Introduction

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The clinical success rate of allogeneic bone marrow transplantation (allo-BMT) has increased steadily over the past several decades and consequently the number of allo-BMTs performed has grown dramatically during this period. In spite of these successes, infectious and noninfectious pulmonary complications and graft-versus-host disease (GVHD) have remained a serious threat to survival after allo-BMT. Over 30% of the deaths that occur after bone marrow transplantation (BMT) (both allogeneic and autologous) have been attributed to pulmonary complications (1), of which up to 85% relate to both infectious and idiopathic forms of interstitial pneumonitis (2). Noninfectious idiopathic pneumonia syndrome (IPS) accounts for approximately 50% of the total cases of post-BMT IPS (4, 5). The main features of IPS include diffuse interstitial pneumonitis, alveolitis, and interstitial fibrosis in the absence of an identifiable infectious agent. Although these disorders are known to occur in the setting of a variety of pulmonary injuries, the etiology of pulmonary inflammation following BMT has not been clearly defined. The type and duration of immunologic defects produced by an underlying malignancy, pretransplant radiation conditioning, chemotherapy, and the development of GVHD may each contribute to the pulmonary abnormalities encountered in these patients.

Animal models that reflect the temporal pattern of pulmonary complications in humans post-BMT are limited. Previous allogeneic BMT models typically revealed an acute onset of mononuclear cell inflammation in the lungs in the setting of rapidly developing and often fatal GVHD. This spectrum does not reflect the development of IPS when mild GVHD is present, as may occur in patients with well-matched marrow transplants. Thus, it has been difficult to evaluate the cellular and molecular mechanisms of IPS after allogeneic BMT due to the absence of a suitable animal model, which would more closely reflect the kinetics and severity of GVHD often observed in patients transplanted under current protocols. In this context, we recently characterized a murine model of IPS involving a semiallogeneic parental F1 transplant strategy (6). In this model, lethally irradiated (DBA/2 X C57BL/6) F1 mice were transplanted with T-cell-depleted C57BL/6 bone marrow mixed with a low number of C57BL/6 spleen cells as a source of alloreactive T cells. This protocol resulted in the induction of a mild form of acute GVHD, which was associated with the development of chronic and progressive pulmonary injury characteristic of IPS in humans (6). A significant loss of body weight occurred during the first 3 wk following allo-BMT, which recovered to normal levels after several weeks. An influx of CD8⁺ T cells, a well-established characteristic of acute GVHD, occurred transiently in the spleen and lungs during the first 3 to 4 wk after transplant. The major T-cell population at 9 to 12 wk posttransplant was the CD4⁺ subset, whereas the CD8⁺ subpopulation was at or below normal, a feature commonly associated with chronic GVHD animal models. In the setting of this mild form of GVHD, these mice developed chronic and progressive interstitial pneumonitis and fibrosis.

In this study we demonstrated first that the lung disease in this murine model of IPS occurred with kinetics, which differed dramatically, both in the acute and chronic phases, from GVHD in other organs, suggesting that the lung may represent a unique target of post-BMT disease. Next, we showed that pretransplant irradiation is essential for the development of lung disease in this model, suggesting an important role of radiation conditioning in induction of IPS after allo-BMT.

Thoracic radiation (TI) conditioning for BMT has evolved clinically within the last two decades as a successful protocol for treatment of several types of lung cancer (7). However, a substantial risk of pulmonary toxicity also exists with this form of treatment, which could potentially contribute to the onset of IPS (8). Given the known sensitivity of the lungs to radiation-induced injury, radiation damage to the lungs during pretransplant conditioning may prime this organ for subsequent development of IPS following allo-BMT. Consistent with this idea, we report that pretransplant TI alone was sufficient to prime the lungs for the development of IPS following BMT, although the severity of IPS was reduced compared to pretransplant conditioning with total body irradiation (TBI). Based on these observations, we propose that radiation exposure is an important cofactor in the lungs for the induction of IPS after allo-BMT.

	Materials and Methods

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Mice

Female C57BL/6 ("parental" strain, H-2^b) and B6D2F1 (F1 strain, H-2^b,d) mice were purchased from the National Cancer Institute (Frederick, MD) and maintained in sterile microisolator cages (Lab Products, Inc., Maywood, NJ) with sterile rodent chow and acidified water ad libitum. Mice were maintained by the Division of Laboratory Animal Resources at the University of Kentucky according to the guidelines in the Animal Welfare Act. Sentinel mice were held in the same room and screened periodically for serologic evidence of infection for a panel of common mouse pathogens. Donor and recipient mice were 6 to 7 wk old at the beginning of each study.

