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Published ahead of print on May 29, 2008, doi:10.1164/rccm.200711-1648OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 379-388, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200711-1648OC

Original Article

Interferon-{gamma} Regulates Idiopathic Pneumonia Syndrome, a Th17+CD4+ T-Cell–mediated Graft-versus-Host Disease

Nora Mauermann1, Julia Burian1, Christophe von Garnier1, Stefan Dirnhofer2, Davide Germano1, Christine Schuett3, Michael Tamm4, Roland Bingisser5, Urs Eriksson1,* and Lukas Hunziker1,*

1 Experimental Critical Care Medicine, Departments of Research and Internal Medicine, and 2 Department of Pathology, University Hospital, Basel, Switzerland; 3 Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany; and 4 Respiratory Medicine, and 5 Emergency Department, University Hospital, Basel, Switzerland

Correspondence and requests for reprints should be addressed to Lukas Hunziker, M.D., Department of Internal Medicine, University Hospital, Petersgraben 4, CH-4031 Basel, Switzerland. E-mail: lhunziker{at}uhbs.ch


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Pulmonary complications of hematopoietic stem cell transplantation include infections and graft-versus-host diseases, such as idiopathic pneumonia syndrome (IPS). Conflicting data exist regarding the role of the interferon (IFN)-{gamma}–producing Th1 CD4+ T-cell subset and IL-17A in IPS.

Objectives: To determine the role of IFN-{gamma} and IL-17A in the establishment of pulmonary graft-versus-host disease.

Methods: A semiallogeneic murine model based on C57BL/6 x BALB/c as recipients with transplantation of BALB/c RAG2–/– bone marrow and transfer of different genetic knockout T cells (T-bet–/–, IFN-{gamma}–/–, IFN-{gamma}R–/–) on a BALB/c background. Lung tissue was examined for parenchymal changes and infiltrating cells by histology and fluorescence-activated cell sorter analysis.

Measurements and Main Results: After transfer of semiallogeneic bone marrow together with donor CD4+ T cells lacking IFN-{gamma} or T-bet—a T-box transcription factor controlling Th1 commitment—we found severe inflammation in the lungs, but no enhancement in other organs. In contrast, wild-type donor CD4+ T cells mediated minimal inflammation only, and donor CD8+ T cells were not required for IPS development. Mechanistically, the absence of IFN-{gamma} or IFN-{gamma} signaling in pulmonary parenchymal cells promoted expansion of IL-17A–producing CD4+ T cells and local IL-17A release. In vivo depletion of IL-17A reduced disease severity.

Conclusions: One mechanism of IFN-{gamma} protection against IPS is negative regulation of the expansion of pathogenic IL-17A–producing CD4+ T cells through interaction with the IFN-{gamma} receptor on the pulmonary parenchymal cell population.

Key Words: idiopathic pneumonia syndrome • graft-versus-host disease • CD4 T cells • IL-17 • antigen-presenting cells



   AT A GLANCE COMMENTARY
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 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Idiopathic pneumonia syndrome is a manifestation of pulmonary graft-versus-host disease that is T-cell mediated, but the nature of the pathologic T-cell response remains obscure.

What This Study Adds to the Field
We provide evidence that idiopathic pneumonia syndrome is mediated by IL-17–producing T-helper cells and that IFN-{gamma} signaling is required to suppress this pathologic T-cell subset.

 
Allogeneic stem cell transplantation is used to treat hematologic malignancies, nonmalignant chronic hematologic diseases, cancer, and autoimmune diseases. Pulmonary complications, including infections and pulmonary graft-versus-host (GvH) disease, affect 30 to 50% of allogeneic stem cell recipients (1). Although obliterative bronchiolitis represents a late manifestation of GvH disease, idiopathic pneumonia syndrome (IPS) can be observed early and late after stem cell transplantation. It is well recognized that the absence of interferon (IFN)-{gamma} is associated with enhanced GvH disease (2) and that treatment with IFN-{gamma} can decrease the manifestation of GvH disease in general (3).

To address the mechanisms responsible for IPS development, several mouse models have been established, and it has been recognized that T cells are critical for disease development (4–6). So far, little is known about the T-cell subsets mediating the IPS phenotype. In particular, we do not understand the role of CD4+ T cells in general and, more specifically, the relevance of the different CD4+ T-cell subsets involved in disease development. However, alloantigen expression on host epithelial cells is not required for acute GvH disease in general, which underlines the important role of CD4 T cells in the disease process (7). Regarding the role of the Th1-specific cytokines, recent direct evidence points to a protective role of IFN-{gamma} (8), whereas the inflammatory cytokine tumor necrosis factor (TNF)-{alpha} aggravates pulmonary GvH disease (8–11). This picture is further clouded by the observation that, apart from "classical" Th1 and Th2 helper cells, another subset of CD4+ T cells characterized by IL-17A production might mediate organ-specific inflammation and recruitment of macrophages (12) or granulocytes (13). We therefore hypothesized that, in the absence of IFN-{gamma}, the IL-17A–producing T helper CD4 T-cell (TH-17) population may become predominant and induce organ-specific (lung) immunopathology.

