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Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation: association between idiopathic pneumonia syndrome and acute graft-versus-host disease
Transplant Immunology 23 (2010) 12–17
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Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation: association between idiopathic pneumonia syndrome and acute graft-versus-host disease☆ Qifa Liu ⁎, Juan Ning, Yu Zhang, Xiuli Wu, Xiaodan Luo, Zhiping Fan Department of Hematology, Nanfang Hospital, Southern Medical University, Guang Zhou 510515, China
a r t i c l e
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Article history: Received 5 October 2009 Received in revised form 18 December 2009 Accepted 22 February 2010 Keywords: Acute graft-versus-host disease Allogeneic hematopoietic stem cell transplant Idiopathic pneumonia syndrome IFN-γ TNF-α
a b s t r a c t Objective: To explore the association between idiopathic pneumonia syndrome (IPS) and acute graft-versushost disease (aGVHD) in allogeneic hematopoietic stem cell transplantation. Methods: Established acute GVHD model of C57BL/6→ BALB/c mice. Chest computed tomography (CT) scans were dynamically performed in recipient mice after transplant. Lung histopathology and cytokine levels (including TNF-α and IFN-γ) were examined in three experimental groups: mice receiving simple irradiation, syngeneic transplants, and allogeneic transplants. Results: All allogeneic transplant mice developed aGVHD. On CT, most aGVHD mice had bilateral diffuse lung infiltrates, while syngeneic transplant mice had normal lungs. On histopathology, aGVHD mice had acute pneumonitis. On immunohistochemistry, the infiltrates were mainly CD4+ T cells during aGVHD onset, but CD8+ T cells predominated during aGVHD progression. Lung TNF-α and IFN-γ levels were higher in the three experimental groups than in normal controls on days +3 and +7 post-transplant. On day +7, TNF-α levels were higher in allogeneic than in syngeneic transplant mice; IFN-γ levels were not different. On days +12 and +16, TNF-α levels were higher but IFN-γ levels were lower in allogeneic mice than in syngeneic transplant mice. Conclusions: The underlying cause of IPS is aGVHD. T cells and TNF-α may play a role in the pathogenesis of aGVHDinduced IPS. IPS progression may be associated with decreasing lung IFN-γ levels. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been used with increasing frequency to treat malignant or nonmalignant hematologic diseases. Although the clinical success rate of allo-HSCT has increased steadily, graft-versus-host disease (GVHD) and pulmonary complications remain serious threats to survival after transplantation [1–6]. Pulmonary complications account for 40–60% of the morbidity and mortality in allo-HSCT patients, and up to 80% of mortality posttransplantation has been reported to result from interstitial pneumonitis (both infectious and idiopathic) [1–4,7,8]. Pulmonary complications are classified as infectious or noninfectious according to the pathogenesis, and also as early- or late-onset depending on whether they appear in the first 100 days after transplantation or later. Idiopathic pneumonia syndrome (IPS) accounts for as many as 50% of cases of interstitial pneumonitis that are noninfectious pulmonary complications after alloHSCT. The frequency of IPS after allo-HSCT ranged from 5 to 25%[9]. As
☆ This study was supported by the National High-Tech Research and Development Plan of China (2006AA02Z4A0) and the Nature Science Foundation of Guangdong Province, China (07005160). ⁎ Corresponding author. Tel./fax: +86 20 61641622. E-mail address: liuqifa@fimmu.com (Q. Liu). 0966-3274/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2010.02.004
IPS usually occurs between 2 weeks and 6 months post-transplantation, it is often classified as an early pulmonary complication [1–4,7,8]. The main pathologic features of IPS are diffuse interstitial pneumonitis and diffuse alveolitis in the absence of an identifiable infectious agent; however, other reported manifestations include interstitial oedema, interstitial fibrosis, lymphocytic bronchiolitis, and alveolar haemorrhage [10–13]. A large body of experimental evidence has demonstrated that late-onset noninfectious lung injury after allo-HSCT is an immunologically mediated disease that shares a similar pathogenesis with chronic GVHD (cGVHD) [3,4,7,8,13]. Traditionally, acute GVHD (aGVHD) usually involves the liver, skin, and gut. In recent years, involvement of other organs, such as the thymus and lung, in aGVHD has also been reported [4,10–15]. With respect to aGVHD and IPS, Beschorner et al. [10] reported a group of patients who developed lymphocytic bronchitis associated with aGVHD after allo-HSCT as early as 1978. Although many studies have reported that IPS was associated with the development of clinical and experimental aGVHD, even when systemic GVHD was absent [12,14], whether the lung is one of the main target organs of aGVHD remains controversial [4,12]. The pathogenesis of how aGVHD induces IPS is unknown. Some authors have suggested that cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) might play a critical role in the process of aGVHD-induced IPS [4,12,16–19].
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To investigate the association between IPS and aGVHD in allo-HSCT, we established a murine model of aGVHD using a C57BL/6 → BALB/c mouse transplant.
2. Methods 2.1. Mice Female C57BL/6(H-2b) and male BALB/c (H-2d) mice were purchased from the Animal Experiment Center of Southern Medical University and transplanted between the ages of 6 and 8 weeks. The mice were subsequently housed in sterilized microisolator cages and received sterile rodent chow and autoclaved hyperchlorinated water after transplant. Ten healthy age-matched male BALB/c (H-2d) mice were served as the normal controls.
2.2. Bone marrow transplantation (BMT) and assessment of GVHD Before transplantation, BALB/c recipient mice received 8 Gy of total body x-ray irradiation (Varian 2100C/D) at a dose rate of 0.5 Gy/min. 6 h after irradiation, 0.2 mL cell mixtures of 1.0 × 107 bone marrow cells and 3.0 × 107 spleen cells, from either syngeneic (BALB/c) (group B) or allogeneic [C57BL/6(H-2b)] (group C) donors, suspended in an RPMI1640 medium were transplanted into BALB/c recipients via tail vein infusion, and a 0.2 mL RPMI-1640 medium was given to the third group of BALB/c recipients via tail vein infusion (simple irradiation group, group A). After the transplantation, the mice were housed under sterile conditions with no additional treatment. GVHD clinical scores were assessed using a scoring system involving five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity, and the histopathology of the liver, gut, and skin were assessed by the histologic criteria for GVHD, as previously described[11].
