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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2008 Apr;14(4):385–396. doi: 10.1016/j.bbmt.2008.01.004

A Role for TNF receptor type II in Leukocyte Infiltration into the Lung during Experimental Idiopathic Pneumonia Syndrome

Gerhard C Hildebrandt 1,2,*, Krystyna M Olkiewicz 1, Leigh Corrion 1, Shawn G Clouthier 1, Elizabeth Pierce 3, Chen Liu 4, Kenneth R Cooke 1,3,$
PMCID: PMC2390587  NIHMSID: NIHMS43827  PMID: 18342780

Abstract

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication following allogeneic stem cell transplantation (allo-SCT). Experimental models have revealed that TNFα contributes to pulmonary vascular endothelial cell (EC) apoptosis, and modulates the infiltration of donor leukocytes into the lung parenchyma. The inflammatory effects of TNFα are mediated by signaling through the type I (TNFRI) or type II (TNFRII) TNF receptors. We investigated the relative contribution of TNFRI and TNFRII to leukocyte infiltration into the lung following allo-SCT by using established murine models. Wild type (wt) B6 mice or B6 animals deficient in either TNFRI or TNFRII were lethally irradiated and received SCT from allogeneic (LP/J) or syngeneic (B6) donors. At week 6 following SCT, the severity of IPS was significantly reduced in TNFRII−/− recipients compared to wt controls, but no effect was observed in TNFRI −/− animals. Broncho-alveolar lavage fluid (BALF) levels of RANTES and pulmonary ICAM-1 expression in TNFRII−/− recipients were also reduced, and correlated with a reduction of CD8+ cells in the lung. Pulmonary inflammation was also decreased in TNFRII−/− mice using an isolated MHC class I disparate model (bm1 → B6), and in bm1 wt mice transplanted with B6 TNFα −/− donor cells. Collectively, these data demonstrate a role for TNFα signaling through TNFRII in leukocyte infiltration into the lung following allo-SCT, and suggest that disruption of the TNFα:TNFRII pathway may be an effective tool to prevent or treat IPS.

Keywords: Stem cell transplantation, TNFα, cytokines, graft-versus-host disease

INTRODUCTION

Pulmonary dysfunction is a major complication following allogeneic stem cell transplantation (allo-SCT) 1–4. Idiopathic pneumonia syndrome (IPS) has been defined as diffuse, non-infectious lung injury that occurs within four months of SCT 1. Although recent studies have reported a lower incidence (5–10%) and an earlier time to onset of IPS than originally described, the typical clinical course involving the rapid onset of pulmonary failure resulting in death has remained unchanged, emphasizing the critical nature of this complication 3,5,6. Risk factors for IPS include myeloablative versus non-myeloablative conditioning regimens, the use of total body irradiation (TBI), the development of acute graft-versus-host disease (GVHD), and older recipient age 2,3,6–9, but the pathophysiology of this disorder remains enigmatic. Data generated from experimental models suggest that the lung is vulnerable to a two-pronged immunologic attack after allo-SCT, involving both alloantigen-specific donor T cell responses 10–13 and the production of inflammatory cytokines 11,14–18. With respect to the latter, previous work has uncovered a causal relationship between TNFα and IPS 14,15,19; hyporesponsiveness of donor cells to lipopolysaccharide (LPS) stimulation 15 and the neutralization of TNFα 14 result in decreased lung injury after SCT. Moreover, we have recently shown that TNFα contributes to pulmonary vascular apoptosis and regulates pulmonary chemokine expression and the subsequent recruitment of inflammatory cells to the lung of allo-SCT recipients 20.

The actions of TNFα are mediated by two receptors: a 55- to 60-kDa type I receptor (TNFRI; p55/60; CD120a) and a 75- to 80-kDa type II receptor (TNFRII; p75/80; CD120b)21–24. These two receptors are co-expressed in almost every cell in the body, but in contrast to the constitutive expression of TNFRI, the expression of TNFRII is strongly modulated by various cytokines including TNFα, IL-1, IFNγ and other inflammatory stimuli like LPS 25–27. In addition, both receptors can be released into the blood following proteolysis where they function as molecules capable of binding circulating TNFα 28,29. TNFRI is generally regarded as the dominant receptor in TNFα biology; it is the high affinity receptor for soluble TNFα (sTNFα), and mediates many of the pro-inflammatory effects of this cytokine 30–34. TNFRI deficiency or TNFRI blockade by using p55-specific monoclonal antibodies results in resistance to endotoxic shock, but enhances susceptibility to infection with certain bacteria such as Listeria monocytogenes 33,35,36. A role for TNFRI has been described in eliciting fulminant alloreactive T cell responses 37,38 and in the inflammatory environment that characterizes acute GVHD early after allo-SCT 39,40. TNFRI also contributes to the development of acute lung injury during hemorrhagic shock 41.

