Failure of bone marrow to reconstitute lung epithelium

Abstract

A new paradigm of epithelial tissue reconstitution has been suggested whereby circulating cells derived from bone marrow contribute to a variety of epithelial cell types. With regard to the lung, several recent reports have used immunofluorescence microscopy to demonstrate engraftment of bone marrow-derived cells as type II pneumocytes, the endogenous progenitors of the lung alveolus. We show here that immunofluorescence microscopy, as has been used in previous reports, cannot reliably identify rare engrafted cells in lung tissue sections after transplantation of bone marrow cells or purified hematopoietic stem cells tracked with ubiquitous labels. We have employed a lineage-specific reporter system based on transgenic mice that express the GFP reporter gene only in lung epithelial cells (surfactant protein C-GFP) to assay for engrafted cells by flow cytometry, histology, and molecular methods. Using this approach to evaluate transplant recipients, including those subjected to bleomycin-induced lung injury, we demonstrate that when autofluorescence, dead cells, and contaminating blood cells are excluded from analysis, there is no detectable reconstitution of lung alveolar epithelial cells by unfractionated bone marrow cells or purified hematopoietic stem cells.

Figures

Figure 1.

Transplantation of unfractionated bone marrow ubiquitously labeled with GFP (B-actin–GFP). Representative lung frozen tissue section from a recipient analyzed 1 yr after transplantation of 10 million unfractionated GFP+ bone marrow cells. At the time of analysis this recipient had 88% GFP+ peripheral blood chimerism. (A) DAPI nuclear counterstaining (blue); (B) immunostaining with an antibody against a specific alveolar type II (AT2) pneumocyte cell marker (Cy3 anti-pro–SP-C; red); and (C) fluorescence of GFP (B-actin–GFP; green) are merged in D, illustrating the 1 cell (of 200 AT2 cells examined on this section) that appeared to express both markers (arrows). The number of cells that are “double-labeled” with GFP fluorescence and AT2-specific red immunostaining in this recipient lung is no different than the number of cells per section that show background autofluorescence on negative control sections (shown in Figure 3 below).

Figure 2.

Hematopoietic reconstitution by a single GFP+ bone marrow stem cell. A single hematopoietic stem cell was purified from the bone marrow of a B-actin–GFP donor mouse, and 1 yr later recipient blood and lung tissue were harvested for analysis. (A) FACS histogram illustrates robust hematopoietic reconstitution of 79% of the recipient's peripheral blood with donor-derived GFP+ cells. (B) FACS-analysis of live cells (PI-negative) from the left lung of this recipient, shows the presence of GFP+ marrow-derived cells (25% of the lung cell suspension). (C) FACS-analysis of the GFP+ and GFP(−) subgates of the lung cell suspension from B demonstrates that 99% of the GFP+ cells (green histogram) express the panhematopoietic marker CD45. In contrast, 22% of GFP(−) cells in this sample are CD45+ (black histogram). An aliquot of this sample stained with a negative control antibody of identical isotype illustrates specificity of the CD45 antibody staining (purple histogram). Similar engraftment results were obtained from recipients that received either unfractionated marrow or 200 purified HSC transplants. (D) A frozen tissue section prepared from the right lung of this recipient was immunostained with Cy3-conjugated anti–pro-SP-C antibody showing that AT2 cells (red) and donor marrow–derived cells (green) are distinct, with the possible artifactual exception of two cells whose tips appear to overlap (arrow).

Figure 3.

Distinguishing autofluorescent artifact from engraftment is not possible with dual channel immunofluorescence microscopy. (A) Lung frozen tissue section from a wild-type mouse that has not been exposed to antibody staining shows rare cells that exhibit equal red and green autofluorescence (arrows). (B) Recipient lung after bone marrow transplantation from a B-actin–GFP mouse (no antibody exposure). Donor-derived cells in this section fluoresce green but not red, indicating true GFP+ fluorescence. Nuclei are counterstained with DAPI.

Figure 4.

Assessment of bone marrow–derived lung engraftment after transplantation of bone marrow cells from the SP-C–GFP lineage-specific reporter mouse. (A) Bright GFP fluorescence only in alveolar type II (AT2) cells is seen in lung frozen tissue sections from the transgenic (positive control) mouse. (B) Representative lung section from a recipient mouse 4 mo after transplantation of 10 million unfractionated bone marrow cells obtained from a donor SP-C–GFP mouse. No GFP+ engrafted cells are seen. (C) Peripheral blood analyzed from this same recipient shows 93% blood chimerism (red box) arising from the donor (CD45.2) transplanted marrow cells (recipient is a CD45.1 congenic mouse). (D) FACS analysis of 1 million live (propidium iodide excluding) lung cells from a SP-C–GFP transgenic mouse (positive control) shows GFP+ fluorescence in 3.56% of cells. (E) Representative FACS of 1 million live lung cells from a recipient transplanted with SP-C–GFP bone marrow shows no GFP+ events. (F) Representative analysis of RNA and DNA extracts from six recipients' lungs shows donor-derived cells are present in recipients' lungs (GFP DNA+), but are not AT2 cells because they do not express GFP mRNA. Controls are labeled as: “+” (SP-C-GFP transgenic lung); WT (wild-type lung); −RT (RNA extract from SP-C–GFP lung minus reverse transcription). RT-PCR of the housekeeping gene β-actin illustrates that cDNA is present in each sample.