We found markedly enhanced pulmonary inflammation after transfer of semiallogeneic bone marrow and donor CD4 T cells lacking IFN-{gamma} or T-bet, a T-box transcription factor controlling Th1 commitment. Importantly, CD8+ T cells played a minor role in IPS. IPS severity directly correlated with the expansion of IL-17A+CD4+ T cells in the absence of IFN-{gamma}, and in vivo depletion of IL-17A reduced IPS severity. Mechanistically, IFN-{gamma} signaling is critically required in the radioresistant pulmonary parenchymal cells of the host to maintain a negative feedback loop and thus prevent the expansion of pathogenic, semiallogeneic CD4+ T cells.


   METHODS
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 METHODS
RESULTS
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Mice
Mice were bred under specific pathogen–free conditions and transferred into sterile isolator cages after irradiation. Sex-matched, 7- to 10-week-old mice were used. All experiments were conducted in accordance with institutional and Swiss national regulations and were approved by the local authorities.

Induction of GvH Disease
For induction of GvH disease, mice were lethally irradiated (1,300 cGy, split into two doses) by a 60Co source. CB6F1 mice received intravenously 1 x 107 of either T-cell–depleted BALB/c bone marrow or recombination-activating gene 2–deficient (RAG2–/–) BALB/c bone marrow 24 hours after irradiation together with, depending on the experimental setting, either 5 x 106 wild-type or IFN-{gamma}–/– splenocytes or 2 x 106 magnetic cell sorted wild-type or IFN-{gamma}–/– donor T cells from BALB/c mice, corresponding to a semiallogeneic or haploidentical transplantation model. BALB/c control mice received the same transplanted cells. Mice were kept on acidified drinking water supplemented with trimethoprim/sulfamethoxazole (Bactrim [trimethoprim 32 mg/L, sulfamethoxazole 160 mg/L]; Roche Pharmaceuticals, Basel, Switzerland) for the duration of the experiments.

Magnetic Cell Sorting
Antibodies and isolation kits were purchased from Miltenyi Biotec GmbH (Cologne, Germany). Magnetically labeled CD90 (Thy 1.2) antibodies were used to deplete T cells from bone marrow. T-cell subpopulations were positively selected from whole splenocyte suspensions using CD90 (Thy 1.2), CD4 (L3T4), or CD8{alpha} (Ly-2) antibodies coupled to magnetic beads following the manufacturer's instructions.

In Vivo Treatment Protocols
Mice were intraperitoneally injected on Days 0, 3, 6, 9, and 12 with 100 µg anti-mouse IL-17 (rat anti-mouse monoclonal antibody, clone 50104; R&D Systems, Minneapolis, MN) (in direct ELISA, this antibody does not cross-react with recombinant murine IL-17E or IL-17F according to the manufacturer's information) or control rat IgG (kindly provided by B. Becher, University of Zurich) in 200 µl phosphate-buffered saline (PBS). For experiments blocking granulocyte recruitment, 100 µg of triggered receptor on myeloid cells (TREM)-1 peptide, a synthetic peptide mimicking a short, highly conserved domain of soluble TREM-1 (LQVTDSGLYRCVIYHPP, synthesized by Anawa, Wangen, Switzerland), was injected intraperitoneally every 12 hours in 200 µl 0.9% NaCl. To deplete granulocytes, mice were treated every 72 hours intraperitoneally by injection of 0.25 mg of a rat anti-mouse myeloid differentiation antigen (Gr-1) (RB6-8C5) antibody (kindly provided by Rolf M. Zinkernagel, Zurich, Switzerland). To neutralize TNF-{alpha}, mice were treated with 40 µg anti–TNF-{alpha} (Enbrel; Wyeth Pharmaceuticals AG, Zug, Switzerland) or vehicle (PBS) alone by intravenous injection on Days 4, 8, and 11.

IL-17A ELISA
An ELISA was performed following the manufacturer's instructions (DuoSet ELISA mouse IL-17, cat no. DY421; R&D Systems) (no cross-reactivity to recombinant IL-17B–F according to the manufacturer's information).

Semiquantitative Evaluation of IPS Severity
Paraffin-embedded lung sections (5 µm) were stained with hematoxylin–eosin. At least 10 sections from individual mice were coded without reference to mouse type or prior treatment regimen and independently examined by a pathologist to establish the following index of injury: grade 0, normal lung tissue; grade 1, small, 1- to 3-cell-diameter periluminal infiltrates (around airways and vessels), 5 to 25% of the lung tissue involved; grade 2, 4- to 10-cell-diameter-thick periluminal infiltrates with 25 to 50% of the lung tissue involved; grade 3, severe generalized (>50% lung tissue) inflammation, including giant cell formation. Images were acquired with an Axio Imager.A1 microscope, an AxioCam ICc3 digital camera, and Axiovision LE Rel.4.6 software (Carl Zeiss AG, Göttingen, Germany).