The absence of TNFRII significantly reduces the dermatotoxic and lethal effects of TNFα 42, and monoclonal antibodies specific for either TNFRI or TNFRII block the development of TNFα-induced skin necrosis in mice 36. TNFRII expression is increased on microvascular endothelial cells (ECs) in the lung during adult respiratory distress syndrome (ARDS) and is associated with the enhanced expression of cell adhesion molecules including ICAM-1, VCAM-1 and of CD14 in this context 43,44. Animals deficient in both TNFRI and TNFRII are protected against the effects of TNFα as a central mediator in bleomycin-induced pulmonary fibrosis 45 and develop delayed pulmonary injury after the infusion of alloreactive T helper 1 (Th1) cells 19.

Given the previously identified role for TNFα in leukocyte infiltration into the lung during the development of IPS, we sought to determine the relative contributions of TNFRI and TNFRII to the inflammation engendered in this context by using well-established mouse SCT models 12,16,46. Alterations in histopathology, lung function, pulmonary vascular EC integrity, and the cellular and cytokine content of broncho-alveolar lavage fluid (BALF) observed in these models correlate very well with that seen in patients with IPS 1,12,20,46–50. Importantly, mechanistic insights provided by these experimental systems are actively being translated back to the clinic in the form of novel treatment strategies for this complication 5. Our results show that TNFα signaling through TNFRII plays a significant role in leukocyte infiltration to the lung during IPS. BALF levels of the inflammatory chemokine RANTES (CCL5) and the pulmonary expression of ICAM-1 were also reduced in TNFRII−/− recipients and correlated with decreases in CD8+ cells in the lung. Finally, the reduction in lung inflammation present in TNFRII−/− recipients is comparable to that observed when wild type (wt) mice are transplanted with allogeneic TNFα deficient donor cells, thereby strengthening the role of TNFα:TNFRII interactions in the pathophysiology of IPS.

MATERIALS AND METHODS

Mice and bone marrow transplantation

Female C57BL/6 (H-2b), LP/J (H-2b), B6.C-H2bm1/ByJ (H-2b), B6.129-Tnfrsf1atm1Mak/J (H-2b; TNFRI−/−, p55 deficient) 33, B6.129S2-Tnfrsf1btm1Mwm/J (H-2b; TNFRII−/−, p75 deficient) 42, B6129SF2/J (H-2b; TNFα+/+) and B6;129S6-Tnftm1Gkl (H-2b; TNFα−/−) 51 were purchased from the Jackson Laboratories (Jax; Bar Harbor, ME) or from the Frederick Cancer Research and Development Center (National Cancer Institute; Frederick, MD). Animals used for SCT and in vitro experiments were between 10 and 14 weeks old. All experiments were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA).

Mice were transplanted according to a standard protocol as described previously 50. When using the MHC matched, minor histocompatibility complex disparate LP/J → B6 system, B6 wt, B6 TNFRI−/− and B6 TNFRII−/− recipients received 13 Gy of TBI (137Cs source) prior to the infusion of 5 × 106 bone marrow (BM) cells and 2 × 106 splenic T cells from either syngeneic (B6) or allogeneic LP/J mice. T cell purification was performed by magnetic bead separation using CD4 and CD8 MicroBeads and the autoMACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s protocol with more than 85% of cells obtained being positive for CD4 or CD8 surface antigens (data not shown). Percentages of purified CD4+ and CD8+ T cells did not significantly differ between donors. Transplanted mice were cared for as previously described 52.

In experiments using the B6 → bm1 or bm1 → B6 SCT system (isolated MHC I-mismatch), host mice received 11 Gy TBI followed by the infusion of 5 × 106 BM and 1.8 – 2.0 × 106 autoMACS-purified (> 85% purity) CD8+ T cells. These changes in transplant parameters were made to insure that sufficient animals would be available for analysis at time points when significant lung injury is present.

