PERSPECTIVE
Mechanisms of Lung Injury after Bone Marrow Transplantation

Rodney J. Folz

Division of Pulmonary and Critical Care, Department of Medicine, Duke University Medical Center, Durham, North Carolina

Dose-intensive chemotherapy combined with localized or total body irradiation (TBI) followed by autologous or allogeneic hematologic stem-cell transplantation is increasingly being used to treat a number of solid tissue and hematologic malignancies. In the case of some hematologic cancers (e.g., AML, ALL, CML, and CLL), which are refractory to conventional dose chemotherapy, several studies now report that allogeneic transplantation offers increased cure rates and increased long-term survival (1). Even patients with nonmalignant chronic conditions, such as aplastic anemia, paroxysmal nocturnal hemoglobinuria, sickle cell, and thalassemias (4), as well as those with autoimmune disorders (7, 8), are being considered for hematogenous stem-cell transplantation. At some centers, autologous stem-cell transplantation after high-dose chemotherapy is frequently performed for advanced stage and high-risk breast cancer (9).

Despite the acclaim and promise of this emerging combined pharmacologic and immunologic technology, side effects remain one of the major limitations to its success. In some cases, those side effects can limit an individual's future therapeutic options. In a majority of regimens, noninfectious forms of pulmonary toxicity (see Table 1) (infectious pneumonias are not addressed in this perspective) all too often inflict unwanted complications, resulting in increased morbidity and mortality. The incidence of noninfectious pulmonary toxicity after bone marrow transplantation has been estimated to be 12 to 70%, and much depends on the specific patient population being analyzed, the details of their respective therapeutic regimen, and the criteria used to define lung toxicity. In 1991, the National Heart, Lung, and Blood Institute held a workshop to review clinical-pathologic features of idiopathic pneumonia syndrome (IPS), a severe form of lung toxicity seen in about 12% of allogeneic marrow transplant recipients (10). The diagnosis of IPS in this setting requires evidence of widespread alveolar injury, as well as an absence of an active lower respiratory tract infection. However, less severe forms of lung toxicity, not meeting this criteria, often occur after autologous transplant. For example, delayed pulmonary toxicity syndrome (DPTS) develops in up to 64% of patients who undergo high-dose chemotherapy and autologous bone marrow transplantation for advanced stage breast cancer, and DPTS is best diagnosed on the basis of reductions in serial DL_CO measurements (11). What challenges a clinical investigator's ability to dissect out important factors in the development of lung toxicity is the myriad of variables specifically assigned to an individual's treatment regimen. Table 2 outlines a partial list of variables that are likely to play important roles in the pathogenesis of post-BMT lung toxicity (12, 13).

Further limiting our understanding of the pathophysiology of lung toxicity has been the lack of animal models. Recently, however, several groups have begun characterizing mouse models of IPS that appear to mimic the human disease process. Shankar and colleagues (14) at the University of Kentucky have developed a mouse model of IPS that utilizes a semi-allogeneic parental  F1 transplant strategy after a conditioning regimen of TBI to induce a mild form of graft versus host disease (GVHD). Several features of this model appear particularly relevant to the human condition. First, these animals develop progressive lung injury over a 3- to 12-wk period that is pathologically characterized by prominent perivascular and peribronchiolar inflammation with diffuse alveolar interstitial mononuclear cell inflammation. Second, the acute phase of this GVHD appears mediated by CD8⁺ cells, whereas CD4⁺ cells are associated with the chronic form of GVHD. Third, these animals develop mild, localized interstitial fibrosis at the later time points analyzed.

What features are necessary for the development of lung disease in this model? Shankar and associates (15) begin to answer this question in this current issue, but in doing so, generate additional questions. First, the kinetics of pulmonary injury seen in this model are unique when compared with that of other tissues (including liver, ear, skin, colon, and tongue) in that the temporal progression develops relatively slowly over a 9- to 25-wk time frame, whereas the other extrapulmonary target organs analyzed showed maximal GVHD within 3 wk posttransplant followed by resolution over the remaining time frame. Second, preconditioning irradiation was found to be necessary for the development of IPS on the basis of several observations. The degree of lung injury showed irradiation dose-dependency. Animals that were not pretreated with conditioning irradiation showed no lung injury, whereas those given increasing irradiation showed progressively increased degrees of lung injury. This requirement for preconditioning irradiation and the subsequent development of lung injury could be overcome, only slightly, by infusing nonirradiated animals with tenfold higher levels of donor spleen T cells, resulting in low level GVHD in the lung. The development of lung injury was not due to radiation lung injury alone because animals treated with the maximal dose of TBI, but not given allogeneic spleen cells, showed no demonstrable lung pathology.

