In this article, we present a new mouse model for the study of BO caused by low-dose allogeneic T-cell infusion in a BMT setting. Lowering the allogeneic T-cell dose in lethally conditioned mice given allogeneic BM results in histology consistent with BO, with many characteristics distinct from the early-onset, acute form that results from high T-cell doses. These characteristics are compared and contrasted in .
DIFFERENCES BETWEEN THE EARLY IDIOPATHIC PNEUMONIA SYNDROME AND LATER OCCURRING BRONCHIOLITIS OBLITERANS IN ALLOGENEIC MURINE BONE MARROW TRANSPLANT
Classically, BO is believed to manifest as airflow obstruction with air trapping; however, evidence demonstrates this to be a heterogeneous disease that can manifest with either obstructive or restrictive physiology (20
). The cause of this heterogeneity is unknown, and in humans, no correlation has been found between the degree of subepithelial collagen deposition and airflow obstruction (20
). In our murine BMT model, the histologic lesion is bronchiolar with some alveolar involvement, yet the physiology is restrictive, demonstrated by an increase in resistance and decreased compliance. A murine model of BO is complicated by an increased percentage of airways with a concomitant fewer number of alveolar units per airway as compared with humans. Subsequently, fewer alveolar units would be subjected to air trapping from small airway occlusion and the murine physiology may not mirror the classical airflow obstruction seen in humans. In our initial description of early-onset, acute mouse IPS post-BMT (13
), we demonstrated that lung dysfunction presents as reduced specific compliance, decreased TLC, and increased wet/dry lung weight ratios. An increase in lung water, as demonstrated by an increase in lung wet/dry ratios, was also seen in this BO model and may contribute to the restrictive physiology.
Typically, rodent models of BO are developed using either heterotopic (22
) or orthotopic (23
) tracheal transplants. The pathology of the airway rejection in these models is similar to that of BO involving T cells and macrophages (24
). However, heterotopic tracheal transplants develop luminal fibrosis, whereas orthotopic tracheal transplants develop subepithelial fibrosis. Orthotopically anastamosed tracheae are contiguous with recipient airways and dendritic cell and lymphocyte trafficking appears to mimic that of the host tissue (26
). The presence of both obstructive disease and subepithelial fibrosis in our current model demonstrates the potential usefulness of this murine model for the study of BO in a transplant setting (albeit, in a reverse scenario in which the lung is host-derived but the immune cells are donor-derived). The presence of an intact, vascularized whole lung in the presence of an allogeneic immune system in a BMT model is a good approximation of the lung transplant setting. The leading hypothesis is that airway epithelium is the primary target of allograft rejection (27
). Epithelial cells and antigens are shed immediately after tracheal transplant (28
) and dendritic cells carry these antigens to the draining lymph nodes where antigen-specific T cells become stimulated (29
). Similarly, we have found that the pre-BMT conditioning regimen used in our mouse model causes epithelial cell injury and that this is most severe in the presence of allogeneic T cells (9
). In lung transplant models, there is much evidence that the early damage is initiated by the ischemia–reperfusion that causes oxidative stress (30
). The conditioning regimen used in our BMT model, that includes cyclophosphamide which depletes glutathione stores, also causes oxidative stress in the peri-BMT period (31
). Therefore, the common denominators in comparing lung transplantation with our BMT model are as follows: (1
) the initial insult of oxidative stress, leading to (2
) pulmonary cell injury in the face of (3
) foreign-tissue immune challenge.
