Evidence that bone marrow cells do not contribute to the alveolar epithelium

Abstract

An ongoing controversy is the role of marrow cells in populating the alveolar epithelium. In this study, we employed flow cytometry and histologic techniques to evaluate this process. Donor bone marrow was harvested from transgenic mice expressing the LacZ or eGFP gene ubiquitously, or under the control of the human surfactant protein (SP)-C promoter, and transplanted into lethally irradiated, neonatal mice. In recipients transplanted with marrow that express eGFP or lacZ ubiquitously, light microscopy revealed cells whose morphology and location were compatible with a type II cell phenotype. Consistent with this, fluorescent microscopy suggested colocalization of eGFP and pro-SP-C proteins in single cells. In mice transplanted with SP-C-eGFP marrow, engraftment was not detectable by histology or flow cytometry. We therefore used deconvolution microscopy to reanalyze histologic sections that were thought to show marrow-derived type II cells. We found that all putative marrow-derived pneumocytes resulted from the overlapping fluorescent signals of an endogenous pro-SP-C+ type II cell and a donor-derived eGFP+ cell. Taken together, our observations underscore the technical difficulties associated with evaluating engraftment in lung, and argue against a contributory role for marrow cells in populating the alveolar epithelium.

Figures

Figure 1.

Light microscopy analysis of alveolar engraftment after transplantation with ubiquitous lacZ or eGFP bone marrow. (A) X-gal staining was performed on lungs from mice 1 mo after transplantion with LacZ bone marrow, to label marrow-derived cells blue. In this representative section, an X-gal+ cell is located in the corner of the alveolus. Its compact, granular morphology is suggestive of the type II epithelial cell. (B) In mice transplanted with actin-eGFP bone marrow, IHC was performed to localize eGFP. In this section taken from a mouse 1 mo after transplantation, an eGFP+ cell is shown whose location and shape is compatible with the type II cell. (C–E) Dual IF staining against pro–SP-C (red) and eGFP (green) was performed in mice transplanted with actin-eGFP bone marrow. Nuclei were counterstained with DAPI. In the composite image, co-localization of the green eGFP+ and red pro–SP-C fluorescent signals results in a yellow color. All sections: 5 μm, ×1,000 magnification. Size bar = 20 μm.

Figure 2.

Flow cytometry analysis of alveolar engraftment after transplantation with ubiquitous eGFP bone marrow. From 1 mo to 3 mo after transplantation, lungs from recipient mice were enzymatically dispersed, fixed with paraformaldehyde, then sequentially incubated with a pro–SP-C polyclonal antibody and a PE-conjugated secondary antibody. In these density dot plots, the circled region denotes eGFP+/pro–SP-C+ cells. In the actin-eGFP mouse (A), 70% of the pro–SP-C+ cells expressed eGFP. In a representative mouse analyzed 1 mo after transplantation (B), 1% of the pro–SP-C cells were eGFP+, suggesting that they derived from the bone marrow.

Figure 3.

Lack of alveolar engraftment by cytocentrifuge analysis. After proteolytic lung digestion, cytocentrifuge slides were prepared and immunostained for pro–SP-C (red) and eGFP (green) in (A) actin-eGFP mouse and (B) recipient mouse transplanted with actin-eGFP marrow. In the actin-eGFP mouse, two eGFP+, pro–SP-C+ cells are identified by white arrows. Note size of cells and the granular staining of pro–SP-C. By contrast, all eGFP+, pro–SP-C+ lung cells in transplanted mice did not have the appearance of a type II cell (hatched arrow in B). These cells were smaller, lacked granular staining, and possessed scant cytoplasm. Two neighboring eGFP-negative cells exhibit size and granular pro–SP-C staining compatible with type II cells. Next, we collected eGFP+ lung cells by flow cytometry and performed PAP staining. While type II cells were readily identified by this method among eGFP+ cells from the actin-eGFP mouse (C), no type II cells were found among eGFP+, marrow-derived lung cells from a mouse transplanted with actin-eGFP marrow (D). Size bar = 10 μm.

Figure 4.

Lack of alveolar engraftment by flow cytometry after transplantation with SP-C–eGFP bone marrow. Bone marrow was procured from donor mice expressing eGFP under the control of the human SP-C promoter. From 2 wk to 3 mo after transplantation, lungs were enzymatically dispersed and treated with PI to exclude dead cells (90% live). During flow cytometry, density dot plot analysis of the PI-negative fraction demonstrated the presence of eGFP-expressing cells in a nontransplanted, SP-C–eGFP mouse (A), but no eGFP+ cells in mice transplanted with SP-C–eGFP bone marrow (B; n = 6) or negative-control mice transplanted with wild-type bone marrow (C; n = 4). Representative images here were taken from mice 1 mo after transplantation.

Figure 5.

Lack of alveolar engraftment by histology after transplantation with SP-C–eGFP bone marrow. From 2 wk to 3 mo after transplantation with SP-C–eGFP marrow, lungs of recipient mice were examined histologically. Lungs were analyzed for endogenous eGFP fluorescence in frozen, unfixed sections (A–C, ×400 magnification; size bar = 50 μm). In paraffin-embedded sections, we performed eGFP immunoperoxidase IHC (D–F, × 400 magnification; size bar = 50 μm) and eGFP immunofluorescent staining (IF) (G–I, × 100 magnification; size bar = 200 μm). By all methods, eGFP+ type II cells were detectable in lung sections from the donor SP-C–eGFP mouse (A, D, G). In mice transplanted with SP-C–eGFP bone marrow, eGFP+ cells were not seen (B, E, H; n = 8). Negative-control mice were transplanted with wild-type bone marrow (C, F, I; n = 6). Representative images here were taken from mice 1 mo after transplantation.

Figure 6.

Comparison of conventional and deconvolution microscopy. Using deconvolution microscopy, we reanalyzed lung sections from mice transplanted with actin-eGFP marrow, that had been thought to show alveolar epithelial engraftment by marrow cells. (A) Viewed under conventional widefield fluorescent microscopy at ×1,000 magnification, the cell in the center appears to co-localize eGFP (green) and pro-SP-C (red) proteins. Size bar = 20 μm. (B) Same section viewed under deconvolution microscopy, size bar = 15 μm. (C) Three-dimensional reconstructed image which has been rotated slightly for better visualization. Fluorescent red signal from pro-SP-C actually is outside the eGFP+ cell.

Figure 7.

Three-dimensional reconstruction by deconvolution microscopy. (A–D) Different rotational views taken of a presumptive marrow-derived type II cell from a mouse transplanted with actin-eGFP bone marrow. Though the eGFP and pro–SP-C signals initially appear merged, C reveals that the pro–SP-C protein actually resides outside the eGFP+ cell (white arrow). The distance between the pro–SP-C protein and the outer edge of the eGPP+ cell was ∼ 300 nm. By contrast, rotational viewing in the SP-C–eGFP mouse (E–H) shows co-localization in all images. For each cell analyzed by deconvolution, 60 rotational views were examined to evaluate for co-localization. Size bar = 15 μm.