γ peaked, respectively, at 6 and 24 hr culture period. These intervals were used for monitoring IL-5 and IFN-γ production by the explants. From 28 to 90 days the production of IFN-γ was significantly higher in BCG-immunized than in control mice (P < 0·05)(a). There were no significant differences in the production of IL-5 among the two groups of mice(b). Results are expressed as mean ± SEM.