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T-bet–/– Donor Splenocytes Promote Severe IPS after Semiallogeneic Bone Marrow Transplantation
We first established a mouse model of semiallogeneic GvH disease. To this end, we transferred 5 x 106 wild-type BALB/c (H-2d) splenocytes together with 1 x 107 RAG2–/– (H-2d) bone marrow cells (B- and T-cell deficient) into BALB/c x C57BL/6 (CB6F1,H-2b,d) recipient mice or syngeneic BALB/c control animals. In all experiments, transplantation was performed across the same major histocompatibility (MHC) class I and II differences, where the donor T cells will mount a major allogeneic response against H-2DbKb and H-2IAb molecules. Similar semiallogeneic transplantation IPS models have been used by different research groups (8, 10). All CB6F1 mice, but not the BALB/c control animals, developed clinical signs of GvH disease with 20 to 25% weight loss and reduced physical activity within 4 to 6 days after transplantation. Later, however, CB6F1 mice regained weight and survived up to 3 to 4 months after transplantation. Histopathology of CB6F1 recipients revealed hepatic and intestinal GvH disease of intermediate severity. In contrast, the lungs were only minimally affected with a few mononuclear infiltrates, mainly around the blood vessels. No relevant inflammation was present in the lungs of the BALB/c control mice.

Next, we specifically addressed the role of Th1 T-cell commitment in the development of IPS. As illustrated in Figure 1A, cotransfer of BALB/c splenocytes lacking T-bet, the T-box transcription factor controlling Th1 development, and RAG2–/– bone marrow resulted in enhanced pulmonary GvH disease in CB6F1 recipient mice within 13 days. Histologic analysis revealed extensive infiltrates around the airways and blood vessels and parenchymal pneumonitis. Infiltrates consisted of mononuclear cells, granulocytes, eosinophils, and several giant cells (Figure 1A). This finding suggests that defective T-bet control of Th1 T-cell development negatively regulates pulmonary GvH disease. These findings, however, specify neither the role of CD4 and CD8 T cells nor the pathogenic role of IFN-{gamma}, because T-bet–/– CD8 T cells are highly competent in their capacity to produce IFN-{gamma} (14). We next repeated the experiment described above but applied IFN-{gamma} starting from Day 4 (5 µg intraperitoneally on Days 4–6 and 10 µg intraperitoneally on Days 7–13). Indeed, the IPS severity was reduced by treating the mice with IFN-{gamma} (Table 1).


Figure 1
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  		Figure 1. Lethal idiopathic pneumonia syndrome (IPS) induced by donor T-cell T-bet deficiency, donor T-cell IFN-{gamma} deficiency, or host IFN-{gamma} receptor deficiency. (A) Representative (n = 4 per group) hematoxylin-and-eosin–stained sections of paraffin-embedded lungs of recipient mice (R), reconstituted with bone marrow from BALB/c RAG2–/– mice and transplanted cells (Tc), and treatment (Tr) as given in the table above the images are shown. The symbols used in (B) are also indicated in the table. Images in the bottom row of (A) (scale, 100 µm) show a close-up of the corresponding image in the top row (scale, 500 µm). (B) The percentage of weight loss or gain of mice in representative groups is plotted as a function of time after reconstitution. Two experimental conditions ({circ}, {triangleup}) resulted in mortality (X) by Day 13. (C) Single-cell suspension of lungs isolated from CB6F1 mice (n = 4 per group), reconstituted with indicated cells, were stained for CD3+, CD8+, and CD4+. Mean values for number of cells and SD values in each quadrant are given. All experiments were performed twice.

 

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  		TABLE 1. DIFFERENT OUTCOMES OF IDIOPATHIC PNEUMONIA SYNDROME BY DESIGNATED RECONSTITUTION OF BONE MARROW, T-CELL, OR ANTIGEN-PRESENTING-CELL POPULATIONS AND/OR TREATMENT STRATEGIES

 
IFN-{gamma}–/– Donor Splenocytes or Defective IFN-{gamma} Signaling in the Semiallogeneic Host Enhances IPS Severity
To specifically address the role of IFN-{gamma} in the pathogenesis of IPS, we transferred 5 x 106 of either wild-type BALB/c or IFN-{gamma}–/– BALB/c splenocytes together with 1 x 107 RAG2–/– bone marrow cells into CB6F1 mice. Similar to the mice transferred with T-bet–/– donor splenocytes, mice transferred with IFN-{gamma}–/– donor splenocytes deteriorated rapidly, lost weight, developed respiratory failure, and had to be killed on Day 13 after transplantation (Figures 1A and 1B) Importantly, the same lethal IPS phenotype developed when CB6F1 mice lacking the IFN-{gamma}–/– receptor (IFN-{gamma}R) were reconstituted with 1 x 107 RAG2–/– bone marrow cells and splenocytes, regardless of IFN-{gamma} deficiency in the T-cell compartment (wild-type or IFN-{gamma}–/– splenocytes; Figures 1A and 1B).