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2.5. Cytokine determination within the lung tissues Mice were sacrificed by exsanguination, and the lung tissues of 10 mice from each group were obtained on days +3, +7, + 12, and +16 post-transplantation. The lung tissues were ground to a powder in liquid nitrogen and weighed, tissue and cell lyses solutions were added for further analysis, the mixtures were centrifuged, and the supernatants were collected. The levels of cytokines, including TNF-α and IFN-γ, were measured by the LiquiChip™ protein suspension array system according to the manufacturer's protocol (LiquiChip Mouse Cytokine Kit, Qiagen, Germany). 2.6. Statistical analysis All values are expressed as means ± standard error. The data were analyzed by the independent-samples T test. P values b 0.05 were considered statistically significant. 3. Results 3.1. Reconstitution of white blood cells (WBCs), aGVHD, and survival times in mice after transplantation None of the mice in group A demonstrated WBC reconstitution. In groups B and C, the times to achieve WBCN 1×109 cells/L post-transplantation were 9.3±2.3 days and 10.3± 1.6 days, respectively (P=0.501). The mean survival times in groups A, B, and C were 9.7± 2.3, 60.0±0.0, and 15.2±1.3 days, respectively. Recipients were assessed daily for systemic aGVHD using the clinical scoring system. The three groups of recipient mice were similar following BMT for the first 6 days. All of them had weight loss with lassitude, depilation, and hunchback, but none had diarrhoea. The mice of group A were observed to be in extremis on day +6, and all of them had died by day +12 post-transplant. The body weights of group B mice began to rise on day +7 and were equal to or above their pre-transplant weights 3 weeks after transplantation, with no symptoms of aGVHD. The body weights of group C mice rose slightly between days +7 and +10, but decreased on days +10 to +12 posttransplantation, accompanied by other manifestations of aGVHD, including diarrhoea, lassitude, depilation, hunchback, and loss of skin integrity. Mice in group C were observed to be in extremis with aGVHD on day +14, and all of them died of GVHD by day +17 posttransplant. Histopathological evaluation of target organ tissues (including skin, liver, and gut) demonstrated characteristic aGVHD pathology. The incidence of aGVHD in group C was 100%, while in groups A and B it was 0%.
2.3. Chest CT scans 3.2. Chest CT findings
Chest high-resolution CT scans were performed on days +3, +7, and +12 after the transplantation in 10 mice from each group that were anaesthetized with 10% chloral hydrate via the abdominal cavity.
2.4. Histopathological and immunohistochemical analyses of lung tissues Ten mice from each group were sacrificed on days +3, +7, +12, and +16 post-transplantation. The lung tissues of each mouse were obtained and analyzed. In order to exclude infection, standard culture and staining methods for bacterial, fungi, viral, and protozoan pathogens were performed, as conventional methods. The residual lung tissues were placed in buffered formalin, and the formalin-preserved specimens were then embedded in paraffin, cut into 5-μm thick sections, and stained with hematoxylin and eosin for histological examination. To characterize the inflammatory cell infiltration and determine the relative distribution of inflammatory cells in the lungs of mice with aGVHD, immunohistochemical analyses for CD4+ and CD8+ T cells and CD68+ macrophages were performed on the lung tissues of recipient mice on days +3, +7, +12, and +16 post-transplantation. For immunohistochemical examination, paraffin-embedded sections were dewaxed, rehydrated, and incubated with 0.5% hydrogen peroxide in methanol to quench endogenous tissue peroxidase. Sections were incubated with pepsin for 45 min for antigen retrieval. After blocking non-specific sites with 1% BSA in PBS, sections were treated with primary anti-CD4, anti-CD8, and anti-CD68 antibodies, as well as the appropriate horseradish peroxidase-conjugated secondary antibodies, for 90 and 45 min, respectively. The definition of IPS regarding the histopathological diagnosis is according to established criteria [11].
Chest CTs were normal in mice of all three groups on days + 3 and + 7 after transplantation. However, two of the 10 mice in group A had diffuse ground-glass attenuation in both lungs on day + 12 (on the brink of death) (Fig. 1B), and seven of the 10 mice in group C had abnormal CT scans on day + 12 post-transplant (2 days after aGVHD appeared) (Fig. 1C). The characteristics of the abnormal CT scans in group C mice were similar to those of group A mice. Chest CT scans were normal in group B mice. A chest CT of a normal control is shown in Fig. 1A. 3.3. Pathogen evaluation of the lung tissues of mice with abnormal CT scans The results of the pathogen evaluation of the lung tissues showed that all group A samples were positive for bacteria and fungi; however, most group C samples were negative, except for one that was positive for fungi. 3.4. Histopathological evaluation of the lung tissues of mice with aGVHD Currently, the presence of IPS after allo-HSCT is mainly determined by examination of the lung histopathology. Histopathological changes in the lungs on day +3 posttransplantation were similar for all three groups; only minor and focal slight oedema of the epithelial cells and alveolar septum, and scattered infiltration by lymphocytes and macrophages were observed (Fig. 2A, B, C). On day +7 post-transplantation, the pathological examination indicated that the changes were exacerbated in group A (Fig. 2D), including alveolar epithelial oedema and hyperplasia, and alveolar septum widening, whereas the changes in group B (Fig. 2E) had diminished compared to those observed on day +3, and, in group C, alveolar epithelial oedema, capillary congestion, alveolar fibrinoid exudation, and lymphocyte and macrophage infiltrations into the alveolar interstitium could be seen (Fig. 2F). Alveolar epithelial necrosis, haemorrhage, and fibrinoid exudation were observed in the alveoli (Fig. 2G) in group A mice on day +12 (on the brink of death); in contrast, the histopathological characteristics had recovered to normal (Fig. 2H) in group B mice on day +12. However, in group C mice on day +12, dense mononuclear cell infiltrates were observed around both pulmonary vessels and bronchioles; acute pneumonitis was present, involving both the interstitial and alveolar spaces; the alveolar infiltrate was composed of macrophages, lymphocytes, epithelial cells,
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Fig. 1. High-resolution CT of the lungs of mice. A, CT findings of normal lungs of mice (both lungs are clear) B and C, diffuse ground-glass attenuation in both lungs in simple irradiation and allogeneic BMT mice, respectively, on day +12 after irradiation or BMT.
Fig. 2. Histopathological changes of lungs of mice in groups A, B, and C (HE, × 400). A, B, and C, only minor and focal slight oedema of epithelial cells and alveolar septum, as well as scattered lymphocyte and macrophage infiltrations on day + 3 in simple irradiation, syngeneic BMT, and allogeneic BMT mice, respectively. D, alveolar epithelial oedema and alveolar septum widening on day +7 in simple irradiation mice. E, the histological lesions on day + 7 are less severe compared to those observed on day + 3 (C) in syngeneic BMT. Alveolar epithelial oedema, capillary congestion, alveolar fibrinoid exudation, and lymphocyte and macrophage infiltrations into the alveolar interstitium is seen (F) on day + 7 in allogeneic BMT mice. G, alveolar epithelial necrosis, haemorrhage, and fibrinoid exudation in the alveoli on day + 12 (on the brink of death) in simple irradiation mice. H, nearly normal histology on day + 12 in syngeneic BMT mice. I and J, dense mononuclear cell infiltrates are observed around pulmonary vessels and bronchioles; acute pneumonitis is present involving both the interstitial and alveolar spaces; the alveolar infiltrate is composed of lymphocytes, macrophages, and scattered polymorphonuclear cells within a fibrin matrix on days + 12 and + 16, respectively, in allogeneic BMT mice. The histological lesions are more severe on day + 16 than on day + 12.
Q. Liu et al. / Transplant Immunology 23 (2010) 12–17 and scattered polymorphonuclear cells within a fibrin matrix (Fig. 2I); and on day +16 the histological lesions were similar to those on day +12, but more severe (Fig. 2J).