Clinical GVHD, survival, and semi-quantitative evaluation of lung histopathology

Survival was monitored daily, and GVHD clinical scores were assessed weekly by a scoring system incorporating five clinical parameters: weight loss, posture (hunching), mobility, fur texture, and skin integrity as described 46. Pulmonary toxicity after SCT was determined by examination of lung histopathology in transplanted animals as previously described 46. Lung sections from individual mice were stained, coded without reference to mouse type or prior treatment regimen, and examined by C.L. to establish an index of injury. Lung tissue was evaluated for the presence of periluminal infiltrates (around airways and vessels) or parenchymal pneumonitis (involving the alveoli or interstitium) using a semi-quantitative scoring system that evaluates both the severity and extent of histopathology present 46.

Broncho-alveolar lavage (BAL)

At the time of analysis, mice were sacrificed by exsanguination and BAL was performed as previously described 46. After centrifugation, supernatants were frozen for subsequent analysis of cytokine and chemokine content, and cell pellets were washed and counted. In some experiments, aliquots of cell suspensions were stained with fluorescent antibodies to cell surface antigens and analyzed by FACS analysis.

Cell surface phenotype and intracellular cytokine analysis

To analyze cell surface phenotype, cells were stained with fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (MoAbs) to Ly9.1 and CD8 or with phycoerythrin (PE) conjugated MoAbs to CD4 and CD8 for flow cytometric analysis as previously described 15. Two color flow cytometric analysis of 1x104 cells was performed using a FACSCalibur (BD Biosciences, San Jose, CA).

Measurement of cytokine and chemokine protein levels by ELISA

Concentrations of specific cytokines and chemokines were measured in the BALF using ELISA kits for TNFα (BioSource #KMC3012, BioSource, Camarillo, CA), RANTES (Quanitkine M, R&D Systems, Minneapolis, MN), MIP-1α (Quanitkine M, R&D Systems, Minneapolis, MN) and for MCP-1 (BioSource #KMC1012, BioSource, Camarillo, CA). Assays were performed according to the manufacturer’s protocol. Assay sensitivity was < 3.0 pg/ml for TNFα, < 2.0pg/ml for RANTES, < 1.5pg/ml for MIP-1α, and < 9pg/ml for MCP-1.

Immuno-histochemical detection of ICAM-1

Immunohistochemical staining was performed on lung tissue five weeks after transplant with goat-anti-mouse CD54/ICAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or Goat-IgG isotype control (Sigma-Aldrich, St. Louis, MO) (both 1:200 dilution) and the VECTASTAIN Elite ABC peroxidase immunostaining kit (Vector Laboratories, Burlingame, CA) plus DAB substrate (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Slides were counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA) and analyzed by light microscopy (Olympus IX71 inverted research microscope; Olympus Corporation, Tokyo, Japan). Photomicrographs were acquired with the OlympusDP controller (Olympus Corporation, Tokyo, Japan) and processed with Adobe Photoshop (Adobe, San Jose, CA).

Real-time quantitative RT-PCR analysis

After perfusion of the pulmonary vascular system with ice-cold PBS, whole lung tissue was retrieved at various time points after BMT and stored at −80°C. mRNA was extracted from whole lungs using TRIzol following the manufacturer’s protocol (GibcoBRL, Grand Island, NY) and quantitated by spectrophotometry. ICAM-1 and GAPDH (an internal control) expression were analyzed by real-time quantitative RT-PCR procedure using an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). All primers and probes used were purchased from Applied Biosystems. ICAM-1 expression was normalized to GAPDH before the fold change in ICAM-1 expression was calculated by comparing values obtained from individual wild-type or TNFRII−/− allo-SCT recipients to the average value measured in syngeneic controls. To this end, ICAM-1 expression in syngeneic mice was given an arbitrary value of 1.

Statistical considerations

All values are expressed as the mean ± SEM. Statistical comparisons between groups were completed using the parametric independent sample t-test when appropriate or the Mann-Whitney test if there were less than five animals per group or if data did not meet the assumptions of normal distribution and equal variance required for a T test. The Wilcoxon rank-test was used for analyzing survival data.