Fluorescence-activated cell sorter (FACS) analysis of the digested lungs of CB6F1 mice transplanted with semiallogeneic RAG2–/– bone marrow revealed an overall increase in the number of CD3+ T cells after donor IFN-{gamma}–/–-splenocyte treatment compared with control animals treated with wild-type splenocytes. As illustrated in Figure 1C, relative numbers of CD3/4+ T cells exceeded numbers of CD3/8+ T cells in both the IFN-{gamma}–/– and wild-type splenocyte–treated semiallogeneic-transplanted animals 13 days after transfer.

Taken together, in the presence of IFN-{gamma}–/– donor splenocytes or in the absence of IFN-{gamma} receptor signaling in the transplanted host, semiallogeneic bone marrow transplantation results in the expansion of CD4 and CD8 T cells in the lungs and severe IPS. These findings suggest that IFN-{gamma} directly mediates the protective effect of the Th1 lineage commitment in IPS development.

Lethal IPS Is a CD4+ T-Cell–mediated Disease
To analyze the specific role of the CD4 and CD8 T-cell subsets in IPS development, we transferred 2 x 106 of either MACS-sorted CD4 or CD8 T cells into CB6F1 recipients. As shown in Figure 2A, IFN-{gamma}–/– CD4 T cells induced severe IPS (grade 2, 2, 3, 3, 3), whereas IFN-{gamma}–/– CD8 T cells induced only minimal disease (grade 0.5, 0.5, 0, 0, 0; Figure 2A). Cotransfer of IFN-{gamma}–/– CD4 T cells and IFN-{gamma}–/– CD8 T cells did not aggravate disease. Again, no relevant IPS could be detected after cotransfer of wild-type CD4 or CD8 T cells. Accordingly, in vivo bromodeoxyuridine incorporation was clearly enhanced in CD4+ T cells, but not in CD8+ T cells of IFN-{gamma}–/– splenocyte–treated mice 13 days after semiallogeneic bone marrow transplantation (Figure 2B).


Figure 2
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  		Figure 2. Idiopathic pneumonia syndrome (IPS) is CD4+ T-cell mediated. (A) Representative (n = 4 per group) hematoxylin-and-eosin–stained sections of lungs from CB6F1 mice, reconstituted with BALB/c RAG2–/– bone marrow, together with indicated sorted cells (2 x 106). The mice were killed 13 days after reconstitution. Images in the bottom row (scale, 100 µm) show a close-up of the corresponding image in the top row (scale, 500 µm). (B) CB6F1 mice transplanted with indicated cells were injected with bromodeoxyuridine (BrdU) every 12 hours for 2 days before being killed on Day 13. Single-cell suspension of lung tissue from each mouse was prepared. The cells were stained using indicated antibodies and analyzed by fluorescence-activated cell sorting. Mean values for number of cells and the SD value in each quadrant is shown. All experiments were performed twice. WT, wt = wild-type.

 
Kinetics of Inflammatory Cell Recruitment and Significance of Neutrophils in IPS
Next, we analyzed the pattern and time course of the recruitment of inflammatory cells in IPS development (15). Six days after CD4+ T-cell transfer, no significant differences were found (Figure E1 of the online supplement). Ten days after transfer, however, macrophages started to accumulate in the lungs of transplanted mice treated with IFN-{gamma}–/– donor CD4+ T cells (Figure E1). Although 13 days after transfer similar amount of myeloid dendritic cells (CD11+/MHC-II+) were observed in IFN-{gamma}–/– donor CD4+ T-cell–treated mice and IFN-{gamma}+/+ CD4+ T-cell–treated control animals, relevant infiltrations of macrophages were only evident in the IFN-{gamma}–/– donor CD4+ T-cell–treated mice (Figure 3). As illustrated in Figure E1, transfer of IFN-{gamma}–/– CD4 T cells resulted in the rapid repopulation of the CD11c-high, MHC class II–intermediate population (positive for CD11b, F4/80, CD105; additional staining not shown), whereas the MHC class II–high, CD11c-low population was almost absent (CD11b negative; additional staining not shown) compared with mice that received wild-type CD4+ T cells, probably representing the B-cell compartment (Figures 3 and Figure E1). In contrast to the FACS analysis of whole lung tissue, bronchoalveolar lavage fluid (BALF) analysis revealed a significant relative increase in neutrophils and concomitant decrease in macrophages when mice received IFN-{gamma}–/– CD4 T cells (Figure E2), whereas the absolute total BALF cell number was increased tenfold in these animals (data not shown).


Figure 3
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  		Figure 3. Lung antigen-presenting cell (APC) repopulation differs in the absence of donor IFN-{gamma} and if lung APCs are of donor origin. (A) Single-cell suspension of dissected lungs from CB6F1 mice (n = 4), reconstituted with BALB/c Rag2–/– bone marrow together with indicated cells were prepared 13 days post-reconstitution. The cells were stained as indicated and analyzed using fluorescence-activated cell sorting. MHC-II = major histocompatibility class II.

