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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1579-1589, (2002)
© 2002 American Thoracic Society

Original Article

Idiopathic Pneumonia Syndrome after Syngeneic Bone Marrow Transplant in Mice

Karan S. Bhalla and Rodney J. Folz

Departments of Medicine and Cell Biology, Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, North Carolina

Correspondence and requests for reprints should be addressed 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Idiopathic pneumonia syndrome is characterized by noninfectious diffuse lung injury after myeloablative chemotherapy and bone marrow transplant. Because little is known about its pathogenesis after autologous-based regimens, we have developed a murine model that closely mimics the human lung disease process. Using an autologous regimen similar to that used for patients with metastatic breast cancer, mice developed pulmonary injury as early as 1 day posttransplant. This lung injury was most dramatically characterized by decreased lung compliance that was associated with an intense monocytic cellular infiltrate of activated macrophages. This influx was preceded by an acute elevation in monocyte chemotactic protein-1 and macrophage inflammatory protein-1{alpha}. The conditioning regimen caused substantial oxidative stress as manifest by elevations in lung lipid peroxidation and oxidized glutathione. To test the hypothesis that oxidation is directly responsible for the lung toxicity, we administered the antioxidant, n-acetylcysteine. These mice showed substantially less lung injury, thus providing direct evidence that oxidative stress plays a distinct role in the development of lung injury in the early periautologous bone marrow transplant period. Attenuation of lung oxidative stress and/or inflammation in patients undergoing autologous bone marrow transplant may reduce the subsequent development of idiopathic pneumonia syndrome.

Key Words: acute lung injury • interstitial pneumonitis • oxidative stress • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose-intensive chemotherapy followed by bone marrow or stem cell transplantation is being increasingly used to treat a number of solid tissue and hematologic cancers, as well as some nonmalignant chronic conditions. However, the success of bone marrow transplantation as a therapeutic modality is significantly limited by a number of transplant-related complications, especially those involving the lung. Idiopathic pneumonia syndrome (IPS) is a term used to describe diffuse lung injury after bone marrow transplant (BMT) for which an infectious etiology is not identified (1). The incidence of IPS varies from 12 to 70% and is largely dependant on the specific patient population being analyzed, the details of their therapeutic regimen, and the criteria being used to define lung toxicity (2). In 1991, a National Institutes of Health workshop described two subtypes of IPS, determined by the source of stem cells for hematopoietic support, either allogeneic or autologous (1). The development of IPS after allogeneic BMT in humans, as well as in animal models, has been directly correlated with the dose of irradiation administered to the lung and with the incidence of graft-versus-host disease (3, 4). In contrast, graft-versus-host disease is presumably not applicable in the setting of autologous regimens, which lack alloreactive stem cell infusions and preconditioning irradiation. Furthermore, the identification of viral agents in autologous transplant patients is considerably less common than after allogeneic BMT. Therefore, IPS after autologous BMT more likely represents the result of pretransplant conditioning regimens consisting of high-dose chemotherapy (HDC), growth factor support, and possibly posttransplant radiation therapy (5). Unfortunately it has been difficult to evaluate the cellular and molecular mechanisms of IPS after autologous BMT in the absence of an animal model that simulates the disease in humans.

We have now developed a murine model of IPS after HDC and syngeneic BMT, designed to closely mimic a human HDC and autologous BMT protocol currently in use for the treatment of advanced stage breast cancer (6). Mice receive high-dose cisplatin, cyclophosphamide, and bischloroethylnitrosourea (BCNU), followed by syngeneic BMT. In this article, we describe the kinetics and potential mechanisms involved in the development of IPS in the early peri-BMT period.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
High Dose Chemotherapy
Female mice of B6C3F1 hybrid (C57BL/6 x C3H) genotype were used throughout. The HDC and BMT (1.75x) protocol consisted of intraperitoneal injections of cyclophosphamide (105 mg/kg) and cisplatin (3.59 mg/kg) on Days -5, -4, and -3. This was followed by a single intraperitoneal injection of BCNU (42.9 mg/kg) on Day -2. Control mice received identical volumes of vehicle.

n-Acetylcysteine (NAC), at 200 mg/kg, was administered to mice, intraperitoneally, on Days -5, -4, -3, -2, and -1 (see online data supplement for detailed methods).

Syngeneic Bone Marrow Transplantation
Bone marrow cells were flushed from femur and tibiae with RPMI-1640 medium supplemented with fetal calf serum, antibiotics, and 2-mercaptoethanol. Reinfusion of bone marrow was performed on Day 0 by intravenous tail-vein injection of 20 x 106 cells in 0.5 ml RPMI-1640. Control mice received an intravenous injection of 0.5 ml RPMI-1640.

Bronchoalveolar Lavage Fluid and Lung Tissue Preparation
Bronchoalveolar lavage fluid (BALF) used for assessment of oxidative stress was supplemented with antioxidants and snap frozen in liquid nitrogen.

Lungs were perfused in situ, weighed, immersed in phosphate-buffered saline containing antioxidants, and homogenized in the presence of a protease inhibitor cocktail.

BALF and peripheral blood total cell counts were performed using a hemacytometer. Differential cell counts were determined after counting a total of 300 cells. BALF total protein, BALF lactate dehydrogenase (LDH), and lung wet/dry ratios were used to assess lung injury and edema. Interleukin-1ß, interleukin-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), and tumor necrosis factor-{alpha} (TNF-{alpha}) levels were determined by ELISA.

Lung Compliance
Lung compliance and pressure–volume curve studies were performed in situ after degassing with 100% oxygen. The lungs were then gradually inflated with air until the pressure reached 30 cm of water. This volume (V30) represents total lung capacity (7). Thereafter, the lungs were serially deflated by volumes of 0.1 ml and the corresponding pressures recorded. Specific lung compliance was calculated by dividing the slope of the deflation portion of the curve, where pressure is usually less than 10 cm of water, by the average lung volume in that portion of the curve (8).