RESULTS

The absence of TNFRI in SCT recipients results in an early reduction of clinical GVHD and improved survival

TNFα has been shown to be a central effector molecule in the development of acute GVHD and IPS 14,15,17,19,20,53–55. To ascertain whether the effects of TNFα in each of these processes are mediated predominantly through TNFRI or TNFRII, we transplanted lethally irradiated wt, TNFRI deficient (TNFRI −/−) or TNFRII deficient (TNFRII −/−) C57/BL6 recipient mice with bone marrow and splenic T cells from either syngeneic B6 or allogeneic LP/J donor mice as described in Material and Methods. SCT recipients were subsequently monitored for survival and the development of clinical GVHD. As shown in figure 1, severe clinical GVHD developed in B6 wt recipients over time in comparison to syngeneic controls (Fig. 1a) and resulted in significant mortality beginning one week after transplant (Fig. 1b). Whereas the severity of GVHD in TNFRII−/− mice receiving allo-SCT did not differ from that observed in allogeneic wt controls, TNFRI−/− recipients demonstrated a reduction in clinical GVHD scores and a significant improvement in overall survival. GVHD severity was specifically decreased during the first four weeks after SCT and correlated with an early reduction in mortality (Fig. 1a and b), confirming previous findings of Speiser et al. 39. In order to ascertain whether the survival advantage observed in TNFRI−/− recipients was in part related to incomplete engraftment of donor cells, T cell chimerism as assessed in surviving animals using antibodies specific for Ly9 allelic differences between donor and host. As shown in figure 1c, donor T cell engraftment after SCT was near complete and comparable in all allogeneic groups and therefore independent of TNF receptor expression in the SCT recipients. Similar findings were observed in splenic T cell populations (data not shown).

Figure 1. The absence of TNFRI in SCT recipients results in an early reduction of clinical GVHD and improved survival.

Figure 1

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BM and T cells from allogeneic MHC matched LP/J donor mice were transplanted into wt (◆, —), TNFRI−/− (▲,…..) or TNFRII−/− (●,---) B6 recipients as described in Materials and Methods. In all experiments, some B6 wt mice also received BM and T cells from syngeneic B6 donors (■, -•-•-•-). Clinical GVHD scores were determined weekly and survival was monitored daily. (A) TNFRI−/− (▲), but not TNFRII−/− (●), mice develop significantly reduced clinical GVHD scores seven to 28 days after allo-SCT when compared to wt controls (◆). Data presented are combined from three comparable experiments; n = 12 to 20 per group; * p < 0.05. (B) The reduction in clinical GVHD correlates with decreased early mortality of TNFRI−/− recipients (…..) after allo-SCT, but no difference in survival is seen between allogeneic wt (—) and TNFRII deficient (---) animals. Data presented are combined from four comparable experiments; n = 21 to 38 per group; * p < 0.05. (C) T cell chimerism was assessed 5 weeks after transplant by FACS analysis for surface expression of the donor marker Ly9.1. Donor T cell engraftment after SCT was independent of the p55 or the p75 TNF receptor expression in the recipient, and comparable in all allogeneic groups. Data presented are from one of similar two experiments; n = 3–4 animals per group.

The extent and severity of lung is significantly reduced in TNFRII−/− but not in TNFRI−/− allo-SCT recipients

Next, we determined the severity of lung injury in surviving mice five weeks after SCT. Consistent with previous work 56, B6 recipients of syngeneic SCT maintained normal histology, whereas, wt and TNFRI−/− recipients of allo-SCT developed significant and comparable lung histopathology (Fig. 2a). In each group, a dense mononuclear cell infiltrate had developed around both bronchial and vascular structures and was accompanied by a mixed leukocytic infiltrate in the interstitial and alveolar spaces. These findings were incorporated into a semi-quantitative scoring system that evaluates the severity and extent of injury present. Using this system, lung histopathology scores were significantly reduced in TNFRII−/− recipients (Fig. 2a and b). This reduction in pulmonary pathology was associated with decreases in total BALF cellularity (Fig. 2c) and BALF CD4+ and CD8+ T cells (Fig. 2d and e), suggesting that TNFRII, but not TNFRI, signaling plays an important role in leukocyte infiltration into the lung following allo-SCT.

Figure 2. Leukocyte infiltration into the lung is significantly reduced in TNFRII −/− but not TNFRI −/− allo-SCT recipients.