 
So far, our kinetic data suggest that the recruitment of macrophages reflects the critical, disease-promoting process in IPS development. Nevertheless, we cannot exclude a major role for granulocytes. To clarify this issue, we therefore depleted granulocytes in groups of semiallogeneic bone marrow transplanted and IFN-{gamma}–/– donor T-cell cotransferred mice with an anti–Gr-1 antibody according to the standard protocol (16). As shown in Table 1, no relevant changes in disease severity were observed in the depleted mice compared with control animals. Similar results were obtained after treatment with a TREM-1 peptide (Table 1) (17).

IFN-{gamma} Negatively Regulates the Expansion of Donor IL-17A+CD4+ T Cells in IPS
Having observed a clear increase in infiltrating granulocytes and macrophages after transfer of IFN-{gamma}–/– T cells, we analyzed the role of CD4+IL-17A+ T cells in the pathogenesis of IPS (18, 19). CD4+IL-17A+ T cells can mediate tissue inflammation in some autoimmune disease models (14, 20). Because in vitro findings suggest that IFN-{gamma} negatively regulates the expansion of CD4+IL-17A+ T cells (21), we compared the fate of IL-17A+ alloreactive CD4+ T cells in semiallogeneic bone marrow transplanted mice after treatment with IFN-{gamma}–/– versus IFN-{gamma}+/+ donor CD4+ T cells.

We first measured IL-17A production in supernatants of in vitro cocultures of CB6F1 dendritic cells and naive BALB/c IFN-{gamma}–/– CD4+ T cells versus IFN-{gamma}+/+ CD4+ T cells. We found that IL-17A was detectable in supernatants of CD4+ IFN-{gamma}–/– T cells only after 96 hours of culture (data not shown). Next, we analyzed the capacity of the lung-infiltrating CD4 T cells to produce IL-17A after overnight restimulation with CB6F1 dendritic cells obtained from animals treated with IFN-{gamma}–/– T cells or IFN-{gamma}+/+ T cells. Again, lung-infiltrating T cells from IFN-{gamma}–/– donor T-cell–treated mice produced significant amounts of IL-17A, whereas no or only minimal IL-17A was detectable in mononuclear pulmonary infiltrates of wild-type donor T-cell–treated control animals (Figure 4A). Similarly, lung-infiltrating T cells from wild-type donor T-cell–treated IFN-{gamma}R–/– mice produced significant amounts of IL-17A (data not shown). In accordance with these findings, FACS analysis of pulmonary infiltrates confirmed markedly enhanced numbers of IL-17A–producing CD4+ T cells in lung-infiltrating CD4 T cells 13 days after transfer of donor IFN-{gamma}–/– CD4 T cells compared with IFN-{gamma}+/+ donor CD4 T cells (Figure 4B).


Figure 4
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  		Figure 4. Up-regulation of IL-17A in severe idiopathic pneumonia syndrome. (A) Lung-infiltrating cells of CB6F1 hosts reconstituted with BALB/c Rag2–/– bone marrow together with indicated cells were isolated 13 days post-reconstitution. Isolated cells were restimulated for 48 hours using C57BL/6 bone marrow dendritic cells in vitro, and the amount of IL-17A secreted by the cells in culture supernatants was quantified by ELISA. Mean values (n = 3 per group) and SD is shown. *P < 0.02. (B) Lung-infiltrating cells, isolated as described in (A) from each group as indicated, were stained for CD4+ and intracellular IL-17A. Stained cells were analyzed by fluorescence-activated cell sorting and the average number of CD4+ T cells (left panels) is indicated. The percentage of IL-17A–expressing cells among the CD4+ T-cell population is shown in the corresponding histograms on the right. (C) Either anti–IL-17A monoclonal antibody (n = 4) or a control IgG (n = 3) was administered (100 µg/mouse) intraperitoneally to CB6F1 hosts reconstituted with BALB/c RAG2–/– bone marrow and BALB/c IFN-{gamma}–/– splenocytes on Days 0, 3, 6, 9, and 12 post-reconstitution. Mice were killed on Day 13 and representative hematoxylin-and-eosin–stained sections of lung tissue are shown. The lower panel shows a magnified image of the corresponding upper panel. Images in the bottom row (scale, 100 µm) show a close-up of the corresponding image in the top row (scale, 500 µm). One representative experiment that was performed twice is shown. APC = antigen-presenting cells; ctrl = control; PE = phycoerythrin; SSC = sideways scatter; WT = wild-type.

 
IL-17A Depletion Ameliorates IPS
IPS development is characterized by extensive infiltrates of granulocytes and macrophages in the injured lung tissue. As the above results suggest, granulocyte infiltration is not an absolute requirement for IPS development. Not only does IL-17A promote neutrophil (13) recruitment to inflamed tissues but it is also chemotactic for pulmonary macrophages (12). We therefore assessed whether IL-17A depletion reduces IPS severity.