Pretransplant Conditioning

B6D2F1 (F1) mice were irradiated (900, 600, or 300 centigray [cGy] as indicated) in a Mark I Cs¹³⁷ irradiator (JL Shepard and Associates, Glendale, CA) at a dose rate of 214 cGy/min and then transplanted within 3 to 4 h. Thoracic irradiation (900 cGy) was performed by placing pentobarbital-anesthetized (Nembutal; Abbott Laboratories, North Chicago, IL) mice individually in a plastic restrainer inside a 1.5-in-thick cerrobend shield (constructed by the Department of Radiation Medicine, University of Kentucky) that had a window (4.5 cm × 1.5 cm) exposing the thoracic region to the radiation source. Mice were kept at room temperature during the irradiation. The shield reduced the radiation dose to protected tissues by greater than 95%.

Bone Marrow Transplantation

The femurae and tibiae of C57BL/6 mice were removed aseptically and the bone marrow cells (BMCs) were flushed out into sterile RPMI-1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 mM 2-mercaptoethanol. T-cell- depleted donor bone marrow was prepared by treating BMC suspensions with anti-Thy 1.2 (HO-13.4.6) for 45 min on ice followed by Low-Tox M rabbit complement (Cedarlane Laboratories, Westbury, NY) for 60 min at 37°C. The percentage of Thy 1⁺ BMCs remaining after such treatment was typically < 1%, as determined by flow cytometry. An appropriate number of spleen cells (5 × 10⁶ unless otherwise indicated) in each experiment was mixed with 1 × 10⁷ T-cell-depleted BMCs and resuspended in endotoxin-free saline before injection. This experimental group that developed GVHD is referred to as "allo-BMT ". As a control group that did not develop GVHD, mice were transplanted with T-cell-depleted BMCs without additional spleen cells. This control group is referred to as "control BMT ". Cells were injected in a volume of 0.1 ml into each recipient via the tail vein. After transplantation, mice were housed in sterile conditions without additional treatment. Body weights were recorded weekly to verify induction of GVHD in the allo-BMT group. Groups of three or four mice were killed at the time points indicated in each experiment. Engraftment of donor cells in spleens of recipient mice was determined by flow cytometry using PE-labeled anti-H-2D^d monoclonal antibody (PharMingen Inc., San Diego, CA) to determine the percentage of H-2D^d-negative donor cells.

Histology

Mice were killed by CO₂ asphyxiation, and several tissue samples were obtained for histologic examination. The dorsal side of each mouse was shaved using clippers and a 1-square-inch piece of full thickness skin was dissected out and placed in neutral buffered formalin (10% formalin, 46 mM Na₂HPO₄, and 29 mM NaH₂PO₄). The tongue, right ear, and pieces of the spleen, liver, and large intestine were removed and also fixed in formalin. For analysis of lung samples, the left main bronchus was first ligated with silk suture thread, then the left lobes of the lungs were removed and placed in supplemented RPMI-1640 for processing for flow cytometry. The right lung lobes were then inflated with neutral buffered formalin via tracheal instillation for 3 min at a pressure of 20 cm Hg. Lungs and heart were then removed en bloc and stored in neutral buffered formalin until further use. Histologic data shown in this report were derived from sections of the right cardiac lobe of the lungs, which were removed from fixative and then embedded in paraffin for sectioning. Multiple 4-µm sections of each organ from groups of three to four mice were stained with hematoxylin and eosin (H&E) by the Histology Services at the University of Kentucky Medical Center. All histology samples were graded on a 0-4 scale by a reader (C.D.J.) in a blind fashion to ensure unbiased observations. A previously established grading scale for liver, GI tract, skin, tongue, and ear, was utilized (9). Lung pathology grading was based on a standard nomenclature for lung graft rejection (10).