Figure 2

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Animals were transplanted as described in Figure 1 and lung injury was assessed six weeks after SCT. (A, B) TNFRII−/− (■), but not TNFRI−/− (■), recipients developed significantly less lung histopathology in comparison to allogeneic wt controls (■). Syngeneic controls maintained normal lung histology (□). (C – E) Decreased lung histopathology scores were associated with a reduction in BALF cellularity and BALF T cells. Data presented are combined from two comparable experiments; n = 10 to 15 per group; ** p < 0.01; * p < 0.05; + p = 0.06 (■) vs. (■).

Reduced histopathology in TNFRII−/− allo-SCT recipients is associated with significantly decreased BALF levels of RANTES

Recent data have shown a role for chemokines in the development of IPS after allo-SCT 57–60. In addition, our group recently demonstrated that pulmonary chemokine expression early after allo-SCT is regulated in part by TNFα 20. To determine whether the abrogation of TNFα signaling through TNFRII results in changes of chemokine expression in the lung, we measured BALF of Mip-1α, RANTES and MCP-1 five weeks after SCT as described in Material and Methods. As shown in Figure 3, BALF chemokine levels were elevated in each scenario after allo-SCT compared to syngeneic controls. While MIP-1α levels did not differ among allogeneic groups, a significant decrease in BALF concentrations of RANTES was observed in B6 TNFRII−/− recipients compared to allogeneic controls (Figure 3b), and correlated with reductions seen in lung histopathology and BALF cellularity (Figure 2a - c). Decreased in BALF MCP-1 levels were also noted TNFRII −/− recipients and trended toward statistical significance (Figure 3c).

Figure 3. Reduction of leukocyte infiltration into the lungs of TNFRII−/− allo-SCT recipients is associated with significantly decreased BALF levels of RANTES.

Figure 3

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Animals received syngeneic or allo-SCT as described in Figure 1, and BALF levels of Mip-1α, RANTES and MCP-1 and were determined 6 weeks later. (A-C) BALF levels of each chemokine were increased following allo-SCT compared to syngeneic controls (□). While no differences in BALF levels of Mip-1α were observed among allogeneic groups, BALF RANTES and MCP-1 levels were reduced in TNFRII−/− (■) but not TNFRI−/− (■) recipients compared to allogeneic controls (■). Data shown are from one experiment; n = 3 to 5 per group; *p < 0.05; +p = 0.07 (■) vs. (■).

Pulmonary expression of ICAM-1 is reduced in allo-SCT recipients deficient in TNFRII

Previous reports have indicated that pulmonary ICAM-1 expression significantly contributes to the development of IPS after allo-SCT 13,56, and ICAM-1 expression on ECs is dependent in part on the expression of TNFRII 43,44. Therefore, we next determined whether differences in ICAM-1 expression could be seen in the lungs of allogeneic wt, TNFRI−/− and TNFRII−/− recipients. As shown in Figure 4a–b, ICAM-1 was barely detectable in the lungs of syngeneic SCT recipients compared to isotype control sections. Pulmonary ICAM-1 expression was strongly increased in lungs from both wild-type and TNFRI−/− allo-SCT recipients in association with robust cellular infiltrates seen in each group (Fig. 4c–d). ICAM-1 staining could be observed both within the pulmonary interstitium (low power) and along the bronchial epithelial lining (high power). By contrast, immuno-histochemical staining was reduced in TNFRII−/− allo-SCT mice to a level comparable to syngeneic controls (Fig. 4e). In an effort to better quantify this apparent reduction, we measured ICAM-1 expression using real time RT-PCR as described in Materials and Methods. As shown in figure 5, similar reductions in whole lung ICAM-1 levels was noted in TNFRII−/− recipients at the mRNA level confirming findings obtained by immuno-histochemical staining. In sum, the absence of TNFRII was associated with a reduction of ICAM-1 expression in the lung following allo-SCT consistent with reports in other non-SCT systems.

Figure 4. Absence of TNFRII is associated with decreased pulmonary ICAM-1 expression following allo-SCT.

Figure 4

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Animals received syngeneic or allogeneic SCT as described in Figure 1 and the pulmonary expression of ICAM-1 was analyzed by immuno-histochemistry as described in Material and Methods. Sections shown are representative of staining observed in lungs from each group. Allogeneic wt and TNFRI−/− recipients (C and D) demonstrate increased signal intensity for ICAM-1 both in the interstitium and the bronchial epithelium when compared to syngeneic SCT controls (B). By contrast, the intensity of ICAM-1 expression in lungs of TNFRII−/− recipients is reduced (E). (900x and 400x magnification; ICAM-1 brown; counter-stain: hematoxylin).