Histopathology and Immunohistochemistry
Lungs were prepared for hematoxylin and eosin staining as described previously (9). Lung MCP-1 immunostaining was performed using either polyclonal rabbit antimurine MCP-1 or preimmune rabbit serum.

Assessment of Alveolar Macrophage Activation
Activation of alveolar macrophage (AM) was determined by measuring in vitro TNF-{alpha} production by stimulating AM with lipopolysaccharide (LPS).

Flow Cytometry
Cells from 12 animals were pooled to allow flow cytometric analysis. The results were expressed first as the percentage of cells that stained positively for each marker after subtracting the percent positive cells in the isotype control and then as mean fluorescence intensity.

Assessment of Oxidative Stress
Lung glutathione peroxidase and glutathione reductase (GR) were determined using commercially available kits. Glutathione content was measured by high-performance liquid cromatography (10). Lipid peroxidation was measured using high-performance liquid cromatography (9). Intracellular reactive oxygen species (ROS) generation in AM was determined by oxidation of the redox-sensitive dye dichlorodihydrofluorescein diacetate and analyzed by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Myeloablative HDC and BMT
To closely mimic the human protocol of HDC and autologous BMT, initial experiments were aimed at titrating the dose of HDC to be myeloablative, necessitating the use of BMT for rescue. The maximal tolerated dose was defined previously as that dosage of chemotherapy that did not induce mortality in mice (60 mg/kg cyclophosphamide, 2.05 mg/kg cisplatin, and 24.5 mg/kg) (11). We termed this as 1x, and dose escalation studies were performed, together with syngeneic BMT, while maintaining the same ratio of drugs. As demonstrated in Figure 1A , a dose twice the maximal tolerated dose, together with BMT (2x HDC + BMT), produced severe myeloablation by Days +3 and +6 (e.g., the mean white blood cell count on Day +6 was 205 ± 37 cells/mm3, n = 6 animals pooled from two independent experiments), which resulted in 100% mortality by Day +6. The 2x HDC without BMT produced 100% mortality by Day +3. However, a dose 1.75 times the maximal tolerated dose was found to be ideal, as mice receiving this dose, together with BMT, had a significant rise in white blood cell counts (mean white blood cell count on Day +11 was 3,210 ± 185 cells/mm3, n = 6) and had 100% survival. When the 1.75x HDC dose was given without BMT, it produced 80% mortality by Day +10 (see Figure 1A). A dose of 1.5x HDC produced nonlethal myeloablation, with 60% survival even without BMT (data not shown). All further experiments were conducted using the 1.75x HDC dose with BMT.



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  		Figure 1. Effects of HDC and BMT on mice. (A) Effect of dose escalation on peripheral white blood cell (WBC) counts. Mice received a 2x dose of HDC plus BMT (2x HDC + BMT, dark squares) or HDC without BMT (2x HDC + RPMI media, open squares). A second group of mice received 1.75x HDC plus BMT (dark triangles) or 1.75x HDC plus RPMI media (open triangles). A control group of mice were treated with vehicle alone (saline + RPMI media, circles). n = 6 at each time point except ("a") (n = 3); ("b") (n = 1). (B) Total cell counts in BALF from 1.75x HDC + BMT–treated (circle) or control (triangle) mice. n = 6 at each time point. (C) Total protein in the BALF from 1.75x HDC + BMT–treated (triangle) or control (circle) mice. n = 6 at each time point. (D) BALF LDH levels were measured in mice receiving 1.75x HDC + BMT (triangle) or control (circle) mice. n = 4 at each time point. *p Value less than 0.05 versus vehicle-treated control mice at the same time point. Values are expressed as mean ± SEM.

 
BALF Cell Counts
BALF total cell counts rapidly and significantly increased after BMT (Figure 1B). By Day +7, the BALF had maximum cellularity. The mean BALF total cell counts on this day were 121.7 ± 29.7 x 104 cells/ml from mice receiving HDC + BMT, compared with 4.7 ± 0.9 x 104 cells/ml (n = 6 in each group, p < 0.05) from control mice. By Day +11 , the BALF cellularity in mice receiving HDC + BMT was reduced to a mean of 42.0 ± 24.1 x 104 cells/ml. However, this was significantly higher than the BALF total cell count in control mice on the same day. This cellular influx into the lung consisted predominantly of AM. At all time points analyzed, 90 to 95% of the BALF cells consisted of AM, in both control mice and in mice receiving HDC + BMT. There were no significant differences in the proportion of cell types in the BALF between control mice and mice receiving HDC + BMT.

Markers of Lung Injury
The permeability and/or cellular injury of the lung elicited by HDC + BMT was measured by an increase in BALF total protein and BALF LDH. HDC resulted in significant increases for both parameters tested (Figures 1C and 1D). BALF total protein was significantly higher in mice receiving HDC + BMT at all time points measured when compared with control mice. The BALF total protein levels in HDC + BMT mice were highest on Day +1 (1161.3 ± 459 µg/ml compared with 105.6 ± 15.3 µg/ml for control mice, n = 6 in each group, p < 0.001). BALF LDH was similarly elevated at all time points measured. On Day +1, BALF LDH in mice receiving HDC + BMT was 362.1 ± 16.3 mU/ml, compared with 111.3 ± 12.4 mU/ml, n = 4 in each group, p value less than 0.001.