Flow Cytometric Analysis

Lungs were dissected into small fragments and incubated for 60 min at 37°C in supplemented RPMI-1640 medium containing 20 U/ml collagenase (Sigma Chemical Co., St. Louis, MO) and 40 µg/ml DNase I (Sigma). Cells were released from the tissue by a 4-min treatment in a Stomacher tissue homogenizer (Seward Medical, London, UK), and then filtered twice through a sterile 74-µm nylon mesh (Small Parts Inc., Miami Lakes, FL) followed by two washes to remove collagenase and DNase. Viable cells were counted by trypan blue exclusion, and 3 × 10⁵ lung cells were stained for flow cytometric analysis with fluorochrome-labeled antibodies. Lung cells from individual mice were assessed by three-color flow cytometry using a cocktail of three monoclonal antibodies: anti-CD45-CY5 (PharMingen), anti-CD3-APC (Sigma), and anti-CD4-FITC (PharMingen). The samples were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer. Data are expressed as the percentage of gated CD45⁺ lymphocytes double-positive for CD3 and CD4 expression.

Statistical Analysis

Significant differences among the groups shown in each experiment were determined by Student's independent (unpaired) t test using SigmaStat software (Jandel Scientific Co., San Rafael, CA). A value of P < 0.05 was considered to be significant.

	Results

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Induction of Murine GVHD and IPS

GVHD and IPS were induced in the allo-BMT group of mice by a single injection of 1 × 10⁷ T-cell-depleted B6 bone marrow (BM) plus 5 × 10⁶ B6 spleen cells, as a source of alloreactive T cells 3 to 4 h after conditioning with a lethal dose of total body irradiation (900 cGy TBI). A separate group of mice (control BMT) received T-cell- depleted bone marrow alone as a control for bone marrow-mediated effects. It has clearly been established that mature T cells are responsible for inducing GVHD, and we have shown recently that mice receiving T-cell- depleted bone marrow alone do not develop IPS or GVHD (6). After transplantation, mice were housed without any additional treatment and were killed at various time points. Survival of mice was nearly 100%, indicating that the level of GVHD induced in the allo-BMT group was nonlethal (not shown). Loss of body weight, a consistent predictor of acute GVHD, was monitored in all groups of mice for 25 wk posttransplant (Figure 1a). Mice transplanted with BM plus T cells (allo-BMT group) displayed an initial loss of body weight during the first 4 wk following transplant after which body weights increased at a rate similar to controls, approaching normal levels after 20 wk posttransplant. Spleen weights of allo-BMT mice were also reduced during the first several weeks after BMT and continued to decrease throughout 20 wk after transplant (Figure 1b). In contrast, control BMT mice maintained similar body and spleen weights as compared with untreated control mice throughout the study. These two indices of acute GVHD (loss of body and spleen weights) demonstrated that animals transplanted with semiallogeneic BM plus T cells developed classical signs of acute GVHD. We have previously shown that IPS occurs in the allo-BMT group of mice: histopathologic hallmarks of IPS, such as interstitial pneumonitis, prominent periluminal mononuclear cell inflammation, and fibrosis were detectable at 9 wk post-BMT and were more severe at week 12 post-BMT (6). Control BMT mice or BM plus syngeneic spleen cells (syngeneic BMT), did not develop IPS, indicating the role of mature alloreactive donor T cells in the induction of this disease (6).

View larger version (27K): [in this window] [in a new window]   Figure 1.   Changes in body and spleen weight following allogeneic bone marrow transplantation. Body weights ( panel a) of untreated normal mice (Normal), mice transplanted with 1 × 107 T-cell-depleted BM (Control BMT) or with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells (Allo-BMT) were recorded at the indicated time points posttransplantation (n = 6 to 15). Spleen weights ( panel b) were determined at the indicated time points after transplant (n = 3). Note that points without error bars had only two mice per measurement. The stippled region in panel b represents the range of spleen weights of normal untreated mice. Data shown represent mean weight ± standard error of the mean (SEM) from one of more than three similar experiments.

Kinetics of Lung Disease Differs from That of GVHD in Other Organs

To compare the temporal development of IPS to GVHD in allo-BMT mice, three to four mice per group were killed at the indicated time points, and tissues (liver, colon, tongue, skin, right ear, and lungs) were evaluated for histopathology as described in MATERIALS AND METHODS. All H&E-stained sections were graded on a 0-4 scale and plotted as a function of time (Figure 2). GVHD was prominent at 3 wk post-BMT in all target organs except the lungs, which did not display any significant pathology at this time compared to untreated normal mice or control BMT mice. GVHD in all target organs except the liver resolved substantially by Week 12 post-BMT. Liver inflammation declined beyond the third week after BMT but never completely resolved through the 25-wk study. In contrast, lung pathology increased progressively through Week 18 and remained elevated up to 25 wk post-BMT. Thus, only the lung demonstrated a progressive development of pathology in allo-BMT mice throughout the 25-wk study, whereas the pathology in all other organs progressively resolved beyond Week 3 posttransplant. These results indicate that a single injection of allogeneic BMCs containing a low number of mature T cells causes the development of progressive interstitial pneumonitis and fibrosis during a period when a mild form of GVHD appears to be resolving.