To this end, we treated groups of mice transplanted with semiallogeneic bone marrow and treated with donor IFN-{gamma}–/– T cells with an IL-17A–depleting antibody. Control groups received an isotype control antibody. IL-17A depletion resulted in less weight loss of transplanted/donor IFN-{gamma}–/– T-cell–reconstituted mice compared with isotype-treated control animals. Furthermore, histology revealed reduced, albeit not absent, pulmonary inflammation in IL-17A–treated mice (Figure 4C).

Requirements for IPS Induction
Next, we addressed what is required to induce IPS and analyzed the origin of lung-infiltrating macrophages and dendritic cells. T-cell–depleted bone marrow of CD45.1 BALB/c mice was transferred together with CD45.1 BALB/c IFN-{gamma}–/– CD4 T cells into lethally irradiated CD45.2 CB6F1 mice. As depicted in Figure 5, almost all infiltrating cells, including macrophages and dendritic cells, were of donor origin, confirming that by Day 10, donor antigen-presenting cells (APCs) had rapidly replaced host APCs in the lungs after bone marrow transplantation. There was no need for continuous allogeneic stimulation in the lung by persisting host APCs to enhance IPS.


Figure 5
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  		Figure 5. Pulmonary dendritic cells and macrophages are donor derived. CB6F1 recipients (CD45.2+) were reconstituted with T-cell–depleted bone marrow from BALB/c CD45.1+ mice together with T cells from either BALB/c wild-type (CD45.2+) (upper panels) or BALB/c IFN-{gamma}–/– CD4+ (CD45.2+) (lower panels) mice. Single-cell suspensions of dissected lungs were prepared 10 days post-reconstitution and stained as indicated. Stained cells were analyzed using fluorescence-activated cell sorting and obtained dot plots (left) with gated regions and mean percentages of total cells in each region are shown. Major histocompatibility class (MHC)-II–high and CD11c-high (R1) and MHC-II–low and CD11c-high (R2) were further analyzed for CD45.1 and CD45.2 expression (middle and right panels, respectively).

 
To address the finding (22) that depletion of host Langerhans cells prevents skin GvH disease, we next analyzed whether prior depletion of host dendritic cells could delay or inhibit IPS development. We used CD11c–diptheria toxin receptor/green fluorescent protein (DTR/GFP) mice (23) on the CB6F1 background and treated them with diphtheria toxin (DT) 1 day before irradiation with 1,300 cGy. FACS analysis 5 days after this procedure showed complete absence of the CD11c population in the lung (not shown). However, DT-treated (CD11c depleted) and nontreated (non–CD11c depleted) CD11c–DTR/GFP on CB6F1 background mice showed no difference in IPS severity when treated with IFN-{gamma}–/– CD4+ T cells (grade 3, 3, 3, 3, 3 vs. 3, 3, 3, 3, 3).

To enhance the chance of complete donor chimerism or donor cell repopulation in the lung in recipient mice, which is a time-dependent process (15), CB6F1 mice were irradiated with 1,300 cGy followed by RAG2–/– bone marrow transplantation. After 30 days, we adoptively transferred 2.5 x 106 of either wild-type or IFN-{gamma}–/– CD4+ T cells. Thirteen days after T-cell transfer, no infiltrates or only minimally peribronchial infiltrates were observed in the group treated with IFN-{gamma}–/– CD4+ T cells (grade 0, 0.5, 0.5, 0.5, 0.5), whereas no immunopathology was seen in the group treated with wild-type CD4+ T cells (grade 0, 0, 0, 0, 0).

Host Parenchymal Cells Mediate the IFN-{gamma}–dependent Negative Feedback Loop
So far, it has not been clear whether the absence of IFN-{gamma}R signaling on lung parenchymal cells or persistent, radioresistant host APCs mediate the negative feedback loop that confines the expansion of pathogeneic CD4+ T cells. Next, we generated bone marrow chimeras by adoptive transfer of CB6F1 bone marrow into CB6F1–IFN-{gamma}R–/– recipients and CB6F1–IFN-{gamma}R–/– bone marrow into CB6F1 recipients. After 4 months, these chimeras were treated with RAG2–/– BALB/c bone marrow and wild-type CD4+ T cells. As illustrated in Figure 6, IPS developed in chimeric mice with the original CB6F1–IFN-{gamma}R–/– background only, independent of whether they were reconstituted with wild-type or IFN-{gamma}R–/– bone marrow. In contrast, only minimal disease was observed in wild-type hosts receiving either wild-type or IFN-{gamma}R–/– bone marrow. These experiments confirm the decisive role of the IFN-{gamma} receptor on lung parenchymal cells in the IFN-{gamma}–dependent negative feedback loop that protects against IPS.