We used lung wet/dry ratio in an attempt to probe pulmonary capillary permeability differences between the two groups of mice. For these studies, lung wet/dry ratios were determined in both control and HDC + BMT mice and found to be 4.53 ± 0.15 versus 4.75 ± 0.04 (Day -2), 4.71 ± 0.11 versus 4.91 ± 0.03 (Day -1), 4.23 ± 0.13 versus 4.95 ± 0.05 (Day 1), 4.35 ± 0.15 versus 5.44 ± 0.44 (Day 2), 4.5 ± 0.30 versus 6.35 ± 0.65 (Day 4), 4.23 ± 0.13 versus 5.02 ± 0.27 (Day 7), and 4.1 ± 0.1 versus 4.9 ± 0.6 (Day 11), respectively. Although the mice receiving HDC + BMT showed a trend toward greater pulmonary edema at all time points, only Days +2 and +4 were significant (p < 0.05).

Lung Compliance
To assess physiologic changes in the lung induced by HDC + BMT, we measured lung capacity and compliance throughout the study. Figure 2A shows typical pressure–volume loops obtained for Day +2 (control and HDC + BMT). It is clear that HDC + BMT increases the elastic recoil of the lung, shifting the curves downward and to the right. Mice receiving HDC + BMT had significantly lower specific lung compliance on Days +2, +4, +7, and +11 (Figure 2B). The mean specific lung compliance post-BMT, for mice receiving HDC + BMT was 0.239 ± 0.06 µl of air/cm of water/µl of lung volume, whereas control mice had a mean compliance of 0.403 ± 0.04 µl of air/cm of water/µl of lung volume, during the same time period (n = 12 in each group, p < 0.01 by Mann–Whitney U test). A similar reduction in total lung capacity, as measured by V30, was observed in mice receiving HDC + BMT (Figure 2C). Post-BMT, mice in the HDC + BMT group, lost an average of 287.5 µl of lung volume, which represents about 25% of their total lung capacity.



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  		Figure 2. Lung compliance. (A) Pressure–volume loops from three randomly chosen sets of lungs from mice receiving HDC + BMT or vehicle control at Day +2. (B) Specific lung compliance of vehicle control mice and mice receiving HDC + BMT. Lung compliance was measured at the lower one-third of the total lung capacity, as these volumes represent the tidal volumes in the mouse. n = 3 at each time point. (C) Total lung capacity. This was measured as the volume of air required to inflate the lung to a pressure of 30 cm of water, which represents maximal physiologic distention of the alveoli in mice. n = 3 at each time point. *p Value less than 0.05 versus vehicle-treated control mice at the same time point. Values are expressed as mean ± SEM.

 
Histopathology
Using light microscopy, on all days examined, the sectioned lungs of the cohort treated with HDC + BMT demonstrated distinct histopathologic abnormalities consistent with cytotoxic drug injury when compared with control lungs. A representative photomicrograph of these histopathologic changes at Day +2 is shown in Figure 3A . These abnormal findings included the presence of large, foamy AM (Figure 3A, 1) and development of septal capillary congestion (Figure 3A, 3). Cytopathologic features in the lungs of mice receiving HDC + BMT was consistent with a reactive proliferation of bronchiolar epithelial cells characterized by an increase in epithelial cell size (Figure 3A, 2) with enlargement of the nuclei (Figure 3A, 5) accompanied by hyperchromatasia and the development of nucleoli. Minimal alveolar edema was evident in localized areas of the lung section from mice receiving HDC + BMT (Figure 3A, 4). Control lungs obtained at Day +2 are shown for comparison (Figure 3B).



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  		Figure 3. Lung histopathology from HDC + BMT and control mice on Day +2. (A) Representative lung section from a mouse receiving HDC + BMT, showing typical features of cytotoxic drug injury. These abnormal findings included the presence of large, foamy AM (1), reactive proliferation of bronchiolar epithelial cells characterized by an increase in epithelial cell size (2) with enlargement of the nuclei (5) and development of septal capillary congestion (3). Minimal alveolar edema was evident in localized areas of the lung (4). (B) Representative lung section from a control mouse receiving vehicle alone displayed no pathologic abnormalities. The lung sections were prepared from formalin-fixed, paraffin-embedded samples and stained with hematoxylin and eosin (x40 magnification). The bar represents 50 µm.

 
Effects of HDC + BMT on BALF and Lung Cytokines
The levels of proinflammatory cytokines (interleukin-1ß, interleukin-6 and TNF-{alpha}) and C–C chemokines (MCP-1 and MIP-1{alpha}) in the BALF and lung of control and HDC + BMT mice were measured. One of our central hypotheses is that local production of cytokines in the early phase of HDC + BMT causes lung injury, and it is essential for monocyte recruitment into the lung. Figure 4A shows significant increase in MCP-1 in the BALF of mice receiving HDC + BMT. This increase was first evident on Day -1, after the mice had received the entire HDC regimen. Interestingly, three cycles of cyclophosphamide and cisplatin alone (Day -2) did not cause any elevation of MCP-1 in the BALF (HDC + BMT = 10.7 ± 3.9 pg/ml, control = 4.8 ± 2.8 pg/ml, n = 3 in each group, p = 0.106), but after BCNU administration (Day -1), MCP-1 levels in the BALF dramatically increased 21-fold, to 93.5 ± 13.8 pg/ml, compared with 4.5 ± 1.2 pg/ml in control mice (n = 3 in each group, p < 0.001). These elevated MCP-1 levels persisted until Day +1 and then gradually returned to control values by Day +7. Changes in BALF MCP-1 correlated with lung parenchymal levels of the C–C chemokine (Figure 4B). We also analyzed the production of MIP-1{alpha}, another chemokine that is a predominant chemoattractant to monocytes. Although MIP-1{alpha} was below detection levels in the BALF (data not shown), lung parenchymal levels of the chemokine were significantly elevated on Days +7 and +11 (Figure 4C). Thus sequential elevation of C–C chemokines in distinct compartments of the lung (alveolar space and parenchyma) is consistent with the striking cellular recruitment seen after HDC and BMT.