View larger version (28K): [in this window] [in a new window]   Figure 2.   Kinetics of tissue histopathology following allo-BMT. Mice were transplanted with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells following TBI conditioning (900 cGy). Tissues from the indicated target organs were collected at the indicated time points and processed for histologic examination by H&E staining. Histopathology was graded on a scale of 0-4 as described in MATERIALS AND METHODS (n = 3 or 4). Note time points at Weeks 21 and 35 had only two mice per measurement. Data shown represent mean histopathologic grade ± SEM from one experiment. The stippled area represents minimal grade pathology observed in normal untreated mice and control BMT mice as determined by a blinded evaluator.

Role of Pretransplant Radiation Conditioning in the Development of IPS

To evaluate the role of pretransplant radiation conditioning in the development of IPS in this model, a group of four B6D2F1 mice were transplanted with T-cell-depleted allogeneic BM plus 5 × 10⁶ allogeneic spleen cells from C57BL/6 mice without any pretransplant conditioning treatment (allo-BMT; no TBI). Lung histopathology of these mice at Week 12 post-BMT was compared to that of mice that were exposed to 900 cGy TBI prior to BMT (allo-BMT + TBI). Prominent peribronchial and perivascular inflammation and focal interstitial pneumonitis were visible in all animals that were exposed to TBI prior to BMT (Figure 3a). In contrast, mice that were transplanted without prior radiation conditioning did not develop any detectable inflammatory lesions in the lungs at Week 12 (Figure 3b). These mice also showed no pathology in the liver and other target organs. Mice that received TBI plus allogeneic BM alone (control BMT), or mice that received BM plus syngeneic spleen cells after TBI conditioning (syngeneic BMT + TBI), also failed to develop IPS (data not shown) indicating that allogeneic T cells are required, and more importantly, that radiation (900 cGy TBI) in the absence of alloreactive T cells fails to induce IPS over this time period.

View larger version (181K): [in this window] [in a new window]   Figure 3.   Role of pretransplant radiation conditioning in the development of lung pathology following allo-BMT. Mice (n = 4 per group) were conditioned with 900 cGy TBI and then transplanted with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells (a) or transplanted similarly without radiation preconditioning (b); alternatively, mice were transplanted with BM plus 5 × 107 allogeneic spleen cells without radiation conditioning (c). One group of mice was pretreated with 900 cGy TI and transplanted with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells (d). Lungs were obtained from mice at 12 wk posttransplant and processed for histologic examination. H&E stained sections are shown at an original magnification of ×125. Images are representative of the pathology seen in all mice per group in two or more similar experiments (a, b, and d) or a single experiment (c).

To determine if the requirement for radiation in the development of IPS could be overcome, another group of mice was transplanted with allogeneic BM plus a 10-fold higher number (5 × 10⁷) of allogeneic spleen cells in the absence of pretransplant radiation conditioning. The rationale for this study was that since radiation insult was determined earlier to be a cofactor in the development of lung disease after allogeneic BMT, it could conceivably be overcome with an increased level of another crucial cofactor, such as a very high number of transplanted mature allogeneic T cells. The lungs from this group of mice did display inflammatory lesions at Week 12 posttransplant (Figure 3c); however, the pathology was substantially less (grade < 1.5) than that observed in lungs of animals that received radiation conditioning and were transplanted with the lower number (5 × 10⁶) of spleen cells (Grade 2 to 2.5; Figure 3a). Thus, although a 10-fold higher number of mature allogeneic T cells could cause a mild form of IPS, this was not equivalent to the combined effect of TBI plus the low dose of allogeneic spleen cells.