3.5. Characterization of the inflammatory cell infiltration of the lungs during aGVHD The lung tissues of all recipient mice displayed mainly CD4+ T cells on days +3 and +7 post-transplantation. The infiltration of CD68+ macrophages was higher on day +7 than on day +3 post-transplantation in irradiation-only mice, but lower on day +7 than on day +3 post-transplantation in syngeneic BMT mice. The infiltration of CD68+ macrophages was the same on days +3 and +7 in allogeneic BMT mice. On days +12 and +16 posttransplantation, the proportion of CD4+ T cells was decreasing and that of CD8+ T cells was increasing, but there were still more CD4+ T cells than CD8+ T cells on day +12, whereas there were fewer CD4+ T cells than CD8+ T cells on day +16 post-transplantation in allogeneic BMT mice. The T cell subset populations did not change at the different time points in syngeneic BMT mice. These data indicated that aGVHD mice developed a progressive interstitial pneumonitis that was dominated by CD4+ T cells early in the course of the disease and was replaced by a prominent infiltration of CD8+ T cells at later time points (Fig. 3).
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3.6. TNF-α and IFN-γ in lung tissues of the recipient mice In an attempt to delineate the mechanisms responsible for IPS occurring after alloHSCT, TNF-α and IFN-γ levels were measured in the lung tissues of recipient mice using multiplex bead-based protein assays. The TNF-α and IFN-γ levels were 23.5 ± 9.0 and 182.5 ± 38.7 pg/mL, respectively, in the lung tissues of normal mice. The posttransplantation TNF-α levels in groups A, B, and C were higher than in normal mice (P b 0.01); they are shown at different time points in Fig. 4. There was no statistical difference among the levels of TNF-α in groups A, B, and C on day + 3, but the TNF-α levels of group C mice were significantly higher than those of group A or group B mice on days + 7, + 12, and + 16 after transplantation (P = 0.001, P = 0.01, P = 0.0001, respectively). The TNF-α levels in groups A and B mice showed a decreasing trend, while those of group C showed an increasing trend on days + 7, + 12, and + 16 after transplantation. As shown in Fig. 5, the post-transplantation IFN-γ levels in all groups were also higher than the level in normal mice (P b 0.01). There were no significant differences in the IFN-γ levels among groups A, B, and C on days +3 and +7, but the IFN-γ levels were significantly higher in group B mice than in group A or C mice on days +12 and +16 after transplantation (P = 0.002, P = 0.0001, respectively).
Fig. 3. Changes in the infiltrating components in the lungs after allogeneic BMT. A, CD3+ T cell infiltration on day + 12 post-transplantation. B, CD4+ T cells on day + 7. C, CD8+ T cells on day + 7. D, CD68+ macrophages on day +7. E, CD4+ T cells on day + 16. F, CD8+ T cells on day + 16. G, changes in the T cell subset and macrophages within the lung tissues of mice with aGVHD.
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Fig. 4. Trends in TNF-α expression after BMT. The TNF-α levels after BMT are higher in each group than in normal mice. TNF-α levels of the simple irradiation group (48.2±7.7 pg/mL on day +3, 34.1±2.7 pg/mL on day +7, 35.2±5.3 pg/mL on day +12) and the syngeneic transplant group (48.7±9.2 pg/mL on day +3, 44.5±6.9 pg/mL on day +7, 42.2±4.3 pg/ mL on day +12, 40.3±5.2 pg/mL on day +16) show a decreasing trend from day +3 posttransplant, while those of the allogeneic transplant group show an increasing trend (46.2± 4.8 pg/mL on day +3, 62.5±6.3 pg/mL on day +7, 68.5±12.2 pg/mL on day +12, 71.5± 10.6 pg/mL on day +16), even with aGVHD.
4. Discussion Potential aetiologies for IPS include pre-transplant chemotherapy and irradiation, aGVHD, occult pulmonary infections, and the release of inflammatory cytokines [2–4,6,14–21]. To investigate the association between IPS and aGVHD in allo-HSCT, we established a murine model of aGVHD using a C57BL/6→ BALB/c mouse transplant and 8 Gy of the total body irradiation as a conditioning regimen, with all the mice achieving haematopoietic engraftment after transplant. The incidence of aGVHD was 100% following allogeneic transplantation and 0% following syngeneic transplantation. Although the dose of irradiation caused histologically detectable, minor interstitial pneumonitis on day +3, the histological changes recovered to normal on day +12 in syngeneic transplants. These results showed that the histological damage to the lungs by 8 Gy of irradiation as a conditioning regimen in syngeneic transplants was reversed. In contrast, the lung tissue from aGVHD mice at
Fig. 5. Trends in IFN-γ expression after BMT. The IFN-γ levels after BMT in each group are higher than in normal mice. IFN-γ levels of the simple irradiation group (319.8 ± 12.5 on day +3, 390.5 ± 37.9 on day +7, 357.5 ± 45.1 pg/mL on day +12) and the syngeneic transplant group (360.0±66.8 on day +3, 380.5±33.2 on day +7, 405.2±23.9 pg/mL on day +12, 417.2 ± 35.2 pg/mL on day +16) show an increasing trend after transplant; the IFN-γ levels of the allogeneic transplant group also increase after transplant (382.0± 29.4 pg/mL on day +3) and peak on day +7 (410.8± 30.0 pg/mL), then show a decreasing trend after aGVHD (303.4± 52.1 pg/mL on day +12, 274.4 ± 39. 0 pg/mL on day +16).