As nicely controlled as these studies are, the reader is left with the burning question of what is/are the biochemical/cellular mechanism(s) by which the preconditioning regimen primes the lung for the subsequent development of lung toxicity after allogeneic BMT. Several hypotheses can be proposed. TBI modulates tissue levels of critical inflammatory cytokines or mediators. Alternatively, TBI exacerbates free radical or oxidative injury in the target organ. To address the question of mechanism, Dr. Blazar and colleagues (16) at the University of Minnesota have developed a slightly different and more acute murine model of IPS whose main difference with that of the previously described University of Kentucky model is the addition of cyclophosphamide to the conditioning regimen. In this model, lung injury manifests at Day 3 posttransplant and is characterized by an influx of T cells, macrophages, and neutrophils. Furthermore, the macrophage activation was shown to be dependent on allogeneic T cells.

To further probe biochemical mechanisms responsible for IPS in this mouse model, Haddad and colleagues, also in this issue (17), proposed the hypothesis that alveolar macrophages are primarily responsible for the development of lung injury via enhanced generation of the toxic compound peroxynitrite. In this scenario, two independent events occur. First, allogeneic T cells are shown to induce nitric oxide synthase levels in alveolar macrophages, resulting in increased NO production. Secondly, cyclophosphamide stimulates superoxide production in alveolar macrophages (18). It is this combination of increased superoxide and nitric oxide levels that favors the formation of peroxynitrite. Peroxynitrite is a strong oxidant and can oxidize a number of biomolecules, including tyrosine-containing proteins, which results in nitrotyrosine formation. Haddad and associates demonstrated markedly increased nitrotyrosine formation in bronchoalveolar lavage (BAL) protein constituents. However, it should be pointed out that alternative pathways exist for the formation of nitrotyrosine independent of peroxynitrite. For example, nitrite can be oxidized by several cellular peroxidases (heme peroxidases, horseradish peroxidase, myeloperoxidase, and lactoperoxidase) in the presence of hydrogen peroxide to form NO·₂, which, in turn, can contribute to nitrotyrosine formation (19).

As intriguing as these allogeneic models are, they do not necessarily address the delayed pulmonary toxicity seen in patients who undergo autologous BMT (11, 20). These autologous regimens lack alloreactive T-cell infusions and preconditioning irradiation, and therefore GVHD does not take place. Yet the mortality and morbidity from pulmonary injury associated with these regimens remain substantial. In these scenarios, it is likely that lung toxicity is directly related to the conditioning chemotherapy. In the example listed above, for instance, the three drugs used for this high-dose chemotherapy regimen (BCNU, cyclophosphamide, and cisplatin) are known to be cytotoxic to the lung; however, the exact mechanism by which these drugs cause lung damage is not known. We, and others, have proposed that these chemotherapeutic agents have a disproportionate effect on the lung and promote an imbalance in oxidative stress and antioxidant reserves. Whether the delayed pulmonary toxicity seen in these patients remains part of the spectrum of IPS or if it is a separate entity still needs to be clarified.

What are some future directions aimed at a better understanding of IPS and a reduction of pulmonary toxicity after BMT? It is clear that animal models of specific transplant-related regimens, such as those described by Haddad and colleagues (17) and by Shankar and coworkers (15) are needed to critically define the contribution of specific variables in the pathogenesis of IPS after BMT. Perhaps adult organ lung culture systems may facilitate this process and minimize the number of animals needed. However, these tissue culture systems have inherent disadvantages and may oversimplify the complex interactions of pulmonary tissues with systemic inflammatory mediators, vascular perfusion, and immunobiology interactions. Transgenic mice manipulating the levels of key lung proteins and/or enzymes, such as nitric oxide synthase or superoxide dismutase, may critically test mechanistic hypotheses in these murine models. Although animal models of IPS are likely to provide insights into its pathogenesis, cleverly designed clinical studies that systematically and safely sample lung tissues (e.g., BAL) are critical to confirm and contrast the molecular, biochemical, and immunologic changes seen in mice with those of the human BMT population.

	Footnotes

Address correspondence to: Rodney J. Folz, M.D., Ph.D., Duke University Medical Center, Box 2620, Room 331 MSRB, Durham, NC 27710. E-mail: rodney.folz{at}duke.edu

(Received in original form April 13, 1999).

Acknowledgments: The author would like to thank Drs. James Vredenburgh, David Rizzieri, Gwynn Long, and Nelson Chao of the Duke University Bone Marrow Transplant Program for helpful discussions. This work was funded in part by National Institutes of Health grants HL55166, ES/HL08698, and American Heart Association Grant in Aid.

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