To determine whether radiation to the lungs alone was sufficient to cause IPS after allogeneic BMT, groups of mice received 900 cGy of radiation to the thoracic region only (TI group). This was accomplished using a shield that reduced radiation exposure to nonthoracic regions to less than 5% of that delivered to the thorax. In spite of the fact that engraftment of donor cells in the spleen was only 6.85% at 12 wk post-BMT (Table 1), TI conditioning led to detectable IPS (Grade 1) 12 wk following transplantation with BM plus a low dose (5 × 10⁶) of allogeneic spleen cells (Figure 3d). The histopathologic features (perivascular and peribronchial inflammation) noted in the lungs of TI-conditioned allo-BMT mice were similar to allo-BMT mice receiving TBI, but the severity was significantly reduced. The pathology shown in Figure 3d reflects inflammation limited to small foci of mononuclear cell infiltrates in contrast to larger foci of such infiltrates plus diffuse interstitial pneumonitis observed in TBI-conditioned mice (Figure 3a). Note that TI-conditioned animals did not develop detectable GVHD in the other target organs following BMT (not shown), and mice that received thoracic irradiation without BMT failed to develop signs of IPS through 12 wk of study (not shown). These observations support a direct effect of radiation on lung tissue as a contributing factor for IPS.

Pretransplant Radiation Conditioning Induces IPS after BMT in a Dose-Responsive Manner

A priming event due to irradiation that promotes IPS after allogeneic BMT could be dependent on the dose of radiation, such that lower doses would promote a less severe form of IPS. Alternatively, priming could be dependent on a critical threshold level of radiation beyond which lung pathology is observed. To address this question, groups of four mice were conditioned with 900, 600, or 300 cGy TBI before transplantation with T-cell-depleted allogeneic BM plus allogeneic spleen cells. Histologic analysis of lung tissue sections in Figure 4 shows that a lethal TBI dose of 900 cGy induced IPS pathology at 12 wk post-BMT similar to that observed in earlier experiments (Figure 4a). A lower dose of 600 cGy also caused lung lesions, but the extent of lung inflammation was reduced (Figure 4b). The lowest dose of 300 cGy did not induce any detectable IPS following allo-BMT (Figure 4c) and the lungs of mice in this group appeared histologically similar to the lungs of mice that received allo-BMT without any pretransplant conditioning (Figure 3b). A similar radiation dose-dependent pathology was noted in nonpulmonary organs including liver, gut, and skin (not shown). The level of engraftment in the spleen varied according to the dose of TBI (Table 1). However, engraftment levels alone cannot explain the occurrence of IPS, as 900 cGy of thoracic irradiation allowed for only 6.85% engraftment and led to IPS, whereas 300 cGy of TBI showed no lung pathology in spite of 52.67% engraftment (Table 1). Finally, all three doses of radiation caused an elevation above normal control mice in the proportion of CD4⁺ T cells in the lungs of transplanted mice (Figure 5), even though histologic lung lesions were not evident in allo-BMT mice receiving only 300 cGy of TBI conditioning.

View larger version (137K): [in this window] [in a new window]   Figure 4.   Effect of radiation dose on the development of IPS. Groups of four mice were administered 900 cGy (a), 600 cGy (b), or 300 cGy (c) of TBI prior to transplant with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells. At 12 wk post-BMT, lung tissues were evaluated for histopathology by H&E staining (original magnification: ×125). Images are representative of the pathology seen in all mice per group in two separate experiments.

View larger version (54K): [in this window] [in a new window]   Figure 5.   Effect of radiation dose on the percentage of CD4+ T cells in the lungs after allo-BMT. Groups of four mice were administered 900, 600, or 300 cGy of TBI, or none, prior to transplant with T-cell-depleted BM plus 5 × 106 allogeneic spleen cells. At week 12 post-BMT groups of four mice were killed and isolated lung cells were analyzed from individual animals by flow cytometry for the percentage of CD4+ T cells. Area within dashed lines represents range of CD4+ T cells in normal untreated mice. Data shown represent mean percentage ± SEM in mice of each group in one of two separate experiments.

	Discussion

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Improved treatment of hematologic and solid tumor malignancies and inherited immunodeficiency diseases through the use of BMT has been due in large part to better control of acute GVHD. However, pulmonary complications following allo-BMT remain a frequent cause of morbidity and mortality. Idiopathic interstitial pneumonitis, or IPS, accounts for as many as 50% of the total cases of interstitial pneumonitis after BMT (2, 4, 5). IPS, which displays pathologic features such as diffuse interstitial pneumonitis, alveolitis, and interstitial fibrosis occurs in humans with a mean onset of 42 to 49 d after allogeneic BMT (11) but can also occur several years after transplant.