days +12 and +16 post-transplantation displayed the histopathologic hallmarks of IPS [11–13], as indicated by the epithelial cell damage, alveolar interstitium thickening, fibrinoid exudation in alveoli, lymphocyte and macrophage infiltrations and perivascular inflammation, and peribronchiolitis. This study demonstrates that BALB/c mice that are conditioned with total body irradiation and undergo transplantation with allogeneic C57BL/6 bone marrow cells containing mature T cells from spleen cells develop IPS. To the best of our knowledge, the study of lung injury using highresolution CT in a murine model of aGVHD has not been previously reported. The characteristic imaging finding in patients with IPS after allo-HSCT is the presence of bilateral lung diffuse infiltrate [2,4,14]. Bolanos-Meade et al. [14] reported that, in one patient, the manifestation of aGVHD of the lung was a diffuse centrilobular nodule on chest CT. Using high-resolution chest CT, we found that diffuse bilateral groundglass changes in the lungs of most of the mice in the allogeneic BMT group had attenuated on day 2 after aGVHD appeared. The characteristic imaging findings in mice with aGVHD were similar to those seen in patients with IPS. But the imaging changes were non-specific because we also observed that there were similar CT manifestations in the irradiation-only mice that suffered from infectious pneumonitis. Our data demonstrated that, in the C57BL/6→ BALB/c mouse transplant model, most recipients might incur early lung injury following aGVHD emergence and IPS associated with aGVHD. Current studies have examined the pathogenesis of GVHD-induced lung injury by demonstrating a role for donor T cells, monocytes/ macrophages, and neutrophils in mediating lung damage that occurs after allo-HSCT, where donor T cells were especially important mediators of lung toxicity [4,12,14–16,22]. Analyses of T cell subsets have revealed that CD8≶ cells are chiefly associated with aGVHD, whereas CD4+ cells are associated with chronic GVHD [22–24] in various GVHD models. In the present study, the immunohistochemical analysis of lung tissue from aGVHD mice showed that CD4+ T cells predominated within the lung tissues only before aGVHD appeared, and their numbers subsequently declined; in contrast, CD8+ T cells tended to increase and predominate within the lung tissues with aGVHD progression. Whether CD4+ T cells and/or the cytokines produced by these cells are responsible for the development of IPS and CD8+ T cells are responsible for the progression of IPS in this model remains to be determined. The activation and proliferation of T cells, as occur in aGVHD, are normally associated with Th1 cytokine secretion. The role of TNF-α in the pathogenesis of aGVHD has been demonstrated both in experimental and clinical settings [11,12,15–17,25–28]. Its role in mediating lung damage has also been demonstrated in an animal model in which systemic GVHD was absent [12]. The lung TNF-α levels increased [11,12,15,22,25,26], and the administration of TNF-α antiserum blocked the development of alveolar haemorrhage and reduced lung injury [25] in animal models with aGVHD. The present results showed that the TNFα levels in lung tissues increased before aGVHD appeared and continued increasing after aGVHD developed in mice that had undergone alloBMT. These results indicate that TNF-α is closely related with the occurrence and progression of aGVHD. Earlier studies demonstrated that activated T cells produced IFN-γ and that the level of IFN-γ in patients receiving allo-HSCT might reflect ongoing GVHD [19,21,29–31]. Such a correlation between high IFN-γ levels and severe GVHD led to a suggestion that IFN-γ might be involved in the pathogenesis of GVHD. In the IPS model of aGVHD, some studies reported that increasing IFN-γ levels in the lung were associated with IPS [11,12,22]. IFN-γ levels tend to be lower in cGVHD compared to aGVHD, especially in patients with accompanying pulmonary fibrosis, so that lower IFN-γ levels are thought to be associated with pulmonary fibrosis during cGVHD [21,22]. However, recent evidence has demonstrated that IFN-γ plays an important role in the maintenance of T cell homeostasis, eliminates activated CD4 and CD8 T cells, and may also downregulate immune responses [32–35]. Studies using anti-IFN-γ
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antibody or IFN-γ gene knockout mice demonstrated that this cytokine could inhibit the development of aGVHD [19,36–38]. Burman et al. [37] found that IFN-γ acts as a positive or negative mediator to aGVHD, mainly depending upon the peculiarity of the target origin, augments aGVHD in the gastrointestinal tract, and prevents the development of IPS. This is the result of the direct effects of IFN-γ on the pulmonary parenchyma to prevent donor cell migration and expansion within the lung. Kim et al. [39] demonstrated that IFN-γ suppresses GVHD by inducing the apoptosis of donor T cells. In the present study, we found that the levels of IFN-γ within the lung tissues of mice who had undergone allo-BMT were significantly elevated before aGVHD appeared compared with untreated controls (normal mice), but they were not significantly different compared with treated controls (mice undergoing simple irradiation or syngeneic BMT). Interestingly, the IFNγ levels in mice undergoing allo-BMT showed a descending trend during aGVHD progression. This result indicates, indirectly, that IPS progression might be associated with decreasing IFN-γ levels within the lung tissues. This result is not in complete accord with some related reports [14– 16,18,22,26]. Some studies have suggested that the descending trend in the IFN-γ levels from aGVHD to cGVHD is the result of Th1 T cells being replaced by Th2 T cells. In this model, the reasons why the IFN-γ levels within the lung tissues decreased during aGVHD progression need further study. We presume that the decrease in IFN-γ levels accompanying the progression of aGVHD results from decreased numbers of T cells producing IFN-γ, is due to the conditioning before aGVHD appeared, which induced the apoptosis of T cells or prevented T cells migrating into, and multiplying within, the lungs. However, this assumption has yet to be proven. In conclusion, aGVHD is one of the primary causes of IPS after alloHSCT. Both lymphocytes and TNF are involved in aGVHD-induced IPS. IPS progression might be associated with decreasing IFN-γ levels within lung tissues. Acknowledgements The authors would like to thank Prof. Zhou Jian-feng from the Department of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for assistance with the immunohistochemical analysis. References [1] Soubani AO, Miller KB, Hassoun PM. Pulmonary complications of bone marrow transplantation. Chest 1996;109(4):1066–77. [2] Afessa B, Litzow MR, Tefferi A. Bronchiolitis obliterans and other late onset noninfectious pulmonary complications in hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;28(5):425–34. [3] Freudenberger TD, Madtes DK, Curtis JR, et al. Association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants. Blood 2003;102(10):3822–8. [4] Yanik G, Cooke KR. The lung as a target organ of graft-versus-host disease. Semin Hematol 2006;43(1):42–52. [5] Bacigalupo A. Management of acute graft-versus-host disease. Br J Haematol 2007;137 (2):87–98. [6] Eapen M, Logan BR, Confer DL, et al. Peripheral blood grafts from unrelated donors are associated with increased acute and chronic graft-versus-host disease without improved survival. Biol Blood Marrow Transplant 2007;13(12):1461–8. [7] Afessa B, Peters SG. Noninfectious pneumonitis after blood and marrow transplant. Curr Opin Oncol 2008;20(2):227–33. [8] Yoshihara S, Yanik G, Cooke KR, et al. Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007;13(7):749–59. [9] Yanik G, Hellerstedt B, Custer J, et al. Etanercept (Enbrel) administration for idiopathic pneumonia syndrome after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2002;8(7):395–400. [10] Beschorner WE, Saral R, Hutchins GM, et al. Lymphocytic bronchitis associated with graft-versus-host disease in recipients of bone-marrow transplants. N Engl J Med 1978;299(19):1030–6.