Figure 5. Reduction in whole lung ICAM-1 mRNA expression confirms immuno-histochemical staining in TNFRII−/− recipients.

Figure 5

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Animals received syngeneic or allogeneic SCT as described in Figure 1 and the pulmonary expression of ICAM-1 was analyzed by RT-PCR as described in Material and Methods. Data are expressed as fold difference in expression compared to that observed in syngeneic controls. n = 4 to 6 animals per group. *p < 0.05.

Absence of either the ligand or the receptor in TNFα:TNFRII interactions results in a comparable reduction of lung injury after allo-SCT across an isolated MHC class I mismatch

TNFRII is only fully activated by membrane bound TNFα (memTNFα) 61, which can be used by cytotoxic T lymphocytes (CTL) to induce target cell lysis and apoptosis 62. Thus, we hypothesized that the role for TNFRII in IPS may be even more evident in a murine SCT system wherein donor and host differ at an isolated MHC class I mismatch and the development of acute lung injury is strictly dependent on the recruitment of alloreactive CD8+ cells to the lung. To test this hypothesis, lethally irradiated B6 wt or B6 TNFRII−/− recipient mice received SCT from bm1 donors and were analyzed for the development of lung injury five weeks after transplant. Syngeneic B6→B6 controls were included as negative controls. The severity of lung injury was significantly reduced in TNFRII−/− recipients compared to allo-SCT controls as measured by lung histopathology, BALF cellularity and BALF CD8+ T cells (Fig. 6a–c), and was associated with decreases in BALF levels of RANTES that did not reach statistical significance (Fig. 6d).

Figure 6. Absence of TNFRII is associated with reduced lung injury following allo-SCT in an isolated MHC class I mismatched system.

Figure 6

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Lethally irradiated B6 wt and B6 TNFRII−/− received SCT from allogeneic bm1 donors. Syngeneic B6→B6 controls were included (□) and lung injury was assessed five weeks after SCT as described in Material and Methods. (A) Allogeneic TNFRII−/− recipients (■) demonstrate a significant reduction in lung histopathology scores compared to allo-SCT controls (■). (B–D) Reduction in lung histopathology in TNFRII−/− recipients (■) was associated with decreased BALF cellularity, BALF CD8+ T cell counts, as well as, a reduction of RANTES levels in the BALF fluid. n = 4 to 7 per group; **p < 0.01; *p < 0.05; #p < 0.09.

In a complimentary set of experiments, we tested whether abrogation of TNFα in donor cells results in a reduction in lung injury similar to that observed in allo-SCT recipients deficient in TNFRII. We used the MHC class I mismatched stain combination B6 → bm1, in order to ensure direct comparability of results. In these experiments, lethally irradiated bm1 mice received SCT from either syngeneic (bm1) or allogeneic (B6 wt or B6 TNFα−/−) donors as described in Material and Methods, and lung injury was again assessed at week five after SCT. As shown in Figure 6a, bm1 mice receiving allo-SCT from B6 wt donors developed severe lung injury compared to syngeneic controls. By contrast, histopathology scores in bm1 animals receiving TNFα−/− donor cells were significantly reduced compared to allo-SCT controls (Fig. 7a) and were comparable to those measured in B6 TNFRII−/− animals receiving allo-SCT from bm1 donors (Fig. 6a). Consistent with previous results, observed decreases in lung pathology were associated with reductions in BALF cellularity, and CD8+ T cells (Fig. 7b–c). In addition, BALF TNFα concentrations were also greatly reduced to levels measured in syngeneic controls (Fig. 6e). Collectively, these data underscore the role of donor derived TNFα and, specifically, TNFRII:TNFα receptor:ligand interactions in the development of IPS.

Figure 7. TNFRII-mediated effects of donor-derived TNFα are important to the development of acute lung injury after allo-SCT.

Figure 7

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Lethally irradiated bm1 recipients were transplanted with BM and T cells from either syngeneic (bm1; □), allogeneic TNFα+/+ B6 (■) or allogeneic TNFα−/− B6 donor mice (■). Lung injury was assessed 35 days after transplant. (A-C) Allogeneic transplantation with TNFα deficient donor cells resulted in a nearly complete abrogation of lung histopathology associated with decreases in BALF cellularity and CD8+ T cells. (D) While TNFα levels in the BALF of allogeneic recipients of TNFα+/+ donor cells were significantly elevated, no differences were found between syngeneic controls and recipients after allo TNFα−/− SCT. All data are from one of two comparable experiments; n = 4 to 7 per group; *p < 0.05.