We have recently characterized a murine model of IPS following allo-BMT (6). Using a P F1 transplant strategy that involved the transplant of a low number of donor spleen cells (as a source of allogeneic T cells) we generated a model of acute GVHD, which was sufficiently mild for the mice to survive beyond 25 wk post-BMT. The occurrence of only transient weight loss, transient immunodeficiency, and overall low mortality was indicative of the GVHD in this model. However, the sustained splenic atrophy suggested that this mild form of GVHD persisted for at least 25 wk after transplant. Against a background of mild GVHD, transplanted mice developed a chronic and progressive lung disease characteristic of IPS, which was histologically evident by week 12 post-BMT.

The current study evaluated histopathology in six target organs for up to 25 wk following transplantation. Pathologic signs of acute GVHD were evident early after allo-BMT (3 wk) in all organs except the lungs. A concurrent rise in the percentage of CD8⁺ T cells, a characteristic of acute GVHD, has also been observed in the spleen during this time period (6). A similar increase in the proportion of CD8⁺ T cells 3 wk after allo-BMT was also seen in the lungs; however, histologic evidence of inflammation was not seen in the lungs at this time. A slow and progressive form of interstitial pneumonitis and diffuse alveolitis developed through Week 18 with no signs of resolution. In contrast, the lesions due to GVHD in all other target tissues began resolving between 3 and 12 wk after transplant. Inflammation of the liver resolved more slowly and continued to show mild, resolving periportal inflammation throughout the 25 wk of study. Thus, the histopathologic trends clearly indicated that GVHD was resolving beyond week 3 in all tissues except the lung. At 12 wk post-BMT, fibrosis was present in the perivascular and alveolar interstitium in lungs of mice that received allogeneic spleen cells (not shown). In contrast, fibrosis was not evident in any other tissue. Thus, the kinetics of inflammatory disease in the lung was unique among all tissues examined after allo-BMT. It is interesting that chronic mild liver pathology was noted in mice with IPS, as a recent study of obstructive lung disease in children treated with BMT showed that the occurrence of liver disease in children with chronic GVHD was a significant predictor of obstructive lung disease (12). Why disease progression in the lungs following allo-BMT differed from other tissues is currently unknown; it may indicate that the disease process in the lungs differs from other tissues undergoing GVHD. A recent study by Cooke and coworkers (13) showed that pulmonary pathology after BMT in a murine model correlated with the presence, but not the severity, of GVHD suggesting that the disease mechanism(s) in the lungs may be independent of the rate of progression of overall GVHD.