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Pneumopathies of the graft-versus-host reaction Alveolitis associated with an increased level of tumor necrosis factor mRNA and chronic interstitial pneumonitis. Lab Invest 1989;61(1):37–45. [27] Remberger M, Ringden O, Markling L. TNF alpha levels are increased during bone marrow transplantation conditioning in patients who develop acute GVHD. Bone Marrow Transplant 1995;15(1):99–104. [28] Nomura S, Ishii K, Inami N, et al. Role of soluble tumor necrosis factor-related apoptosis-inducing ligand concentrations after stem cell transplantation. Transpl Immunol 2007;18(2):115–21. [29] Das H, Imoto S, Murayama T, et al. Kinetic analysis of cytokine gene expression in patients with GVHD after donor lymphocyte infusion. Bone Marrow Transplant 2001;27(4):373–80. [30] Mowat AM. Antibodies to IFN-gamma prevent immunologically mediated intestinal damage in murine graft-versus-host reaction. Immunology 1989;68(1):18–23. [31] Niederwieser D, Herold M, Woloszczuk W, et al. Endogenous IFN-gamma during human bone marrow transplantation analysis of serum levels of interferon and interferon-dependent secondary messages. Transplantation 1990;50(4):620–5. [32] Badovinac VP, Tvinnereim AR, Harty JT. Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma. Science 2000;290(5495):1354–8. [33] Chu CQ, Wittmer S, Dalton DK. Failure to suppress the expansion of the activated CD4 T cell population in interferon gamma-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J Exp Med 2000;192(1):123–8. [34] Dalton DK, Haynes L, Chu CQ, et al. Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J Exp Med 2000;192(1):117–22. [35] Refaeli Y, Van Parijs L, Alexander SI, et al. Interferon gamma is required for activation-induced death of T lymphocytes. J Exp Med 2002;196(7):999–1005. [36] Asavaroengchai W, Wang H, Wang S, et al. An essential role for IFN-gamma in regulation of alloreactive CD8 T cells following allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2007;13(1):46–55. [37] Burman AC, Banovic T, Kuns RD, et al. IFNgamma differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract. Blood 2007;110(3):1064–72. [38] Murphy WJ, Welniak LA, Taub DD, et al. Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice. J Clin Invest 1998;102(9):1742–8. [39] Kim JH, Choi EY, Chung DH. Donor bone marrow type II (non-Valpha14Jalpha18 CD1d-restricted) NKT cells suppress graft-versus-host disease by producing IFNgamma and IL-4. J Immunol 2007;179(10):6579–87.
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Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation: association between idiopathic pneumonia syndrome and acute graft-versus-host disease☆ Qifa Liu ⁎, Juan Ning, Yu Zhang, Xiuli Wu, Xiaodan Luo, Zhiping Fan Department of Hematology, Nanfang Hospital, Southern Medical University, Guang Zhou 510515, China
a r t i c l e
i n f o
Article history: Received 5 October 2009 Received in revised form 18 December 2009 Accepted 22 February 2010 Keywords: Acute graft-versus-host disease Allogeneic hematopoietic stem cell transplant Idiopathic pneumonia syndrome IFN-γ TNF-α
a b s t r a c t Objective: To explore the association between idiopathic pneumonia syndrome (IPS) and acute graft-versushost disease (aGVHD) in allogeneic hematopoietic stem cell transplantation. Methods: Established acute GVHD model of C57BL/6→ BALB/c mice. Chest computed tomography (CT) scans were dynamically performed in recipient mice after transplant. Lung histopathology and cytokine levels (including TNF-α and IFN-γ) were examined in three experimental groups: mice receiving simple irradiation, syngeneic transplants, and allogeneic transplants. Results: All allogeneic transplant mice developed aGVHD. On CT, most aGVHD mice had bilateral diffuse lung infiltrates, while syngeneic transplant mice had normal lungs. On histopathology, aGVHD mice had acute pneumonitis. On immunohistochemistry, the infiltrates were mainly CD4+ T cells during aGVHD onset, but CD8+ T cells predominated during aGVHD progression. Lung TNF-α and IFN-γ levels were higher in the three experimental groups than in normal controls on days +3 and +7 post-transplant. On day +7, TNF-α levels were higher in allogeneic than in syngeneic transplant mice; IFN-γ levels were not different. On days +12 and +16, TNF-α levels were higher but IFN-γ levels were lower in allogeneic mice than in syngeneic transplant mice. Conclusions: The underlying cause of IPS is aGVHD. T cells and TNF-α may play a role in the pathogenesis of aGVHDinduced IPS. IPS progression may be associated with decreasing lung IFN-γ levels. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been used with increasing frequency to treat malignant or nonmalignant hematologic diseases. Although the clinical success rate of allo-HSCT has increased steadily, graft-versus-host disease (GVHD) and pulmonary complications remain serious threats to survival after transplantation [1–6]. Pulmonary complications account for 40–60% of the morbidity and mortality in allo-HSCT patients, and up to 80% of mortality posttransplantation has been reported to result from interstitial pneumonitis (both infectious and idiopathic) [1–4,7,8]. Pulmonary complications are classified as infectious or noninfectious according to the pathogenesis, and also as early- or late-onset depending on whether they appear in the first 100 days after transplantation or later. Idiopathic pneumonia syndrome (IPS) accounts for as many as 50% of cases of interstitial pneumonitis that are noninfectious pulmonary complications after alloHSCT. The frequency of IPS after allo-HSCT ranged from 5 to 25%[9]. As
☆ This study was supported by the National High-Tech Research and Development Plan of China (2006AA02Z4A0) and the Nature Science Foundation of Guangdong Province, China (07005160). ⁎ Corresponding author. Tel./fax: +86 20 61641622. E-mail address: liuqifa@fimmu.com (Q. Liu). 0966-3274/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2010.02.004
IPS usually occurs between 2 weeks and 6 months post-transplantation, it is often classified as an early pulmonary complication [1–4,7,8]. The main pathologic features of IPS are diffuse interstitial pneumonitis and diffuse alveolitis in the absence of an identifiable infectious agent; however, other reported manifestations include interstitial oedema, interstitial fibrosis, lymphocytic bronchiolitis, and alveolar haemorrhage [10–13]. A large body of experimental evidence has demonstrated that late-onset noninfectious lung injury after allo-HSCT is an immunologically mediated disease that shares a similar pathogenesis with chronic GVHD (cGVHD) [3,4,7,8,13]. Traditionally, acute GVHD (aGVHD) usually involves the liver, skin, and gut. In recent years, involvement of other organs, such as the thymus and lung, in aGVHD has also been reported [4,10–15]. With respect to aGVHD and IPS, Beschorner et al. [10] reported a group of patients who developed lymphocytic bronchitis associated with aGVHD after allo-HSCT as early as 1978. Although many studies have reported that IPS was associated with the development of clinical and experimental aGVHD, even when systemic GVHD was absent [12,14], whether the lung is one of the main target organs of aGVHD remains controversial [4,12]. The pathogenesis of how aGVHD induces IPS is unknown. Some authors have suggested that cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) might play a critical role in the process of aGVHD-induced IPS [4,12,16–19].
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To investigate the association between IPS and aGVHD in allo-HSCT, we established a murine model of aGVHD using a C57BL/6 → BALB/c mouse transplant.
2. Methods 2.1. Mice Female C57BL/6(H-2b) and male BALB/c (H-2d) mice were purchased from the Animal Experiment Center of Southern Medical University and transplanted between the ages of 6 and 8 weeks. The mice were subsequently housed in sterilized microisolator cages and received sterile rodent chow and autoclaved hyperchlorinated water after transplant. Ten healthy age-matched male BALB/c (H-2d) mice were served as the normal controls.
2.2. Bone marrow transplantation (BMT) and assessment of GVHD Before transplantation, BALB/c recipient mice received 8 Gy of total body x-ray irradiation (Varian 2100C/D) at a dose rate of 0.5 Gy/min. 6 h after irradiation, 0.2 mL cell mixtures of 1.0 × 107 bone marrow cells and 3.0 × 107 spleen cells, from either syngeneic (BALB/c) (group B) or allogeneic [C57BL/6(H-2b)] (group C) donors, suspended in an RPMI1640 medium were transplanted into BALB/c recipients via tail vein infusion, and a 0.2 mL RPMI-1640 medium was given to the third group of BALB/c recipients via tail vein infusion (simple irradiation group, group A). After the transplantation, the mice were housed under sterile conditions with no additional treatment. GVHD clinical scores were assessed using a scoring system involving five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity, and the histopathology of the liver, gut, and skin were assessed by the histologic criteria for GVHD, as previously described[11].
