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Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17+ cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1β, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wk and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-β, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17+ cells. At 30 days, SM inhalation promoted the accumulation of IL-17+ cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-β, fibrosis, and IL-17+ cells in the lung. IL-17+ cells likely play an important role in the pathogenesis of SM-induced lung injury.
Sulfur mustard (SM; 2-bis-chloroethyl-sulfide) is a highly toxic vesicant that has been used in war settings, causing injuries and deaths to military and civilian personnel [1, 2]. Because of the ease of production and stability, SM represents a potential terrorist threat . SM exposure occurs primarily through inhalation and absorption through skin and the anterior surface of the eye, making the lungs, the skin, and the eye as major targets of SM toxicity . The severity of damage depends largely on the dose and duration of exposure. Acute effects may include skin blisters, eye irritation, and breathing discomfort (chest tightness, hacking cough, rhinorrhea); however, at sublethal doses these effects are relatively temporary. During the Iran-Iraq war, immediate mortality among the Iranian soldiers exposed to SM was only 3–4%, but decades later nearly half of the victims developed chronic respiratory complications (chronic bronchitis, airway hyperreactivity, lung fibrosis, and bronchopneumonia)–the primary cause of morbidity and mortality among these soldiers [5–9].
The mechanism(s) of SM-induced respiratory toxicity are not clear. Altered immune/inflammatory responses such as decreased natural killer cell numbers , increased cytokine production , and possible changes in cytotoxic T cells [12, 13] have been reported in Iranian SM victims. Increased expression of proinflammatory cytokines has been observed in the rat skin  and lungs , and human lung cell cultures [16, 17] after SM exposure. A single lung exposure to the nitrogen mustard analogue melphalan in mice induced an acute inflammatory response with increased IL-1 and IL-6 in the bronchoalveolar lavage (BAL) as well a chronic respiratory impairment characterized by lymphocytic infiltration and lung fibrosis . In a recent study exposure of mice to nitrogen mustard induced T cell-dependent long-term lung pathology .
The role of T cells in SM-induced lung injury is not well delineated. We have demonstrated that hairless euthymic guinea pigs dermally exposed to SM developed a SM-specific, delayed-type hypersensitivity response, suggesting that SM can activate specific T cell-mediated immune responses . Th17, a subset of CD4+ T cells, has recently been implicated in a number of inflammatory, autoimmune, and chronic fibrotic lung diseases , and a strong relationship between the profibrotic cytokines and Th17 has been established . In this communication, we show that SM exposure causes acute and delayed responses. The delayed lung responses include lung fibrosis and the presence of IL-17+ cells in the inflamed regions of the lung, suggesting that IL-17+ cells may have an important role in the development of chronic fibrosis in SM-exposed lungs.
Except where noted, all the chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Female F344 rats (Charles River Labs, Wilmington, MA, 11–13 wk, 170–190 g) and cynomolgus monkeys (Lovelace Respiratory Research Institute monkey colony) were quarantined for a minimum of 2 wk prior to use. All animal studies were approved by the Institutional Animal Care and Use Committee, conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and carried out in compliance with the Guide for the Care and Use of Laboratory Animals. Animals were provided water and food (Harlan-Teklad, Madison, WI) ad-libitum.
Sulfur mustard, which was synthesized by proprietary methods and analyzed by gas chromatography-mass spectrometry, gas chromatography-flame ionization detector, and nuclear magnetic resonance, was determined to be greater than 99% pure . Rats were exposed by tracheal intubation as described . Briefly, rats were anesthetized with isoflurane (5% induction, 2% maintenance). Anesthetized rodents were intubated with a 14-gauge Teflon catheter (approximately 5 cm in length). The catheter was inserted into the trachea to a terminal location approximately half way between the bifurcation of the trachea and the larynx. The placement and seal of the catheter were verified by observing air displacement in a groundglass, gas-tight syringe during an inhalation/exhalation cycle. Intubated rodents were transferred to the exposure plenum. An airflow consisting of SM + vehicle or vehicle alone constantly ran through the exposure plenum. Rats were exposed to a single dose of 150 mg/m3 SM for 10 min and allowed to recover for observations.
Cynomolgus monkeys were restrained by the non-human primate cage-squeeze mechanism; prior to exposures, monkeys were administered ketamine as a pre-anesthetic to isoflurane and transported to the SM exposure suite. Monkeys were anesthetized with isoflurane in oxygen (5% induction) and maintained (~2.5% maintenance) during the exposure period. The monkeys were intubated and placed prone on an exposure sled; isoflurane was constantly administered to maintain anesthesia. The setup for exposure of monkeys was similar to the rodent exposure system as described . Briefly, air, O2, isoflurane, and SM were delivered to the monkeys in a flow-pass manner, where the animal inhales from an exposure atmosphere “stream” maintained at a constant flow (~ 2 L/min) and concentration of SM (150 mg/m3). Oxygen was added to the system to maintain a concentration of 19% to 22% and monitored just downstream of the breathing zone. Isoflurane was kept at a maintenance level of 1 to 3%. Pulse oximetry was utilized to assist the monitoring of vital signs. Monitoring was continued after the exposures until the animals recovered from the anesthesia. The awakened monkeys were returned to their cages, which were placed in a temporary off-gassing chamber. The animals were observed continuously until the concentration of SM returned to safe levels at which time the NHPs were removed from the temporary off-gassing chamber and returned to their home rooms.