DISCUSSION

IPS is a frequently fatal complication after allo-SCT. The pathophysiology of IPS is complex, but data obtained from experimental models have helped to understand how the recruitment of cellular effectors and the production of inflammatory cytokines contribute to IPS 10–12,14,18–20,58–60. With respect to the latter, TNFα has been specifically identified as a critical mediator of IPS. TNFα levels are increased in the BALF during experimental and clinical IPS 18,63. In addition, the neutralization of TNFα significantly reduces the severity of lung injury after SCT in mice 14, and has been associated with improvements in pulmonary dysfunction in humans 5. TNFα production by donor, BM derived, accessory cells and mature T cells contributes to the development of IPS 20. However, TNFα produced by donor T cells plays a prominent role by regulating 1) pulmonary chemokine expression early after SCT and 2) the subsequent recruitment of donor effector cells to the lung 20. We now investigated whether the observed effects of TNFα on IPS development are mediated primarily through TNFRI and/or TNFRII.

We have previously examined the kinetics of lung injury using the LP → B6 system 56 and found that histopathology scores are relatively low at two weeks following allo-SCT and then increase sharply between weeks 4 and 6. In the current studies, the severity of systemic GVHD was comparable in each allogeneic group at the time of analysis. We speculated that animals would continue to succumb to GVHD over time thereby making inter-group comparisons at later time points more challenging and therefore elected to evaluate lung injury in all animals at week 5 after SCT. While it is possible that animals with more severe lung injury could have died prior to the time of analysis, this would be inconsistent with our previous data and otherwise would be expected in all allogeneic groups. Thus, while one could hypothesize that the reduction in lung injury observed between TNFRII−/− and TNFRI−/− mice could be the result of selection bias, the same argument cannot be made when comparing TNFRII−/− recipients to the “true” wild-type controls. In this context, our results show that interactions between TNFRII and TNFα contribute to the leukocyte infiltration to the lung following allo-SCT. In fact, the reduction in lung injury in TNFRII −/− allo-SCT recipients directly mirrors the outcome observed when TNFα −/− mice were used as SCT donors. These results are in accord with those recently reported using a fully MHC mismatched SCT model. Shukla and colleagues demonstrated that absence of TNFRI in allo-SCT recipients resulted in improved early post-BMT survival that was associated with decreased pulmonary edema and improved lung compliance on day 7 after SCT. However, cellular infiltration into the lung along with BALF levels of pro-inflammatory cytokines and chemokines were actually higher in BALF from TNFRI−/− SCT recipients compared with TNFRI+/+ controls 40.

Both sTNFα and memTNF are able to activate TNFRI, but TNFRII can only be fully activated by memTNF 62. TNFRII contributes to the cytocidal effects of TNFα by its own signaling, and also by regulating the access of this cytokine to TNFRI thereby enhancing TNFRI signaling 65–69. The absence of either of one of these TNFRII functions may contribute to the reduction in lung histopathology observed in TNFRII−/− recipients. However, the development of lung injury in TNFRI−/− recipients with a severity and extent that is comparable to the one seen in wt recipients, suggests that the “ligand-passing” function of TNFRII to TNFRI, or cooperative cell death signaling, does not play a significant role. Rather, effects of TNFα:TNFRII interactions that are independent of TNFRI appear to be operative.

Previous work has shown that lung injury is delayed in mice lacking both TNFRI and TNFRII following the transfer of cloned alloreactive Th1 cells 19. In those experiments, sTNFα is secreted by both Th1 cells and host macrophages, and leads to perivascular infiltration, interstitial pneumonitis and vascular injury 19. Cytotoxic CD8+ T cells are not involved in the pathophysiology of lung injury in that model, and no cytolytic potential was seen for the CD4+ T cells transfused 19. It is likely that TNFα-mediated effects in that model are propagated through TNFRI, and the delayed presence of pulmonary injury in the absence of TNFRI and TNFRII involves other inflammatory cytokines including IFNγ or IL-1. In contrast, the recruitment of donor CD8+ T cells plays a critical role in murine models of lung injury after allo-SCT including the ones used in this study 50,60. Both CD4+ and CD8+ T cells have been shown to contribute to systemic GVHD when LP mice are used as donors for irradiated B6 recipients 64, whereas GVHD develops almost exclusively in response to CD8+ activation in the BM1 → B6 model due to an isolated class I MHC mismatch that exists between these two mouse strains 60. CD8+ T cells predominantly express memTNFα 70 and may initiate injury via direct cytotoxic effects on alveolar epithelial cells, which are also uniquely sensitive to memTNFα-mediated injury 71.