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2.5. Cytokine determination within the lung tissues Mice were sacrificed by exsanguination, and the lung tissues of 10 mice from each group were obtained on days +3, +7, + 12, and +16 post-transplantation. The lung tissues were ground to a powder in liquid nitrogen and weighed, tissue and cell lyses solutions were added for further analysis, the mixtures were centrifuged, and the supernatants were collected. The levels of cytokines, including TNF-α and IFN-γ, were measured by the LiquiChip™ protein suspension array system according to the manufacturer's protocol (LiquiChip Mouse Cytokine Kit, Qiagen, Germany). 2.6. Statistical analysis All values are expressed as means ± standard error. The data were analyzed by the independent-samples T test. P values b 0.05 were considered statistically significant. 3. Results 3.1. Reconstitution of white blood cells (WBCs), aGVHD, and survival times in mice after transplantation None of the mice in group A demonstrated WBC reconstitution. In groups B and C, the times to achieve WBCN 1×109 cells/L post-transplantation were 9.3±2.3 days and 10.3± 1.6 days, respectively (P=0.501). The mean survival times in groups A, B, and C were 9.7± 2.3, 60.0±0.0, and 15.2±1.3 days, respectively. Recipients were assessed daily for systemic aGVHD using the clinical scoring system. The three groups of recipient mice were similar following BMT for the first 6 days. All of them had weight loss with lassitude, depilation, and hunchback, but none had diarrhoea. The mice of group A were observed to be in extremis on day +6, and all of them had died by day +12 post-transplant. The body weights of group B mice began to rise on day +7 and were equal to or above their pre-transplant weights 3 weeks after transplantation, with no symptoms of aGVHD. The body weights of group C mice rose slightly between days +7 and +10, but decreased on days +10 to +12 posttransplantation, accompanied by other manifestations of aGVHD, including diarrhoea, lassitude, depilation, hunchback, and loss of skin integrity. Mice in group C were observed to be in extremis with aGVHD on day +14, and all of them died of GVHD by day +17 posttransplant. Histopathological evaluation of target organ tissues (including skin, liver, and gut) demonstrated characteristic aGVHD pathology. The incidence of aGVHD in group C was 100%, while in groups A and B it was 0%.
2.3. Chest CT scans 3.2. Chest CT findings
Chest high-resolution CT scans were performed on days +3, +7, and +12 after the transplantation in 10 mice from each group that were anaesthetized with 10% chloral hydrate via the abdominal cavity.
2.4. Histopathological and immunohistochemical analyses of lung tissues Ten mice from each group were sacrificed on days +3, +7, +12, and +16 post-transplantation. The lung tissues of each mouse were obtained and analyzed. In order to exclude infection, standard culture and staining methods for bacterial, fungi, viral, and protozoan pathogens were performed, as conventional methods. The residual lung tissues were placed in buffered formalin, and the formalin-preserved specimens were then embedded in paraffin, cut into 5-μm thick sections, and stained with hematoxylin and eosin for histological examination. To characterize the inflammatory cell infiltration and determine the relative distribution of inflammatory cells in the lungs of mice with aGVHD, immunohistochemical analyses for CD4+ and CD8+ T cells and CD68+ macrophages were performed on the lung tissues of recipient mice on days +3, +7, +12, and +16 post-transplantation. For immunohistochemical examination, paraffin-embedded sections were dewaxed, rehydrated, and incubated with 0.5% hydrogen peroxide in methanol to quench endogenous tissue peroxidase. Sections were incubated with pepsin for 45 min for antigen retrieval. After blocking non-specific sites with 1% BSA in PBS, sections were treated with primary anti-CD4, anti-CD8, and anti-CD68 antibodies, as well as the appropriate horseradish peroxidase-conjugated secondary antibodies, for 90 and 45 min, respectively. The definition of IPS regarding the histopathological diagnosis is according to established criteria [11].
Chest CTs were normal in mice of all three groups on days + 3 and + 7 after transplantation. However, two of the 10 mice in group A had diffuse ground-glass attenuation in both lungs on day + 12 (on the brink of death) (Fig. 1B), and seven of the 10 mice in group C had abnormal CT scans on day + 12 post-transplant (2 days after aGVHD appeared) (Fig. 1C). The characteristics of the abnormal CT scans in group C mice were similar to those of group A mice. Chest CT scans were normal in group B mice. A chest CT of a normal control is shown in Fig. 1A. 3.3. Pathogen evaluation of the lung tissues of mice with abnormal CT scans The results of the pathogen evaluation of the lung tissues showed that all group A samples were positive for bacteria and fungi; however, most group C samples were negative, except for one that was positive for fungi. 3.4. Histopathological evaluation of the lung tissues of mice with aGVHD Currently, the presence of IPS after allo-HSCT is mainly determined by examination of the lung histopathology. Histopathological changes in the lungs on day +3 posttransplantation were similar for all three groups; only minor and focal slight oedema of the epithelial cells and alveolar septum, and scattered infiltration by lymphocytes and macrophages were observed (Fig. 2A, B, C). On day +7 post-transplantation, the pathological examination indicated that the changes were exacerbated in group A (Fig. 2D), including alveolar epithelial oedema and hyperplasia, and alveolar septum widening, whereas the changes in group B (Fig. 2E) had diminished compared to those observed on day +3, and, in group C, alveolar epithelial oedema, capillary congestion, alveolar fibrinoid exudation, and lymphocyte and macrophage infiltrations into the alveolar interstitium could be seen (Fig. 2F). Alveolar epithelial necrosis, haemorrhage, and fibrinoid exudation were observed in the alveoli (Fig. 2G) in group A mice on day +12 (on the brink of death); in contrast, the histopathological characteristics had recovered to normal (Fig. 2H) in group B mice on day +12. However, in group C mice on day +12, dense mononuclear cell infiltrates were observed around both pulmonary vessels and bronchioles; acute pneumonitis was present, involving both the interstitial and alveolar spaces; the alveolar infiltrate was composed of macrophages, lymphocytes, epithelial cells,
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Fig. 1. High-resolution CT of the lungs of mice. A, CT findings of normal lungs of mice (both lungs are clear) B and C, diffuse ground-glass attenuation in both lungs in simple irradiation and allogeneic BMT mice, respectively, on day +12 after irradiation or BMT.