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  		Figure 4. BALF and lung cytokine levels. These assays were performed at the time points indicated. n = 4 at each time point. Values are expressed as mean ± SEM. *p Value less than 0.05 versus vehicle-treated control mice at the same time point.

 
Of the proinflammatory cytokines measured, interleukin-6 was significantly elevated in the BALF after BMT (Figure 4D). No significant difference was detected between the levels of BALF interleukin-1ß among the two treatment arms. BALF TNF-{alpha} was below detection limits in both groups of animals.

Immunohistochemistry
Owing to the dramatic elevation of MCP-1 in the lung parenchyma and BALF, we attempted to probe the cellular source of this chemokine in the lung using immunohistochemistry. On Day +1, control mice demonstrated no active MCP-1 production (Figure 5B) , whereas mice receiving HDC + BMT had intense positive staining in AM, as well as in alveolar epithelial cells (Figure 5A). A section of the lung from both groups of mice was stained using normal rabbit serum in place of the primary antibody, as a negative control, and showed no positive staining (not shown).



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  		Figure 5. MCP-1 immunostaining. (A) Representative lung section from mice receiving HDC and BMT, on Day +1. Intense positive staining was present in AMs (1) as well as in alveolar epithelial cells (2). This finding is temporally associated with elevated MCP-1 levels in BALF and lung. (B) Lung section from a saline control mouse on Day +1. No MCP-1 immunostaining was detected. (x40 magnification). The bar represents 50 µm.

 
Assessment of AM Activation
Our aim in these experiments was to determine the activation state and immunophenotye of the vigorous AM influx into the lung that occurs after HDC and BMT. Activation state was determined by in vitro TNF-{alpha} release by AM stimulated with LPS. The mean LPS-induced TNF-{alpha} release by AM from mice receiving HDC + BMT was 813.8 ± 421.2 pg/ml. Similarly treated AM from control mice, released 392.9 ± 105.4 pg/ml (n = 9 mice in each group, p = 0.002). There was no measurable spontaneous release of TNF-{alpha} by AM from either treatment arms. AMs are exquisitely sensitive to endotoxin, and the absence of TNF-{alpha} release by unstimulated AM suggests that the BALF contained extremely minimal, if any, amounts of endotoxin. We controlled for the lower viability of AM from HDC + BMT mice by culturing equal number of viable cells from both treatment arms. Viability of BALF cells from HDC + BMT mice was 60%, compared with 95% for cells from control mice.

Immunophenotyping of AM was done to assess the activation and maturation state of the infiltrating mononuclear phagocytes. Mean results from flow cytometric analysis in the post-BMT period are shown in Table 1 . The expression of constitutive adhesion molecules, CD11a, CD11c and CD18, was unchanged after HDC + BMT. Expression of the adhesion molecules CD11b and CD54 was significantly increased after HDC + BMT. Mac-3, a marker of macrophage activation, was also significantly elevated after HDC + BMT. The enhanced LPS responsiveness of AM from mice receiving HDC + BMT may be partly caused by a significant upregulation of the LPS receptor on AM, CD14 (mean fluorescence intensity of control = 0.05, compared with mean fluorescence intensity of HDC + BMT = 3.30, p = 0.005). In addition to these findings, there was significant downregulation of CD71, the transferrin receptor. No changes in the expression of CD13 (aminopeptidase N) and the macrophage scavenger receptor (as detected by antibody clone 2F8) were noted. This AM finding (CD11bhigh, CD54high, CD71low) is characteristic of an immature macrophage phenotype.


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  		TABLE 1. Results of flow cytometric analysis of alveolar macrophage

 
Assessment of Oxidative Stress
Figure 6A demonstrates the inhibition of lung GR produced by BCNU. On Day -1, 24 hours after the mice received intraperitoneal BCNU, the lung GR activity was reduced by 50% to 34.1 ± 4.0 mU/ml, compared with a normal GR activity in control mice of 66.9 ± 3.4 mU/ml (p < 0.001, n = 4). The GR activity remained persistently depressed for several days, gradually returning to normal levels by Day +4 (HDC + BMT GR activity = 62.8 ± 8.2 mU/ml, control GR activity = 68.7 ± 2.1 mU/ml, p = 0.297, n = 4). There was no change in lung glutathione peroxidase activity through the entire course of the study (data not shown).



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  		Figure 6. Lung oxidant/antioxidant analysis. (A) Effect of HDC + BMT on lung GR activity. The assay was performed at the time points indicated. n = 4 at each time point. (B) Analysis of lipid peroxidation in the BALF after HDC + BMT. Lipid peroxidation was assayed using thiobarbituric acid reactive substance method, by the high-performance liquid cromatography measurement of MDA. n = 4 at each time point. *p Value less than 0.05 versus vehicle-treated control mice at the same time point. (C) Analysis of glutathione content in BALF of mice receiving HDC + BMT. The left ordinate represents the absolute quantities of reduced GSH (lower stack), free oxidized GSH (GSSG, middle stack), and protein-bound oxidized GSH (GSS protein, upper stack), expressed in µM. The height of all individual bars represents the total glutathione content or GSH-T (where GSH-T = GSH + 2GSSG + GSS protein) in the BALF (n = 5, on each day). The bar representing "control" is the mean values of the respective GSH components from saline control mice used during the entire course of the study (n = 35). The right ordinate represents the fractional percentage each source of glutathione contributes to GSH-T (% GSSG, triangles), GSS protein (% GSS protein, circles), and total oxidized glutathione (% [GSSG + GSS protein], squares). (D) Analysis of GSH and its various fractions in lung homogenate. Figure legends and explanations of abbreviations are the same as in C. n = 3, on each day. Values are expressed as mean ± SEM.

