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. 2014 Mar 15;192(6):2821-9.
doi: 10.4049/jimmunol.1302269. Epub 2014 Feb 17.

In vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: implications for understanding lung disease in humans

Yanhui Cai  1 , Chie Sugimoto, Mariluz Arainga, Xavier Alvarez, Elizabeth S Didier, Marcelo J Kuroda
Affiliations

In vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: implications for understanding lung disease in humans

Yanhui Cai et al. J Immunol. .

Abstract

Alveolar macrophages (AMs) obtained by bronchoalveolar lavage (BAL) are commonly used to study lung macrophage-mediated immune responses. Questions remain, however, about whether AMs fully represent macrophage function in the lung. This study was performed to determine the contribution of interstitial macrophages (IMs) of lung tissue to pulmonary immunity and that are not present in BAL sampling. In vivo BrdU injection was performed to evaluate the kinetics and monocyte/tissue macrophage turnover in Indian rhesus macaques (Macaca mulatta). Lung macrophage phenotype and cell turnover were analyzed by flow cytometry and immunohistochemistry. AMs and IMs in lungs of rhesus macaques composed ∼70% of immune response cells in the lung. AMs represented a larger proportion of macrophages, ∼75-80%, and exhibited minimal turnover. Conversely, IMs exhibited higher turnover rates that were similar to those of blood monocytes during steady-state homeostasis. IMs also exhibited higher staining for TUNEL, suggesting a continuous transition of blood monocytes replacing IMs undergoing apoptosis. Although AMs appear static in steady-state homeostasis, increased influx of new AMs derived from monocytes/IMs was observed after BAL procedure. Moreover, ex vivo IFN-γ plus LPS treatment significantly increased intracellular expression of TNF-α in IMs, but not in AMs. These findings indicate that the longer-lived AMs obtained from BAL may not represent the entire pulmonary spectrum of macrophage responses, and shorter-lived IMs may function as the critical mucosal macrophage subset in the lung that helps to maintain homeostasis and protect against continuous pathogen exposure from the environment.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Fig 1
Fig 1. Identification of AM in BAL
Results in Panel A demonstrate the differential cell counts of BAL specimens from 11 healthy adult rhesus macaques. Cells recovered from BAL were stained, analyzed and sorted into three populations (P1, P2 and P3) by 3-laser FACSAria (Becton Dickinson) based on expression of HLA-DR and CD11b (myeloid cell markers) as shown in Panel B. Cytospins were prepared from each sorted fraction, stained with Wright-Giemsa, and imaged under 400X magnification. Results shown are representative of studies from three monkeys.
Fig 2
Fig 2. Macrophages are predominant cells of the immune system in healthy lung
Panel A shows the gating strategy for analyzing cells isolated from lung tissue of healthy rhesus macaques. CD11b+ staining cells were considered to be of myeloid lineage. Granulocytes (CD11b+, HLA-DR−) were separated after first excluding lymphocytes that stained with CD3/20/8. Then HLA-DRhi, CD11b+, CD163+, CD206+ cells were defined as AM. IM were identified as HLA-DRhi, CD11bhi, CD163+ cells. mDC were identified as CD11c+, HLA-DR+, CD163−, CD206− and pDC were identified as CD123+, HLA-DR+, CD163−, CD206−. Lymphocytes were small cells (SSC−) and were further divided into CD3/20+ cells including CD4+ T cells, CD8+T cells and B cells (CD4−, CD8−, HLA-DR+), whereas CD3/20− cells comprised CD8+, CD16+ NK cells and CD8+, CD16− NK cells. A small subset of cells not identified with these markers was labeled UN (unidentified cells). The mean percent values (± st. dev.) of each cell population were determined from lung tissues of five rhesus macaques as shown in Panel B.
Fig 3
Fig 3. Confocal microscopy for the identification of AM and IM in healthy rhesus macaque lung tissue