Fig. 2. Histopathological changes of lungs of mice in groups A, B, and C (HE, × 400). A, B, and C, only minor and focal slight oedema of epithelial cells and alveolar septum, as well as scattered lymphocyte and macrophage infiltrations on day + 3 in simple irradiation, syngeneic BMT, and allogeneic BMT mice, respectively. D, alveolar epithelial oedema and alveolar septum widening on day +7 in simple irradiation mice. E, the histological lesions on day + 7 are less severe compared to those observed on day + 3 (C) in syngeneic BMT. Alveolar epithelial oedema, capillary congestion, alveolar fibrinoid exudation, and lymphocyte and macrophage infiltrations into the alveolar interstitium is seen (F) on day + 7 in allogeneic BMT mice. G, alveolar epithelial necrosis, haemorrhage, and fibrinoid exudation in the alveoli on day + 12 (on the brink of death) in simple irradiation mice. H, nearly normal histology on day + 12 in syngeneic BMT mice. I and J, dense mononuclear cell infiltrates are observed around pulmonary vessels and bronchioles; acute pneumonitis is present involving both the interstitial and alveolar spaces; the alveolar infiltrate is composed of lymphocytes, macrophages, and scattered polymorphonuclear cells within a fibrin matrix on days + 12 and + 16, respectively, in allogeneic BMT mice. The histological lesions are more severe on day + 16 than on day + 12.
Q. Liu et al. / Transplant Immunology 23 (2010) 12–17 and scattered polymorphonuclear cells within a fibrin matrix (Fig. 2I); and on day +16 the histological lesions were similar to those on day +12, but more severe (Fig. 2J).
3.5. Characterization of the inflammatory cell infiltration of the lungs during aGVHD The lung tissues of all recipient mice displayed mainly CD4+ T cells on days +3 and +7 post-transplantation. The infiltration of CD68+ macrophages was higher on day +7 than on day +3 post-transplantation in irradiation-only mice, but lower on day +7 than on day +3 post-transplantation in syngeneic BMT mice. The infiltration of CD68+ macrophages was the same on days +3 and +7 in allogeneic BMT mice. On days +12 and +16 posttransplantation, the proportion of CD4+ T cells was decreasing and that of CD8+ T cells was increasing, but there were still more CD4+ T cells than CD8+ T cells on day +12, whereas there were fewer CD4+ T cells than CD8+ T cells on day +16 post-transplantation in allogeneic BMT mice. The T cell subset populations did not change at the different time points in syngeneic BMT mice. These data indicated that aGVHD mice developed a progressive interstitial pneumonitis that was dominated by CD4+ T cells early in the course of the disease and was replaced by a prominent infiltration of CD8+ T cells at later time points (Fig. 3).
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3.6. TNF-α and IFN-γ in lung tissues of the recipient mice In an attempt to delineate the mechanisms responsible for IPS occurring after alloHSCT, TNF-α and IFN-γ levels were measured in the lung tissues of recipient mice using multiplex bead-based protein assays. The TNF-α and IFN-γ levels were 23.5 ± 9.0 and 182.5 ± 38.7 pg/mL, respectively, in the lung tissues of normal mice. The posttransplantation TNF-α levels in groups A, B, and C were higher than in normal mice (P b 0.01); they are shown at different time points in Fig. 4. There was no statistical difference among the levels of TNF-α in groups A, B, and C on day + 3, but the TNF-α levels of group C mice were significantly higher than those of group A or group B mice on days + 7, + 12, and + 16 after transplantation (P = 0.001, P = 0.01, P = 0.0001, respectively). The TNF-α levels in groups A and B mice showed a decreasing trend, while those of group C showed an increasing trend on days + 7, + 12, and + 16 after transplantation. As shown in Fig. 5, the post-transplantation IFN-γ levels in all groups were also higher than the level in normal mice (P b 0.01). There were no significant differences in the IFN-γ levels among groups A, B, and C on days +3 and +7, but the IFN-γ levels were significantly higher in group B mice than in group A or C mice on days +12 and +16 after transplantation (P = 0.002, P = 0.0001, respectively).
Fig. 3. Changes in the infiltrating components in the lungs after allogeneic BMT. A, CD3+ T cell infiltration on day + 12 post-transplantation. B, CD4+ T cells on day + 7. C, CD8+ T cells on day + 7. D, CD68+ macrophages on day +7. E, CD4+ T cells on day + 16. F, CD8+ T cells on day + 16. G, changes in the T cell subset and macrophages within the lung tissues of mice with aGVHD.
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Fig. 4. Trends in TNF-α expression after BMT. The TNF-α levels after BMT are higher in each group than in normal mice. TNF-α levels of the simple irradiation group (48.2±7.7 pg/mL on day +3, 34.1±2.7 pg/mL on day +7, 35.2±5.3 pg/mL on day +12) and the syngeneic transplant group (48.7±9.2 pg/mL on day +3, 44.5±6.9 pg/mL on day +7, 42.2±4.3 pg/ mL on day +12, 40.3±5.2 pg/mL on day +16) show a decreasing trend from day +3 posttransplant, while those of the allogeneic transplant group show an increasing trend (46.2± 4.8 pg/mL on day +3, 62.5±6.3 pg/mL on day +7, 68.5±12.2 pg/mL on day +12, 71.5± 10.6 pg/mL on day +16), even with aGVHD.
4. Discussion Potential aetiologies for IPS include pre-transplant chemotherapy and irradiation, aGVHD, occult pulmonary infections, and the release of inflammatory cytokines [2–4,6,14–21]. To investigate the association between IPS and aGVHD in allo-HSCT, we established a murine model of aGVHD using a C57BL/6→ BALB/c mouse transplant and 8 Gy of the total body irradiation as a conditioning regimen, with all the mice achieving haematopoietic engraftment after transplant. The incidence of aGVHD was 100% following allogeneic transplantation and 0% following syngeneic transplantation. Although the dose of irradiation caused histologically detectable, minor interstitial pneumonitis on day +3, the histological changes recovered to normal on day +12 in syngeneic transplants. These results showed that the histological damage to the lungs by 8 Gy of irradiation as a conditioning regimen in syngeneic transplants was reversed. In contrast, the lung tissue from aGVHD mice at
Fig. 5. Trends in IFN-γ expression after BMT. The IFN-γ levels after BMT in each group are higher than in normal mice. IFN-γ levels of the simple irradiation group (319.8 ± 12.5 on day +3, 390.5 ± 37.9 on day +7, 357.5 ± 45.1 pg/mL on day +12) and the syngeneic transplant group (360.0±66.8 on day +3, 380.5±33.2 on day +7, 405.2±23.9 pg/mL on day +12, 417.2 ± 35.2 pg/mL on day +16) show an increasing trend after transplant; the IFN-γ levels of the allogeneic transplant group also increase after transplant (382.0± 29.4 pg/mL on day +3) and peak on day +7 (410.8± 30.0 pg/mL), then show a decreasing trend after aGVHD (303.4± 52.1 pg/mL on day +12, 274.4 ± 39. 0 pg/mL on day +16).