 
The most dramatic changes in the pulmonary glutathione system were witnessed in the BALF after HDC + BMT. BALF from control mice had a consistent distribution of the various fractions of glutathione (see Figure 6C), throughout the course of the study. In control mice, the mean total glutathione (GSH-T) content was 1.84 ± 0.6 µM, mean reduced glutathione (GSH) content was 1.31 ± 0.4 µM, mean free oxidized glutathione (GSSG) content was 0.39 ± 0.1 µM and mean protein-bound oxidized glutathione (GSS protein) content was 0.14 ± 0.02 µM. This corresponded to an oxidized glutathione fraction (2GSSG + GSS protein)/GSH-T) of 28.7 ± 3.7% (n = 35). As shown in Figure 6C, HDC + BMT caused a twofold increase in GSH-T in the BALF as early as Day -2 (4.05 ± 0.8 µM with 31.1 ± 0.3% being in the oxidized form). Before BMT, on Days -2 and -1, this increase in GSH-T was in large part due to an increase in reduced GSH, whereas oxidized glutathione remained at control levels (Day -2 = 31.3 ± 0.2%, Day -1 = 30.7 ± 1.1%, n = 5). However, after BMT, there was distinct evidence for enhanced oxidation of GSH, with both GSSG and GSS protein fractions increasing significantly, accompanied by a further increase in GSH-T. The GSH-T on Day +2 increased ninefold (16.64 ± 2.4 µM), whereas the oxidized glutathione increased 14-fold (7.54 ± 1.1 µM (45.3%)) over control levels. By Day +11, the GSH-T in BALF from mice receiving HDC + BMT had reduced to 3.43 ± 0.8 µM. Strikingly, 60% was still in the oxidized form, which signifies severe and persistent oxidative stress. This pattern of glutathione oxidation in lung homogenate was similar to that seen in the BALF (Figure 6D). However, in contrast to the substantial increase in GSS protein in the BALF, the oxidation of GSH in lung tissue was manifest primarily as GSSG. For example, on Day +4, control lung GSSG levels were 6.34 ± 0.8 µM, whereas HDC + BMT lung GSSG was 23.21 ± 1.2 µM (p < 0.001, n = 3). On the same day and from the same group of animals, the control lung GSS protein was 1.4 ± 0.8 µM, compared with a GSSG of 2.3 ± 1.1 µM (p = 0.095, n = 3) in the HDC + BMT lung.

In addition to the data presented, we also analyzed the effect of HDC + BMT on lipid peroxidation by measuring levels of malonyldialdehyde (MDA) in the BALF (Figure 6B). The MDA levels in control BALF measured 36.5 ± 7.1 pM, compared with 59.7 ± 7.3 pM (p < 0.001, n = 5) in BALF from mice receiving HDC + BMT on Day -2. After HDC + BMT, the BALF MDA levels peaked at 96.2 ± 12.7 pM on Day +2 and decreased thereafter. At all time points analyzed, the BALF MDA from mice receiving HDC + BMT was significantly higher than control BALF.

Analysis of intracellular production of oxidants by AM in the BALF was performed by flow cytometry using the redox sensitive dye dichlorodihydrofluorescein diacetate. Dead and apoptotic AM were excluded using 7-AAD. The mean fluorescence of viable AM from control mice was 59.5 ± 8.5 (arbitrary fluorescence units, n = 9 mice, data pooled from 3 separate experiments), compared with a mean fluorescence of 170.9 ± 15.5 in viable AM from mice receiving HDC + BMT (n = 6 mice, data pooled from analysis of mice on Days +1, +2 and +4), indicating enhanced oxidant production.

To better understand the immediate oxidative effects of chemotherapy on the lung, and with the view to design an antioxidant supplementation regimen, we measured the glutathione profile in the lung and BALF from mice, at very early time points after chemotherapy. In the first group of experiments, mice received their first dose of cyclophosphamide and cisplatin. As shown in Figure 7A within 1 hour of receiving the drugs, the total glutathione content in BALF of these mice was reduced by 40% to 1.1 ± 0.5 µM (compared with unmanipulated control mice, p = 0.057, n = 3). Of this total, 61.1% was oxidized, signifying acute depletion of GSH and enhanced oxidative stress. Subsequently, the BALF GSH-T increased to above normal control levels and by 8 hours peaked to 5.2 ± 1.8 µM and remained elevated 24 hours later. During this period of augmented GSH production, the lung was able to maintain a normal GSH oxidation profile. A similar profile was assayed in lung homogenate (data not shown).



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  		Figure 7. Assessment of acute GSH oxidation produced by chemotherapeutic drugs. (A) Mice received a single intraperitoneal dose of cyclophosphamide and cisplatin, and glutathione content was analyzed in the BALF 1, 4, 8, and 24 hours later. Time 0 represents unmanipulated mice. The left ordinate represents total glutathione in the BALF, expressed in µM (open circles). The right ordinate represents the fraction percent contribution of GSSG to total glutathione displayed at various time points (triangles). (B) Mice received a single intraperitoneal dose of BCNU at time 0 hour, before which they had received three cycles of daily cyclophosphamide and cisplatin. Analysis of GSH in the BALF was performed at the time points indicated. n = 3 at each time point. Values are expressed as mean ± SEM.