Figure 6
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  		Figure 6. Long-term bone marrow chimera reveals importance of host lung parenchyma. CB6F1 or CB6F1 IFN-{gamma}R–/– mice were reconstituted with either BALB/c wild-type (WT) or BALB/c IFN-{gamma}R–/– bone marrow (n = 3 for each group). After 4 months, all the mice were irradiated a second time and reconstituted with BALB/c RAG2–/– bone marrow and BALB/c CD4+ T cells. Mice were killed 13 days after the second reconstitution. Representative hematoxylin-and-eosin–stained sections of the lungs are shown. Images in the right column (scale, 100 µm) show a close-up of the corresponding image in the left column (scale, 500 µm). One representative experiment that was performed twice is shown.

 
Because it is known that IL-17+CD4 T cells produce TNF-{alpha} (24), we neutralized TNF-{alpha} by intravenously administering 40 µg of a commercially available anti–TNF-{alpha} (etanercept) antibody 4, 7, and 10 days after transfer of either wild-type or IFN-{gamma}–/– T cells. Etanercept is well known to inhibit the biological activity of murine TNF-{alpha} (8, 9, 11). Etanercept and IFN-{gamma}–/– T-cell–treated mice had reduced numbers of neutrophils and higher macrophage counts in BALF (Figure E3A). The severity of pulmonary GvH disease was reduced and more localized but not completely absent in the etanercept-treated mice (Figure E3B).


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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a mouse model of semiallogeneic GvH disease, we found severe inflammatory lung injury after transfer of semiallogeneic bone marrow together with donor CD4+ T cells defective in IFN-{gamma} production. In contrast, wild-type donor CD4+ T cells mediated only minimal pulmonary inflammation, and donor CD8+ T cells played a minor role. Mechanistically, the observed disease phenotype strictly depended on the absence of either IFN-{gamma} production by CD4+ donor T cells or IFN-{gamma} signaling on pulmonary parenchymal cells, and was characterized by accumulation of IL-17A–producing CD4+ T cells in the lungs and local IL-17A release promoting recruitment of neutrophils (13) and macrophages (12). Neither IFN-{gamma}R–deficient donor T cells nor IFN-{gamma}R–deficient APCs were relevant for the observed lung infiltration. Of note, enhanced inflammation was restricted to the lungs.

Interestingly, CD8+ T cells were not critical for IPS development. Rather, our data suggest that pulmonary GvH disease represents a CD4+ T-cell–mediated disease (7). The question arises as to whether this observation can also be extended to the human system. If CD4+ T cells are also critical in human IPS, our data might be of considerable interest for the development of innovative therapeutic strategies.

The role of persisting host APCs in inducing GvH disease is still a matter of debate (22, 25). According to our results with the CD11c-depleted recipient mice (DTR/GFP–CD11c mice) and the experiment with the delayed T-cell transfer after bone marrow transplantation (Day 30), T-cell presence at the irradiation time seems to be a prerequisite for the induction of severe pulmonary GvH disease. One can argue that the irradiation-induced apoptosis results in initial massive host antigen presentation by donor APCs. Therefore, the donor CD4+ T cells would recognize foreign peptides in a cognate T-cell receptor–, peptide-, and MHC class II–dependent manner. The observed pulmonary phenotype in our model might well represent a minor allogeneic immune response. This would also correlate with the rather late appearance of the pulmonary immunopathology around Day 12. However, it is well recognized that donor lymphocyte infusion is associated with GvH disease and graft-versus-leukemia effects (26) in a time- and dose-dependent manner. As shown recently, once complete donor chimerism is established, donor lymphocyte infusion does not induce GvH disease at the cost of an absent GvL effect (27). Here we show that even the strong phenotype-associated IFN-{gamma}–deficient T cells could be abrogated by delayed transfer. This is probably also a consequence of complete donor chimerism being established 30 days after bone marrow transplantation (Figure 5).

Our findings are in line with several observations suggesting a T-cell–modulating role of the classical proinflammatory IFN-{gamma}. Mice lacking IFN-{gamma}, for example, show high mortality due to persistent T-cell activation after infection with noncytopathic viruses (28–30). In fact, it appears that IFN-{gamma} regulates the extent of CD4 and CD8 T-cell expansion and contraction upon antigen challenge (31–33). Similarly, exacerbated disease and persistence of activated, self-antigen–specific CD4+ T cells were found in IFN-{gamma}–deficient or IFN-{gamma}R–deficient mice in the context of several CD4+ T-cell–mediated autoimmune disease models (34–36). More specifically, recent in vitro findings suggest that Th1 T-cell responses and IFN-{gamma} negatively regulate the generation of a specific CD4+ T-cell subset characterized by IL-17A production (14, 21). In contrast to the conclusion of Murphy and colleagues (2), that the absence of IFN-{gamma} enhanced GvH disease and the absence of IL-4 decreased GvH disease, making the Th2 response the major contributor to GvH disease, newer studies using T-bet x IL-4 double-knockout mice indicate a Th1- or Th2-independent polarization (14, 20, 37). However, we have not formally ruled out in our study that IL-4 or Th2 polarization may have a role.

