Local origin of mesenchymal cells in a murine orthotopic lung transplantation model of bronchiolitis obliterans

Abstract

Bronchiolitis obliterans is the leading cause of chronic graft failure and long-term mortality in lung transplant recipients. Here, we used a novel murine model to characterize allograft fibrogenesis within a whole-lung microenvironment. Unilateral left lung transplantation was performed in mice across varying degrees of major histocompatibility complex mismatch combinations. B6D2F1/J (a cross between C57BL/6J and DBA/2J) (Haplotype H2b/d) lungs transplanted into DBA/2J (H2d) recipients were identified to show histopathology for bronchiolitis obliterans in all allogeneic grafts. Time course analysis showed an evolution from immune cell infiltration of the bronchioles and vessels at day 14, consistent with acute rejection and lymphocytic bronchitis, to subepithelial and intraluminal fibrotic lesions of bronchiolitis obliterans by day 28. Allografts at day 28 showed a significantly higher hydroxyproline content than the isografts (33.21 ± 1.89 versus 22.36 ± 2.33 μg/mL). At day 40 the hydroxyproline content had increased further (48.91 ± 7.09 μg/mL). Flow cytometric analysis was used to investigate the origin of mesenchymal cells in fibrotic allografts. Collagen I-positive cells (89.43% ± 6.53%) in day 28 allografts were H2Db positive, showing their donor origin. This novel murine model shows consistent and reproducible allograft fibrogenesis in the context of single-lung transplantation and represents a major step forward in investigating mechanisms of chronic graft failure.

Supplemental Figure S1

Loss of club cells in airways during allograft fibrogenesis. Immunofluorescent staining for club cell secretory protein (CCSP) was used to evaluate epithelial changes in allografts. Representative images of the allografts (B6D2F1/J to DBA/2J) at various time points (days 7, 14, 28, and 40) after transplantation are shown. CCSP staining is shown in red. Scale bars: 100 μm. Original magnification: ×200.

Figure 1

Development of bronchiolitis obliterans in murine lung allografts. A: Comparison of murine lung allografts transplanted across varying degrees of major histocompatibility complex mismatch. Representative gross anatomy and hematoxylin and eosin images at day 28 after transplant. B: Time course of histologic changes. Histopathology of isografts (DBA/2J to DBA/2J) and allografts (B6D2F1/J to DBA/2J) studied at various time points (days 7, 14, 28, and 40) after transplantation. Changes consistent with mild acute rejection and lymphocytic bronchitis (mononuclear cell infiltration in the perivascular and peribronchial areas) were noted at day 7. Day 14 images show marked perivascular, intraluminal, and peribronchial infiltrate of lymphocytes, neutrophils, and some eosinophils; intraepithelial lymphocytic infiltration also is noted. Epithelial denudation with subepithelial fibrosis is seen at day 28. This section also shows intraluminal fibrosis in the airway. Bronchiolitis obliterans with peribronchial fibrosis, smooth muscle hypertrophy, and luminal narrowing is seen in allografts at day 40. Isografts showed normal histology at all time points. Original magnification: ×40 (A); ×200 (B).

Figure 2

Characterization of kinetic changes in immune cell infiltration in the transplanted lung grafts. A: Immunohistochemical staining for T cells (CD3) and macrophages (F4/80) in the allograft (B6D2F1/J to DBA/2J) model at various time points (days 7, 14, 28, and 40) after transplantation. T-cell and macrophage infiltration of the peribronchiolar area is noted along with perivascular and intraluminal infiltration. B: Immunophenotyping of infiltrating immune cell populations in allografts (B6D2F1/J to DBA/2J). Single-cell suspension of day 14 isografts (n = 3; black bars), day 14 allografts (n = 3; white bars), and day 28 allografts (n = 4; grey bars) were stained and analyzed by flow cytometric analysis to quantitate infiltrating CD45⁺ leukocytes, CD4⁺ T cells (CD4⁺), CD8⁺ T cells (CD8⁺), and B cells (CD19⁺). To identify myeloid cells, initial gates eliminated CD3⁺ and CD19⁺ lymphocytes and Ly-6G⁺ granulocytes. Subsequent gates identified CD11b⁺ dendritic cells (FSC^{moderate/high} nonautofluorescent CD11c⁺ CD11b⁺), monocyte-derived exudate macrophages (FSC^high autofluorescent CD11c⁺ CD11b⁺), and alveolar macrophages (FSC^high autofluorescent CD11c⁺ CD11b⁻). Total cell numbers were obtained by multiplying the cell frequency by the total number of CD45⁺ leukocytes for each lung. ^∗P < 0.05, ^∗∗P < 0.01. All comparisons were made with isografts other than when specified by the bar below the asterisk. Original magnification: ×200 (A).

Figure 3

Time course of fibrogenesis in the lung allografts. A: Masson’s trichrome staining was used to evaluate collagen expression in lung sections. Representative images of the allografts (B6D2F1/J to DBA/2J) at indicated time points after transplantation. Collagen staining (blue) is noted, centered around the airways and vessels, by day 28. Subepithelial collagen expression is noted in both the larger and the smaller terminal bronchioles. Some airways show intraluminal collagen expressing fibrous plugs. Collagen appears denser at day 40. B: Collagen content quantitation by hydroxyproline assay. Lung grafts were harvested on specified days and analyzed for hydroxyproline content. Results are reported as the mean concentration (μg/mL) ± SEM (n = 3 to 5 mice in each group with experiments repeated in duplicates). ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

Morphometric assessment and determination of donor versus recipient origin of collagen-expressing cells in the lung grafts. A: Morphometric analysis of airway wall thickness in transplanted lungs. The difference in the area, delimited by the basement membrane and the outer edge of the airway adventitia, divided by the length of subepithelial basement membrane, was measured for all airways in lung sections from individual grafts. Data from each individual graft are shown as floating bars from minimum to maximum, with a line at the mean. Data from five isografts (B6D2F1/J to B6D2F1/J), five minor major histocompatibility complex mismatch transplants (C57BL10 to C57BL6), and 10 F1 to parent transplants (B6D2F1/J to DBA/2J). B: Morphometric analysis of airway obliteration in transplanted lungs. A conventional point counting method was used to quantitate open airway lumens in a lung field. The number of points intersecting airway lumens in the graft and native lung in the same section were counted and expressed as a fraction. Data from each individual transplanted mouse are shown as the mean of the data from four separate fields. N = 5 isografts, 5 C57BL/10 to C57BL/6 allografts, and 10 B6D2F1/J to DBA/2J allografts. C: Donor origin of collagen-expressing cells in fibrotic lung allografts. Unilateral left lung transplantation was performed with B6D2F1/J [a cross between C57BL/6J (H2-Db) and DBA/2J (H2-Dd)] as donors and DBA/2J (H2-Dd) as recipients. Flow cytometric analysis for expression of H2-Db, a major histocompatibility complex class I molecule expressed only in donor mouse in this combination, was used to investigate donor versus recipient origin of collagen-expressing cells in the allografts. Flow cytometric analysis of single-cell suspension of the lungs from the three species confirms a lack of H2-Db expression in the recipients and its positive expression in the donor B6D2F1/J lungs. Expression of H2-Db on collagen-expressing cells was quantified in native lungs and allografts at day 28. Collagen⁺ cells are predominantly H2-Db positive in the allografts. n = 3 transplanted animals. ^∗∗∗∗P < 0.0001. APC, allophycocyanin; FSC, forward side scatter; PE, phycoerythrin.