 
A second group of experiments were aimed to ascertain the acute effects immediately after BCNU on lung oxidation. Mice used in these experiments had received three doses of cyclophosphamide and cisplatin before the injection of BCNU. At 1, 4, and 8 hours after BCNU administration, lung GR activity was reduced to 34.1 ± 0.2 mU/L, 17.6 ± 1.8 mU/L, and 16.7 ± 1.2 mU/L, respectively, compared with a control value of 66.9 ± 3.4 mU/ml. Thus, 8 hours after BCNU, lung GR activity was reduced to 25% of control lung GR activity. As shown in Figure 7B before receiving BCNU (0 hour), the BALF GSH-T was elevated (5.7 ± 1.8 µM), due to the fact that these mice had already received three doses of cyclophosphamide and cisplatin. After administration of BCNU, there was a dramatic increase in GSH-T in BALF. At 1, 4, 8, and 24 hours later, the GSH-T was 15.7 ± 1.4 µM, 19.1 ± 3.6 µM, 17.2 ± 2.1 µM, and 10.5 ± 2.8 µM, respectively. Consistent with inhibition of GR by BCNU, 90.5% of the glutathione was oxidized by 8 hours. A similar profile of GSH-T elevation and oxidation was assayed in lung homogenate (data not shown).

Antioxidant Supplementation Therapy
Preliminary experiments were aimed at determining the effect NAC had on glutathione in the lungs of unmanipulated mice. As early as 1 hour after a single NAC administration, GSH-T in the BALF increased 3.5-fold to 6.3 ± 3.5 µM (control = 1.8 ± 0.6 µM, p < 0.001, n = 3) and remained elevated 4 hours (8.3 ± 2.5 µM) and 8 hours (11.9 ± 4.6 µM) later. Twenty fours later, the BALF GSH-T returned to control levels (2.0 ± 0.9 µM). During this period of augmented glutathione in the BALF, the percentage of oxidized glutathione decreased, with the oxidized fractions representing 14.3, 16.2, and 18.5% at 1, 4, and 8 hours respectively, as compared with 28.7% in controls.

Figure 8 demonstrates the protective effects of NAC on the development of lung injury after HDC + BMT. Most parameters of lung injury analyzed showed significant improvement with NAC administration. First, BALF total cell counts were dramatically reduced at all three time points analyzed. On Day -1, the BALF total cell counts in mice receiving HDC + BMT and NAC supplement were 9.9 ± 0.96 x 104 cells/ml, compared with 16.8 ± 1.2 x 104 cells/ml in mice receiving HDC + BMT alone (p < 0.001, n = 3). After HDC + BMT (on Days +1 and +4), there was a significant reduction in the recruitment of cells (by 50%) into the epithelial lining fluid of mice receiving HDC + BMT and NAC, versus HDC + BMT alone.



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  		Figure 8. Administration of NAC results in attenuation of lung injury after HDC + BMT. (A) NAC protection resulted in significantly lower BALF total cell counts. (B) Lower BALF total protein indicates that NAC administration reduced lung injury produced by HDC + BMT. (C) Mice receiving NAC also had lower cytotoxicity as measured by BALF LDH. (D) Attenuation of lipid peroxidation by NAC is consistent with its antioxidant properties. Administration of NAC alone produced no evidence of lung injury. n = 3 at each time point. *p value less than 0.05 versus HDC + BMT–treated mice at the same time point. Values are expressed as mean ± SEM.

 
Mice supplemented with NAC also showed a significantly diminished degree of lung injury caused by HDC + BMT. As shown in Figures 8B and 8C, there was a significant reduction in total protein and LDH in the BALF of mice receiving HDC + BMT and NAC, versus HDC + BMT alone. The BALF total protein in mice receiving HDC + BMT and NAC remained constant at around 600 µg/ml at all the time points analyzed. On Day +1, BALF LDH was significantly reduced in mice receiving HDC + BMT + NAC to 210.2 ± 5.6 U/ml, as compared with 360.0 ±11.1 U/ml in HDC + BMT mice (p = 0.008, n = 3). At Days -1 and +4, NAC-treated mice showed no significant differences in BALF LDH levels.

Administration of NAC also reduced lipid peroxidation in the lungs of mice receiving HDC + BMT (Figure 8D). At all time points analyzed, markers of lipid peroxidation were significantly lower in the BALF of mice receiving HDC + BMT and NAC supplement. BALF MDA levels in mice receiving HDC + BMT and NAC were 53.5 ± 8.5 pM, 58.2 ± 1.6 pM, and 62.6 ± 4.5 pM, compared with 83.7 ± 11.3 pM, 92.2 ± 10.3 pM, and 86.1 ± 11.4 pM from mice receiving only HDC + BMT, on Days -1, +1, and +4, respectively (on each day p < 0.01, n = 3). Administration of NAC alone, showed no evidence of lung injury in all parameters analyzed (Figure 8).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the most common nonhematologic complications of dose-intensive chemotherapy and autologous BMT is pulmonary toxicity with a reported incidence of 39 to 64% (12–14). This form of IPS, under some series, appears to respond well to corticosteroids when recognized and treated early (12–14). However, morbidity and mortality remains a major concern, and severe IPS can limit further treatment options for patients with cancer. We have developed a murine model of IPS, modeled after a commonly used HDC and autologous BMT protocol (6). This strategy closely mimics human protocols and may allow better extrapolation of results to human studies. In this study, we have focused on the critical, early proinflammatory events occurring in the peri-BMT period that may promote the generation of IPS. Viewed collectively, our data indicate that the early phases of IPS were associated with an increase in lung AM population, induction of inflammatory proteins conducive to the recruitment and activation of macrophages, reduced lung compliance, and a striking oxidative stress profile.