IL-17, first described by Rouvier and coworkers (38), and IL-17A–producing CD4 T cells are pathogenic for experimental allergic encephalitis (39, 40), collagen-induced arthritis (41), and experimental autoimmune myocarditis (14). In addition, IL-17A may play a direct role in allograft rejection (42, 43) and is a relevant factor for neutrophil and macrophage accumulation in the lung (12, 13). Our data provide in vivo evidence that IFN-{gamma}–/– CD4 T cells promote the expansion of IL-17A–producing donor CD4 T cells in GvH disease. Furthermore, we have shown that IL-17A+CD4 T cells accumulate in the lung. Because we did not observe enhanced GvH disease in other organs, we believe that the pathologic accumulation of IL-17A+CD4 T cells is lung specific.

Several mechanisms might explain the pathogenic capacity of IL-17A+CD4+ T cells in IPS. Our data suggest a direct pathogenic role for IL-17A because IL-17A depletion ameliorated IPS severity after semiallogeneic bone marrow transplantation and IFN-{gamma}–/– donor T-cell cotransfer. In accordance with our findings, transgenic overexpression of IL-17A in lung epithelial cells has been shown to lead to severe lung inflammation (37). IL-17A release in tissues results in the recruitment of granulocytes and macrophages (12, 13). In IPS, however, depletion of granulocytes by an anti–Gr-1 antibody or blocking TREM-1 function with a competitive peptide, which attenuates cytokine production and protects septic or LPS-treated mice from death (17), did not decrease the IPS severity in our experimental setup. We cannot absolutely rule out a contribution of neutrophils or neutrophil-derived compounds in IPS development, due to the limitations of depleting antibodies or insufficient TREM-1 blocking.

IL-17A–mediated effects are far more complex and might involve recruitment of many other inflammatory cells as well as alternative pathways of tissue infiltration. As studies in humans with lung transplantations suggest, IL-17 is increased in the BALF during lung rejection episodes (44), and it is discussed as an important factor in the pathophysiology of acute lung rejection (44). IL-17A depletion, however, did not completely abrogate IPS in our study, either because our antibody or our depletion protocol was not efficient enough or because IL-17A is only one of several important players in disease pathogenesis. In addition, other non–T-cell sources of IL-17 production that are less accessible to antibodies could be relevant in disease pathogenesis. However, to our knowledge, CD4 T cells are currently accepted as the major source for IL-17A (45).

Several factors might explain the increased expansion of CD4+ T cells in the absence of IFN-{gamma}. Possible mechanisms are the IFN-{gamma}–dependent induction of inducible nitric oxide synthetase (iNOS) (34, 46) and/or indolamine 2,3-dioxygenase (IDO) (47, 48) in APCs via lung parenchymal cells or in lung parenchymal cells themselves, resulting in release of NO or IDO metabolites, mediating growth arrest and/or apoptosis of activated T cells. Semiquantitative reverse transcriptase–polymerase chain reaction of RNA isolated from digested lung 13 days after transfer of either IFN-{gamma}–/– or wild-type CD4 T cells showed comparable iNOS mRNA levels in the two situations (data not shown). Treatment with the NOS inhibitor L-NAME (34) did not increase the IPS score in mice receiving wild-type CD4 T cells (data not shown). In contrast, we found no IDO mRNA expression after transfer of IFN-{gamma}–/– CD4 T cells, whereas IDO mRNA was present after transfer of wild-type CD4 T cells. In our own preliminary results using mass spectrometry, decreased levels of kynurenine with a decreased kynurenine/tryptophan ratio in the sera of IFN-{gamma}–/– T-cell–treated mice compared with wild-type T-cell–treated animals were observed (data not shown). Overall, we did not observe any relevant involvement of NO in our experimental setting, but given the preliminary results, we cannot exclude IDO as an important factor. However, a recently published study by Burman and colleagues largely excluded a relevant role of NO and IDO in the prevention of IPS by using knockout mice (8).

Similar to the results of Burman and coworkers, our findings confirm the essential role of IFN-{gamma} signaling on pulmonary parenchymal cells in protecting the lung from T-cell–mediated immunopathology (8). Which mechanisms are essential and which cell types (epithelial cells, endothelial cells, or fibrocytes) are the key players in this process remain unresolved. In addition, we can provide evidence that IL-17A–producing T cells play an important role in IPS. Blocking IL-17A, possibly in combination with TNF-{alpha} antagonists, might be a valuable option for treating IPS.


   FOOTNOTES
 
The Swiss Life Jubiläumsstiftung supports this project (L.H.). U.E. holds a Swiss National Foundation professorship.

* These authors shared last authorship. Back

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

Originally Published in Press as DOI: 10.1164/rccm.200711-1648OC on May 29, 2008

Conflict of Interest Statement: N.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.B. received a research grant from AstraZeneca (20,000 CHF) in 2005. C.v.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. U.E. received a research grant from AstraZeneca (20,000 CHF) in 2006. L.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 7, 2007; accepted in final form May 29, 2008


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 METHODS
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 DISCUSSION
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