days +12 and +16 post-transplantation displayed the histopathologic hallmarks of IPS [11–13], as indicated by the epithelial cell damage, alveolar interstitium thickening, fibrinoid exudation in alveoli, lymphocyte and macrophage infiltrations and perivascular inflammation, and peribronchiolitis. This study demonstrates that BALB/c mice that are conditioned with total body irradiation and undergo transplantation with allogeneic C57BL/6 bone marrow cells containing mature T cells from spleen cells develop IPS. To the best of our knowledge, the study of lung injury using highresolution CT in a murine model of aGVHD has not been previously reported. The characteristic imaging finding in patients with IPS after allo-HSCT is the presence of bilateral lung diffuse infiltrate [2,4,14]. Bolanos-Meade et al. [14] reported that, in one patient, the manifestation of aGVHD of the lung was a diffuse centrilobular nodule on chest CT. Using high-resolution chest CT, we found that diffuse bilateral groundglass changes in the lungs of most of the mice in the allogeneic BMT group had attenuated on day 2 after aGVHD appeared. The characteristic imaging findings in mice with aGVHD were similar to those seen in patients with IPS. But the imaging changes were non-specific because we also observed that there were similar CT manifestations in the irradiation-only mice that suffered from infectious pneumonitis. Our data demonstrated that, in the C57BL/6→ BALB/c mouse transplant model, most recipients might incur early lung injury following aGVHD emergence and IPS associated with aGVHD. Current studies have examined the pathogenesis of GVHD-induced lung injury by demonstrating a role for donor T cells, monocytes/ macrophages, and neutrophils in mediating lung damage that occurs after allo-HSCT, where donor T cells were especially important mediators of lung toxicity [4,12,14–16,22]. Analyses of T cell subsets have revealed that CD8≶ cells are chiefly associated with aGVHD, whereas CD4+ cells are associated with chronic GVHD [22–24] in various GVHD models. In the present study, the immunohistochemical analysis of lung tissue from aGVHD mice showed that CD4+ T cells predominated within the lung tissues only before aGVHD appeared, and their numbers subsequently declined; in contrast, CD8+ T cells tended to increase and predominate within the lung tissues with aGVHD progression. Whether CD4+ T cells and/or the cytokines produced by these cells are responsible for the development of IPS and CD8+ T cells are responsible for the progression of IPS in this model remains to be determined. The activation and proliferation of T cells, as occur in aGVHD, are normally associated with Th1 cytokine secretion. The role of TNF-α in the pathogenesis of aGVHD has been demonstrated both in experimental and clinical settings [11,12,15–17,25–28]. Its role in mediating lung damage has also been demonstrated in an animal model in which systemic GVHD was absent [12]. The lung TNF-α levels increased [11,12,15,22,25,26], and the administration of TNF-α antiserum blocked the development of alveolar haemorrhage and reduced lung injury [25] in animal models with aGVHD. The present results showed that the TNFα levels in lung tissues increased before aGVHD appeared and continued increasing after aGVHD developed in mice that had undergone alloBMT. These results indicate that TNF-α is closely related with the occurrence and progression of aGVHD. Earlier studies demonstrated that activated T cells produced IFN-γ and that the level of IFN-γ in patients receiving allo-HSCT might reflect ongoing GVHD [19,21,29–31]. Such a correlation between high IFN-γ levels and severe GVHD led to a suggestion that IFN-γ might be involved in the pathogenesis of GVHD. In the IPS model of aGVHD, some studies reported that increasing IFN-γ levels in the lung were associated with IPS [11,12,22]. IFN-γ levels tend to be lower in cGVHD compared to aGVHD, especially in patients with accompanying pulmonary fibrosis, so that lower IFN-γ levels are thought to be associated with pulmonary fibrosis during cGVHD [21,22]. However, recent evidence has demonstrated that IFN-γ plays an important role in the maintenance of T cell homeostasis, eliminates activated CD4 and CD8 T cells, and may also downregulate immune responses [32–35]. Studies using anti-IFN-γ
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antibody or IFN-γ gene knockout mice demonstrated that this cytokine could inhibit the development of aGVHD [19,36–38]. Burman et al. [37] found that IFN-γ acts as a positive or negative mediator to aGVHD, mainly depending upon the peculiarity of the target origin, augments aGVHD in the gastrointestinal tract, and prevents the development of IPS. This is the result of the direct effects of IFN-γ on the pulmonary parenchyma to prevent donor cell migration and expansion within the lung. Kim et al. [39] demonstrated that IFN-γ suppresses GVHD by inducing the apoptosis of donor T cells. In the present study, we found that the levels of IFN-γ within the lung tissues of mice who had undergone allo-BMT were significantly elevated before aGVHD appeared compared with untreated controls (normal mice), but they were not significantly different compared with treated controls (mice undergoing simple irradiation or syngeneic BMT). Interestingly, the IFNγ levels in mice undergoing allo-BMT showed a descending trend during aGVHD progression. This result indicates, indirectly, that IPS progression might be associated with decreasing IFN-γ levels within the lung tissues. This result is not in complete accord with some related reports [14– 16,18,22,26]. Some studies have suggested that the descending trend in the IFN-γ levels from aGVHD to cGVHD is the result of Th1 T cells being replaced by Th2 T cells. In this model, the reasons why the IFN-γ levels within the lung tissues decreased during aGVHD progression need further study. We presume that the decrease in IFN-γ levels accompanying the progression of aGVHD results from decreased numbers of T cells producing IFN-γ, is due to the conditioning before aGVHD appeared, which induced the apoptosis of T cells or prevented T cells migrating into, and multiplying within, the lungs. However, this assumption has yet to be proven. In conclusion, aGVHD is one of the primary causes of IPS after alloHSCT. Both lymphocytes and TNF are involved in aGVHD-induced IPS. IPS progression might be associated with decreasing IFN-γ levels within lung tissues. Acknowledgements The authors would like to thank Prof. Zhou Jian-feng from the Department of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for assistance with the immunohistochemical analysis. References [1] Soubani AO, Miller KB, Hassoun PM. Pulmonary complications of bone marrow transplantation. Chest 1996;109(4):1066–77. [2] Afessa B, Litzow MR, Tefferi A. Bronchiolitis obliterans and other late onset noninfectious pulmonary complications in hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;28(5):425–34. [3] Freudenberger TD, Madtes DK, Curtis JR, et al. Association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants. Blood 2003;102(10):3822–8. [4] Yanik G, Cooke KR. The lung as a target organ of graft-versus-host disease. Semin Hematol 2006;43(1):42–52. [5] Bacigalupo A. Management of acute graft-versus-host disease. Br J Haematol 2007;137 (2):87–98. [6] Eapen M, Logan BR, Confer DL, et al. Peripheral blood grafts from unrelated donors are associated with increased acute and chronic graft-versus-host disease without improved survival. Biol Blood Marrow Transplant 2007;13(12):1461–8. [7] Afessa B, Peters SG. Noninfectious pneumonitis after blood and marrow transplant. Curr Opin Oncol 2008;20(2):227–33. [8] Yoshihara S, Yanik G, Cooke KR, et al. Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007;13(7):749–59. [9] Yanik G, Hellerstedt B, Custer J, et al. Etanercept (Enbrel) administration for idiopathic pneumonia syndrome after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2002;8(7):395–400. [10] Beschorner WE, Saral R, Hutchins GM, et al. Lymphocytic bronchitis associated with graft-versus-host disease in recipients of bone-marrow transplants. N Engl J Med 1978;299(19):1030–6.
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