The presence of increased numbers of AM in the epithelial lining fluid of HDC + BMT damaged lungs may have important implications for understanding the pathogenesis of IPS and for designing therapeutic modalities that could alter its development. There are two mechanisms by which the expansion of macrophage populations within an inflamed lung may occur: in situ proliferation and recruitment of monocytes into the lung. Although these two mechanisms may not be mutually exclusive, we favor the latter for several reasons. First, the elevation of two known monocyte chemoattractants, MCP-1 and MIP-1{alpha}, in the lung and their temporal relation to BALF cell counts implicates these molecules as responsible, at least in part, for the observed recruitment of monocytes. Second, treatment with myeloablative HDC would be predicted to all but eliminate the proliferative capacity of host monocytes. Third, the phenotype of AM from mice receiving HDC + BMT, CD11bhigh, CD54high, and CD71low, represents an immature monocyte-like phenotype (15). These findings suggest that the rapid expansion of the AM population was predominantly caused by an influx of monocytes.

We attempted to probe the potential stimuli that may be responsible for this cellular influx, as the elevated expression of key C–C chemokines in the lungs may have crucial ramifications in the development of IPS. We found sequential elevation of two key chemokines, MCP-1 and MIP-1{alpha}, in the lung and BALF. Both proteins are known to be chemotactic to mononuclear cells in a wide variety of lung diseases (16). Our data confirms AM as a major source of MCP-1 expression. MCP-1 has previously been shown to be sufficient for the transmigration of monocytes across the alveolar capillary interface (17). Peripheral blood monocytes do not normally constitutively express MCP-1, but this is strongly induced by transendothelial migration (18) and interaction with extracellular matrix proteins (19).

A key feature of MCP-1 expression in our murine model was its acute elevation before BMT, when there is a relative paucity of AM in the lungs. Alveolar epithelial cells may initiate the induction of MCP-1 expression in the lung after HDC. There may be several potential signaling mechanisms for inducing MCP-1 production. First, it has been shown that intracellular glutathione redox status modulates MCP-1 expression by the activation of redox-sensitive transcription factors, nuclear factor-kappa B and activator protein-1 (20). The enhanced oxidation of GSH that occurs after BCNU administration may be pivotal. Second, cyclophosphamide is known to cause early induction of monocyte chemotactic factors in the lungs of rats (21). Thus, both metabolic products of cyclophosphamide and altered GSH redox status may combine, leading to the initial stimulus for increasing MCP-1.

The presence of large numbers of immature AM in the lung raises the question of their contribution to the subsequent development of lung injury in the post-BMT period. An important finding in this study was the observation that AM from mice receiving HDC + BMT express significant quantities of CD14 on their cell surface. CD14 is a glycosylphospatidylinositol-linked cell surface receptor centrally involved in LPS recognition by myeloid and nonmyeloid cells (22). Its expression is rapidly upregulated in response to inflammatory activation by LPS, proinflammatory cytokines, and chemokines (23). Expression of CD14 was absent on AM from control mice as it has been shown that this antibody stains only macrophages activated by thioglycollate, culture, or mouse macrophage cell lines (24). Furthermore, CD14 expression is absent on mouse monocytes, unstimulated macrophages, and dendritic cells, suggesting that CD14 expression may differ in mice and humans (24, 25). Interestingly, CD14 upregulation in our model was associated with a heightened response to LPS challenge as evidenced by the twofold increase in TNF-{alpha} release by AM from mice receiving HDC + BMT, compared with that by AM from control mice. In addition, we found upregulation of Mac-3 on AM from mice receiving HDC + BMT. The Mac-3 antigen is expressed on thioglycollate-elicited peritoneal macrophages but not on unelicited macrophages and is considered a nonspecific marker of macrophage activation (26, 27). The data presented makes it reasonable to suppose that such activation of monocytes entering the alveolar compartment may contribute to lung inflammation after HDC + BMT. In addition, we have demonstrated that these AMs have a significantly higher oxidative burst, as detected by the redox-sensitive dye DCFH. Although initial characterization suggested that DCFH was specific for hydrogen peroxide, it may be oxidized by other free radicals including nitric oxide or products of nitric oxide interaction with superoxide anion (28). Enhanced respiratory burst in AM, and the accompanied secretion of hydrogen peroxide and superoxide anion, can damage endothelial cells, generate chemotactic factors, inhibit normal serum inhibitors of leukocytic proteases, and can release elastases, myeloperoxidases, and other enzymes that may contribute to the generation of enhanced oxidative stress in the epithelial lining fluid (29).

We have used the measurement of lung compliance as a tool to assess the pathophysiologic effects of HDC + BMT. Alterations in lung compliance have been described in mice exposed to hyperoxia, residual oil-fly ash, bleomycin, and also in a murine model of IPS after allogeneic BMT (7, 30, 31). Changes in lung compliance can reflect both lung injury and alterations in surfactant function. Endothelial cell injury with consequent alveolar and interstitial edema will stiffen the lungs, thereby decreasing total lung capacity and pressure–volume relationships. Modest changes in lung wet/dry ratios after HDC + BMT indicate that pulmonary edema probably plays only a small role in the reduction of lung compliance, and the early time points studied would mostly preclude a significant role of lung fibrosis. However, several mechanisms could account for a functional surfactant deficiency in this setting of lung injury. These include altered phospholipid content as a result of phospholipase activity, cell membrane breakdown with release of nonsurfactant phospholipids, decreased apoprotein content due to protease degradation, inhibition of function due to elevated serum protein levels resulting from microvascular leakage, and acyl group peroxidation by free radicals released from inflammatory cells (32). Of these potential causes, surfactant function inhibition after HDC + BMT is likely due to the very high levels of proteins in the BALF and the pro-oxidant milieu of the epithelial lining fluid. Preliminary electron microscopy indicated that the constituents of surfactant in the alveoli appeared morphologically normal in distribution and number (data not shown). However, in the absence of a detailed morphometric study of electron micrographs of lungs from mice receiving HDC + BMT, a true estimate of surfactant composition is precursory.