Antigen recognition on alveolar epithelial cells by antigen-specific CD8+ T cells has been shown to result in epithelial cell activation, inflammatory chemokine induction and cytolysis through memTNFα 71. This CD8+ T cell-mediated lung injury occurs in the absence of perforin and Fas and is completely abrogated by neutralizing TNFα 72. TNFα neutralization is also associated with a reduction of both experimental and clinical lung injury after allo-SCT 5,14, suggesting therefore that similar mechanisms may be used by alloantigen-specific CTLs that contribute to pulmonary damage after allo-SCT.

We and others have shown that TNFRI:TNFα receptor:ligand interactions contribute to the toxicity incurred early after allo-SCT 39. TNFRI is known as the high affinity receptor for sTNFα and sTNFα production is directly related to inflammation engendered by pre-transplant conditioning and donor T cell activation and expansion early after allo-SCT. However, a role for TNFRII in leukocyte infiltration into inflamed tissues is supported by both experimental and clinical data. Up-regulation of TNFRII, but not TNFRI, has been observed in pulmonary microvascular ECs from patients with ARDS and is associated with increases in CD14, ICAM-1 and VCAM-1 expression in this context 43. Similar findings were reported in a murine model of cerebral malaria where ICAM-1 and TNFRII, but not TNFRI, expression are significantly increased in brain microvasculature 44. Mice lacking TNFRII−/−, but not TNFRI−/−, were resistant to mortality caused by this infection 44. Interestingly, ICAM-1 up-regulation was not detected in the brain of TNFRII−/− mice, indicating a close correlation between TNFRII, ICAM-1 and disease development 44. Our group recently confirmed and extended the findings by Panoskaltsis-Mortari and co-workers, and demonstrated a role for ICAM-1 expression on pulmonary vascular ECs in the development of IPS 13,61. In the current study, we show for the first time a potential association between TNFRII, ICAM-1 expression and pulmonary disease after allo-SCT. ICAM-1 expression was decreased in TNFRII−/− recipients after allo-SCT within the alveolar wall, which most likely reflects decreased ICAM-1 on alveolar epithelial cells and the alveolar capillary endothelium. Reductions in protein expression in TNFRII−/− allo-SCT recipients as assessed by immuno-histochemical staining are supported by similar reductions in whole lung mRNA levels (fig. 5). Therefore, the observed reduction in lung injury in TNFRII−/− recipients in this study may not only involve the lack of TNFRII mediated cell death, but also be due to a decreased expression of adhesion molecules like ICAM-1.

Finally, reduced lung histopathology in TNFRII−/− recipients shown in this study was also associated with decreased RANTES levels in the BALF. We have recently shown that donor T cell-derived RANTES contributes in part to leukocyte recruitment to the lung following SCT 59. However, chemokine production by non-hematopoietic cells in the lung may also be influenced by TNFα:TNFRII interactions, as we recently demonstrated a regulatory role of donor T cell TNFα in pulmonary chemokine expression during IPS 20. Therefore, it remains unresolved whether the reduction in BALF RANTES levels in TNFRII−/− allo-SCT recipients is secondary to a decreased number of infiltrating donor T cells, a reduction of RANTES secretion due to abrogation of TNFα:TNFRII signaling, or both.

Taken together, our data demonstrate a role for TNFRII in the inflammation engendered during the development of IPS, and suggest a link between TNFα:TNFRII signaling, ICAM expression and leukocyte infiltration to the lung that occurs in this context. Clinically, IPS develops during the administration of systemic immunosuppression and is poorly responsive to the addition of conventional anti-inflammatory therapy. Thus, a better understanding of the mechanisms involved in lung injury following allo-SCT is essential to improving outcomes of this frequently fatal complication. Our data further support the use of novel approaches targeting TNFα:TNFRII receptor:ligand interactions in addition to standard immunosuppressive therapy to treat or prevent IPS after allo-SCT.

Footnotes

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