Although a kinetic analysis of the progression to BO in the lungs of the mice shown in this study was not performed, a comparison can be made among bronchioles with varying degrees of occlusion within the same lung. We observed that nonoccluded bronchioles were surrounded by inflammatory cells similar in number and phenotype to those that surrounded partially occluded airways (shown in ) (i.e., CD4+
T cells, CD8+
T cells, with a large macrophage component and few numbers of B cells and neutrophils). In many ways, this resembles the early-onset murine IPS model, with the exception of the presence of B cells, in which there is peribronchiolar cuffing. However, BO was never observed in the early-onset, acute IPS model. It has also not been reported in other mouse models of IPS (32
), including one model in which total body irradiation–only conditioning and low allogeneic T-cell doses led to a delayed onset of IPS (32
). Another difference in our model and the aforementioned ones (32
) is that our BO model is induced in a setting in which the donor is MHC class I and II disparate with the recipient. This may underscore the role of MHC in the pathogenesis of BO similar to clinical findings (1
). It should be noted that our findings were reproducible in the reverse strain combination as well—that is, C57BL/6 mice as donors and B10.BR mice as recipients. We have also successfully replicated this model using BALB/c and FVB/N mice as recipients of B6 donor cells. Therefore, the pathology seen is not mouse strain specific and should lend itself well to the study of BO using various deletional mutant mice on a C57BL/6 genetic background as either donors or recipients.
Histologically, acute murine IPS was associated with injured alveolar type II cells and increased frequencies of cytotoxic T cells (9
). In our previous studies, bronchoalveolar lavage fluid and sera of mice with acute IPS contained elevated levels of inflammatory cytokines. A comparative kinetic analysis of the inflammatory mediators in early IPS injury versus late-onset disease with BO showed that the systemic and lung-localized increase in cytokines that is induced in the peritransplant period (using this same strain combination) and that is associated with acute, early-onset IPS (9
) has subsided by the time the mice developed BO in the current model. We found significant differences between the two T-cell–dose groups for five mediators (CXCL10, CXCL8, CCL2, IL-5, IL-6). Paradoxically, these mediators were expressed at higher levels at the early Day 7 post-BMT time point in mice given the lower dose of T cells and that go on to develop BO (compared with the mice given high-dose T cells). This is contrary to what one might expect because these mediators are usually considered as inflammatory and one would predict that they would be found at higher levels in the mice given high-dose T cells that die early post-BMT. CXCL8 (KC in the mouse) was the only mediator that we found to increase with time in the post-BMT lungs. CXCL8 may play a role in neutrophil recruitment and/or vascular remodeling in airway fibroobliteration (35
). Consistent with what has been found in human lung transplant recipients with BO (36
), we did not find increased levels of TNF-α or TGF-β in the lungs of mice with BO (data not shown). This does not mean that inflammatory mediators play no role in BO, but rather, once established, BO may not be dependent on elevated levels of cytokines, at least not those that we were able to analyze. Furthermore, the temporal and spatial relationship of these cytokines may be important in the genesis of BO. Therefore, the roles of chemokines, lipid mediators, and their receptors that have been demonstrated to play roles in the pathogenesis of BO (24
), and the migration of circulating fibrocytes that mediate fibrosis (39
) in tracheal transplant models, warrant investigation in the current mouse model.
The striking loss of the Clara cell protein in the epithelium supports epithelial cells being a target for injury in our model. In addition to the phenotypic change of the epithelium, this loss of Clara cell protein likely contributes to disease progression. Clara cell protein is an inhibitor of phospholipase A2 and is believed to have both antiinflammatory and antiproliferative properties (40
). Therefore, the loss of this protein could contribute to further epithelial cell injury and cellular proliferation. In our model, proliferating cells are mostly seen in the peribronchiolar region, not in the epithelium. This increase in basal cell proliferation is consistent with the appearance of CK5 staining at the epithelial surface in the lungs with BO. CK5 is a cytokeratin associated with basal cells that lie beneath the epithelium (41
). Few CK18-positive cells were seen in the airways and may represent the remaining epithelial cells attempting to repopulate the airways or could be indicative of epithelial–mesenchymal transitional changes (42
). Further injury results in the loss of Clara cell protein and proliferation of the basal cells that subsequently repopulate the epithelial layer with eventual obliteration of the airway by fibroblasts.
In conclusion, we present a new mouse model of BO with the hallmarks of obstructive airway disease and fibrosis that will be helpful for the study of late post-HSCT pulmonary complications and lung transplant rejection.