The most striking feature of this murine model was the intense oxidative stress produced in the lung. BCNU is known to inhibit GR (33), thus depleting free GSH levels (34) and leading to higher levels of oxidized glutathione. Cyclophosphamide has also been shown to deplete glutathione stores (35), increase the generation of reactive oxygen species by AM (21), inhibit prostaglandin E2 production by AM (36), and modulate epithelial lining fluid cell populations (21). Acrolein, a toxic metabolite of cyclophosphamide, can also react directly with GSH, forming irreversible adducts (37). Cisplatin has been shown to increase the production of superoxide anion (38) and to decrease the activity of lipid peroxide–protecting enzymes, such as catalase, Cu, Zn–superoxide dismutase, glutathione peroxidase and glutathione-S-transferase, in the liver and kidney of rats (39).

The lung is especially prone to oxidative injury as it has the largest endothelial surface area of any organ in the body, making it vulnerable to circulating toxins. To combat this, the lung has developed a battery of antioxidant defense systems. For example, lung tissue has the highest extracellular glutathione concentration among all tissues in the body, with normal epithelial lining fluid glutathione levels varying from 200 to 400 µM (40). Assessment of the glutathione profile in lung diseases is an accurate method to analyze pulmonary oxidative stress. Administration of a single combination dose of cyclophosphamide and cisplatin caused an acute depletion of GSH-T, accompanied by intense GSH oxidation (Figure 7A). Subsequently, there is a rebound increase in GSH-T, and GSH-T levels remained elevated throughout the entire course of the study. This is a well-known adaptive response to oxidative stress, wherein cells once exposed to an oxidant stimulus augment the production of GSH to combat further oxidation. Upregulation of GSH production can occur acutely (within minutes), without de novo synthesis, by a release of GSH bound to inactive {gamma}-glutamylcysteine synthetase. This enzyme is the rate-limiting step in the synthesis of GSH and is converted to an active form once GSH is released. Furthermore, the promoter region of the {gamma}-glutamylcysteine synthetase gene is regulated by redox-sensitive transcription factors, nuclear factor-kappa B and activator protein-1, indicating that oxidants can directly upregulate the gene for GSH synthesis (41). Thus our data indicate that after the first course of chemotherapy (Day -5), the lung is exposed to oxidative stress and tries to achieve an oxidative balance by augmenting GSH levels. However, the oxidative stress produced by chemotherapy would appear overwhelming as evidenced by enhanced lipid peroxidation and glutathione oxidation in the lung after HDC but before BMT. Our data demonstrate that oxidized glutathione in BALF before BMT is within normal limits, but coinciding with the influx of AM (Day +1), there is amplified GSH oxidation.

To test our hypothesis that the development of IPS was driven, at least in part, by oxidative stress, we supplemented mice receiving HDC + BMT with the antioxidant NAC. Administration of NAC significantly attenuated HDC + BMT–induced lung injury as manifest by fewer AM in the air spaces, reduced microvascular leak, reduced cytotoxicity, and marked attenuation of lipid peroxidation in the BALF. Although the protection was striking, NAC was unable to completely abrogate lung toxicity indicating one of two possibilities. First, at the dose used, NAC may be unable to combat the potent oxidative stress present in this model, or second, mechanisms other than oxidative stress may be involved in the pathogenesis of IPS. The antioxidant action of NAC has been attributed to direct oxidant scavenger function because of its sulfhydryl groups and to its glutathione precursor function (42). We were able to demonstrate significant increases in BALF GSH after administration of NAC to unmanipulated mice, indicating that the attenuation of oxidative stress and the consequent amelioration of lung injury was likely caused by the antioxidant effect of NAC in the lungs itself. Previously published data support our findings (43).

Our model of IPS after autologous BMT bears resemblance and also some dissimilarities from IPS seen after allogeneic BMT. Both types of IPS display the same kinetics of lung injury regarding cytokine and chemokine production, cellular influx, and surfactant dysfunction (31, 44). Both show increased lung oxidative stress (45), whereas allogeneic models also implicate reactive nitrogen species (46, 47). However, differences also exist in several respects. First, as expected, there was an absence of graft-versus-host disease, in our model, a phenomenon that likely plays a key role in the development of IPS after allogeneic BMT (48). Not only is the lung not a site for graft-versus-host disease, but other organs examined such as, liver, spleen, kidney, thymus, and intestine, displayed no graft-versus-host disease and minimal injury (data not shown). This suggests that the lung is preferentially injured in our model. Second, the prominent absence of lymphocytes in the early cellular influx into the airspaces is in stark contrast to allogeneic IPS models, where T cells have been show to play a key role in development and progression of the disease (31). We hypothesize, that this absence of lymphocytic influx is due to the lack of allogenicity in our IPS model.

In summary, in this murine model of HDC and autologous BMT, lung injury begins with an initial, severe oxidative stress, which produces tissue damage and further recruits an influx of monocytes into the lung, which we postulate may further exacerbate the lung damage. We have shown that NAC has the potential to attenuate the development of IPS, and this effect may warrant human trials. Furthermore, measures to block the adhesion, extravasation, activation, or differentiation of lung monocytes in the peri-BMT period are alternative strategies that may alleviate the incidence and severity of IPS after autologous BMT.


   Acknowledgments
 
The authors are indebted to Dr. Nelson Chao and Benny Chen for assistance with BMT. The technical expertise of Mike Cook and members of the Duke University Comprehensive Cancer Center Flow Cytometry Facility is gratefully acknowledged. The authors are especially grateful to Jordan Savov for his expertise with electron microscopy of the lung. They also thank Stephen Young for critical review of this manuscript.


   FOOTNOTES
 
Supported in part by National Institutes of Health Grants HL55166 and ES/HL-086988 and by an unrestricted research grant in memory of Doris M. Knauff to R.J.F.

This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form January 25, 2002; accepted in final form August 30, 2002


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