Tissue sections from three different areas of the lung were analyzed: alveolar/interlobular (A&D), peribronchovascular (B&E) and subpleura (C&F). CD163 (macrophage marker, green), caveolin-1 (endothelium marker, red) and ToPro-3 (nucleic acid, blue) were used in Panels A, B, and C. CD206 (mannose receptor (AM marker), red), CD163 (green) and ToPro-3 (blue) were used in Panels D, E, and F. White arrows (→) indicate IM (CD163+ cells) outside the vessels. Stars (*) indicate AM (CD163+CD206+) that were located exclusively in the alveolar space. Sections shown are representative of three monkeys and were imaged using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems) at 400x final magnification under oil (40X objective, fluotar/NA 1.0).
Fig. 4
Fig. 4. Phenotype differences between AM, IM and monocytes
Blood monocytes, IM and AM were stained with antibodies (Supplemental Table IA) for flow cytometry analyses. Black lines represent isotype control antibody staining and the filled gray lines indicate specific antibody staining. The histograms are representative of at least three healthy rhesus macaques. The results demonstrate that monocytes, IM and AM could be discerned from each other, but monocyte and IM were relatively similar to each other.
Fig 5
Fig 5. IM but not AM exhibit high turnover during steady state homeostasis
Cell turnover was reflected by measuring the uptake of BrdU by AM, IM or monocytes in relation to the total population. As shown in Panel A, BrdU staining was highest in monocytes and IM 48 hr after BrdU injection and was low in AM after 48 hr and in monoctyes after 24hr (n=4). Staining 24 hr after BrdU injection was determined to be a good measure for the production of monocytes and emigration into the blood from bone marrow, so this time point was used to define monocyte turnover (19). Confocal microscopy in Panel B confirmed that high turnover of IM and negligible turnover of AM occurs during steady state based on triple-label confocal microscopy staining for CD163 (red), CD206 (green) and BrdU (that identifies recently-arrived cells, blue). Stars (*) indicate CD163+CD206+ AM with no BrdU staining. Arrows (→) indicate CD163 single positive IM stained with BrdU. This experiment was performed using samples collected from four different uninfected monkeys necropsied two days after BrdU injection. Results in Panel C indicated that a significantly higher percent of IM than AM were undergoing apoptosis as measured by TUNEL staining. A confocal microscopy image in Panel D shows apoptotic (blue), CD163+ (green) macrophages in normal lung tissue and demonstrates high turnover of IM in relation to increased apoptosis. Arrows (→) indicate CD163 single positive IM stained with TUNEL. Stars (*) indicate AM (CD163+) with no TUNEL staining. Confocal images were acquired under an oil objective (63X, fluotar/NA 1.0) and are representative of studies from four monkeys (A&C).
Fig 6
Fig 6. Monocytes/IM are precursors to AM
Rhesus macaques were injected with BrdU or EdU nucleic acid analogues and stained for uptake and macrophage markers as indicated. In the first experiment, BAL was performed on day 2 to remove AM and again on day 7 (i.e. five days later) to follow the turnover of the AM that were repopulating the alveolar space. Results in Panel A (n=8) demonstrated significant increases in repopulating AM turnover 5 days after mechanical removal of AM via BAL. If no initial BAL is performed on day 2, AM turnover was observed to remain low on day 7 as shown in Panel B (n=6). A macrophage undergoing transition from IM to AM is shown in Panel C and exhibits expression of macrophage markers CD68 (blue), CD206 (green) and CD163 (red). The image was captured under oil immersion (63X, fluotar/NA 1.0).
Fig 7
Fig 7. Ex vivo macrophage activation signaling with IFN-γ plus LPS significantly increased TNF-α expression in IM but not in AM
Adherent single cell isolates from the lung of uninfected rhesus macaque were stimulated with LPS and IFN-γ ex vivo for 4hr prior live/dead and antibody staining for flow cytometry analysis. Results in Panel A demonstrated the gating strategy for AM and IM, as well as representative histograms for the intracellular staining of TNF-α in AM and IM post stimulation from three uninfected monkeys. Results in Panel B show the mean fold-change (± st. dev.) in intracellular expression of TNF-α in AM and IM after LPS and IFN-γ treatment compared to untreated or IFN-γ alone controls (n=3).
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