The induction of IPS has been suggested to be more closely associated with pretransplant conditioning rather than GVHD since lung disease can occur after either autologous or allogeneic BMT (14). In fact, lung shielding during radiation conditioning in humans has been shown in one study to reduce but not eliminate the mean incidence of IPS (15), suggesting the possibility that radiation-induced lung damage may participate in the initiation of this syndrome. The severity of GVHD across both major and minor histocompatibility barriers in murine BMT models has been related to the extent of pretransplant radiation conditioning (16). Direct target organ injury due to irradiation can play an important role in localized GVHD. For example, Desbarats and coworkers demonstrated that irradiated skin was more permissive to lesion formation than unirradiated skin in the setting of systemic GVHD (17). Down and associates (18) also showed that GVHD-associated mortality and lung dysfunction after TBI conditioning and allogeneic BMT correlated with the radiation dose as well as the number of allogeneic T cells transplanted. Indeed, modulation of pretransplant radiation conditioning paradigms has been shown to ameliorate post-BMT mortality and injury to target organs (19). Radiation dose fractionation is a suggested option at several BMT centers, as it has been demonstrated to reduce the incidence of restricted ventilation and impaired gas exchange (22). Although high-dose TI without BMT can cause pulmonary inflammation and fibrosis, lower doses of up to 1000 to 1100 cGy in the absence of BMT have been shown to be relatively nontoxic (23, 24). In contrast, studies utilizing animal models have shown that pretransplant radiation conditioning in this range, when combined with allo-BMT, significantly enhanced GVHD-associated mortality (25, 26). The present report shows for the first time that under conditions of sublethal GVHD the occurrence of IPS also is dependent on pretransplant radiation conditioning in an animal model. Classical radiation-induced pneumonitis did not occur in our animal model, as radiation-conditioned mice transplanted with T-cell-depleted BM without allogeneic T cells, or with BM plus syngeneic spleen cells, did not develop IPS (6). Moreover, lung disease did not occur in the absence of pre-BMT radiation treatment under BMT conditions known to induce mild acute GVHD in this model (6).Whether mild GVHD in the absence of pretransplant conditioning would eventually lead to IPS beyond the 12-wk observation period is not known. However, it is clear from these studies that radiation conditioning plays a significant role in the development of lung disease in our model (6). Although pretransplant TBI conditioning led to similar pathology in all GVHD target organs including the lungs, TI conditioning led to lung disease in the absence of detectable pathology in nonpulmonary organs. Our findings provide supportive evidence for an earlier report by Keane and coworkers (27) who correlated the occurrence of IPS among patients treated by BMT with the absolute dose of radiation to the lung. Pretransplant radiation conditioning is known to aid in the engraftment of donor BMCs. In the present study, an examination of engraftment following various pretransplant conditioning regimens (Table 1) showed that IPS correlated with the level of myeloablative treatment. However, it is unlikely that the extent of stem cell engraftment determines the onset of IPS for several reasons. First, the onset of IPS was dependent on the presence of mature spleen cells, rather than marrow cells. Second, survival of mature alloreactive T cells in recipients is probably less dependent on the extent of myeloablation. Finally, 900 cGy of TI, which led to IPS post-BMT, displayed only 6.85% marrow engraftment, whereas 300 cGy of TBI, which caused no lung pathology after BMT, displayed 52.6% engraftment. Thus, in this model, the percent engraftment could be dissociated from the onset of IPS. Thus, in addition to promoting better marrow engraftment, radiation conditioning may also "prime" the lungs of the recipient via unknown mechanisms that promote the ability of allogeneic T cells to establish IPS.

Future studies in our laboratory are aimed at addressing the role of lung tissue priming through experiments involving thoracic shielding, which should alleviate or lessen lung pathology while not affecting GVHD in other target organs if radiation is causing direct effects in the lungs. The mechanism by which pretransplant conditioning could prime the lungs for IPS is presently unknown. It is possible that radiation may be involved in the lowering of the "threshold" through a common mechanism involving increased local and systemic levels of proinflammatory and T-cell-activating cytokines and chemokines. Although such cytokines have been found to be induced early postirradiation, and can persist for prolonged periods (28), it has also been shown that the early wave of radiation-induced cytokines is most critical for the induction of GVHD, as BMT 4 to 7 d following TBI preconditioning reduced the severity of acute GVHD (29). Gastrointestinal damage by radiation and subsequent endotoxemia has been proposed as a mechanism by which radiation conditioning can augment GVHD (16) and IPS (13) via induction of proinflammatory cytokines. The observation that TI was sufficient to induce IPS indicates that direct radiation injury to the gut is not required for IPS. However, it is clear from the present study that radiation injury to tissues other than the lung, such as the gut, can enhance the severity of IPS, possibly by augmenting proinflammatory cytokine production. There are several direct effects of radiation on lung tissue that could promote IPS. Ionizing radiation exposure to the lungs has been demonstrated to induce synthesis of both proinflammatory cytokines, growth factors, and vascular adhesion molecules (30). Moreover, direct radiation of several cell types in vitro can increase expression of histocompatibility molecules (35) and adhesion molecules such as E-selectin, ICAM-1, and VCAM-1 (36), which could potentially promote better extravasation into lung tissues and enhance allorecognition by T cells. Finally, gamma irradiation can enhance the ability of stimulated monocytes to produce hydrogen peroxide (39) and nitric oxide (40), which may suggest an increased capacity for causing tissue damage. The unique temporal development of IPS in this model, compared to the development of GVHD in other organs, is intriguing and correlates with the known sensitivity of the lungs to irradiation. Why the lungs are uniquely sensitive to radiation could potentially be related to its oxygen-rich environment. Radiation injury is mediated via generation of reactive oxygen species (ROS) in the irradiated tissues, and the levels of ROS generated is dependent on the concentration of oxygen in the surrounding environment (41, 42). Elevated oxygen levels in the lungs compared to other tissues would be expected to lead to greater radiation injury in lung tissue, which may increase the sensitivity of the lungs to IPS or allow for a more prolonged period of injury. Additional studies in thoracic-irradiated mice will be required to distinguish between these possible effects.