Rats were deeply anesthetized and exsanguinated by clipping the vena cava. A 16-gauge needle was introduced into the trachea and secured using 3/0 suture. The thoracic cavity was then opened to expose the trachea and lung. The left lung was lavaged three times with 3 ml aliquots of sterile saline. The lavage fluid was pooled, centrifuged at 300×g for 10 min and the supernatant collected and stored at −80°C until analyses. The left lobe along with the right cranial, medial, caudal, and accessory lobes of the lung were removed, snap frozen in liquid nitrogen, and stored at −80°C. A similar procedure was used to collect BAL and lung tissues from the monkeys.
Total RNA was isolated from lung tissues using TRI-Reagent (Molecular Research Center, Cincinnati, OH) as described . Briefly, lung tissues were homogenized in 1 ml Tri-Reagent containing 100 μl BCP (Molecular Research Center), and the homogenates were centrifuged at 13,000× g for 10 min at 4°C. The aqueous layer was collected and mixed with 600 μl of isopropanol. After 15 min at room temperature, samples were centrifuged (13,000× g; 10 min) and the pellets resuspended in 75% ethanol, centrifuged, and air dried. The air-dried samples were resuspended in diethyl-pyrocarbonate-treated water (55°C for 10 min to dissolve RNA) and quantitated spectrophotometrically. The real-time polymerase chain reaction (qPCR) analysis was performed on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix (Applied Biosystems, Foster City, CA). The relative expression of each mRNA was calculated by the method described earlier . mRNAs were normalized with 18S rRNA or GAPDH; both gave similar results. All primer/probe sets for cytokines/chemokines and GAPDH were purchased from Applied Biosystems.
For immunohistochemistry (IHC), lung sections were made as described . Briefly, the lung was inflated for 2 h at a constant hydrostatic pressure of 25 cm with 10% neutral buffered formalin and immersed in the same solution for 48 h. The fixed lung was trimmed, embedded in paraffin, and cut into 5 μm sections. The endogenous peroxidase was quenched by incubating the slides containing tissue sections in 2% hydrogen peroxide diluted in methanol for 1 min. The slides were washed with deionized water followed by washes with Dulbecco’s polybuffered saline (pH 7.4) containing 0.05% Brij. Proteins were unmasked by incubating the tissue sections with trypsin solution (Zymed Laboratory, San Francisco, CA) at 37°C for 10 min. After blocking nonspecific binding by 1% horse serum containing 2% BSA and 0.1% Triton X-100, the slides were incubated overnight at 4°C in a humidified chamber with primary antibody diluted in blocking buffer, followed by incubation with biotinylated secondary antibody (VECTASTAIN® Elite ABC kit, Vector Laboratories, Burlingame, CA). Binding was visualized using an avidin-biotinylated enzyme complex (VECTASTAIN® Elite ABC kit) with 3, 3′-diaminobenzidine (DAB) as substrate.
Terminal deoxynucleotidyl transferase (TUNEL) staining and making the positive and negative controls were performed on paraffin-embedded lung sections by the TACS 2 TdT-DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) as per manufacturer’s instructions.
All statistical calculations were performed using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA). Results are expressed as mean values ± standard deviations. Statistical comparisons were determined by the unpaired Student’s t-test. The differences with P values of ≤ 0.05 were regarded as significant.
To investigate whether inhalation of SM induces an acute proinflammatory response in the lung, we determined the expression of pro-inflammatory cytokines and chemokines in the lung tissues and BAL fluid of control and SM-exposed rats by qPCR and ELISA. At 3 days after SM exposure, qPCR analysis indicated that compared to vehicle control, SM induced significant increases in the lung expression of proinflammatory cytokines (IL-1β, TNF-α, IL-2, and IL-6) (Fig. 1a) and proinflammatory chemokines (Fig. 1b), including CCL2 (MCP-1), CCL3 (MIP-1α), CCL11 (eotaxin), and CXCL1 (Gro-α). ELISA determinations of BALF indicated that SM exposure caused a significant rise in the immunoreactive protein content of TNF-α (19.2 ± 4.4 PG/ML) compared to control (3.1 ± 2.2 PG/ML) and IL-1β (11.5 + 2.4 PG/ML) compared to control (4.3 ± 2.0 PG/ML). Interestingly, the expression (qPCR and ELISA analysis) of the profibrotic cytokines (e.g., TGF-β, IL-13) was not significantly different between the control and SM-exposed lung at this time point (not shown). These results suggest that SM inhalation exposure leads to early upregulated expression of proinflammatory cytokines and chemokines but not profibrotic cytokines in the rat lung. Histopathology of the lung sections from SM-exposed rats at 3 days post exposure also indicated a significant increase in neutrophilic infiltration in the lung (not shown). Similarly, histopathology of the lungs from monkeys at 2 wk post SM exposure showed significant increases in neutrophilic infiltration and proliferative changes in mucosal epithelium (Fig. 2). Thus, the early lung inflammatory response is the predominant feature of SM exposure.
Early apoptotic cell death has been suggested as the critical event in the initiation of a number of fibrotic diseases, including lung fibrotic diseases . To ascertain whether SM-induced lung injury involved apoptotic cell death, we performed a TUNEL assay on lung sections from control and SM-exposed rats at 3 days post exposure. Results showed that SM induced a significant increase in TUNEL-positive cells in the lung (Fig. 3). Thus, as with other fibrotic lung diseases, prior to a significant fibrotic response, SM exposure triggers an early apoptotic cell death.
Lung fibrosis is the major cause of morbidity and mortality in SM-exposed Iranian victims . To determine whether SM exposure leads to lung fibrosis, we determined whether SM inhalation causes lung fibrosis in rats at various times following SM exposure. Pro-fibrotic changes as judged by histopathology were minimal up to 14 days post SM exposure (not shown); however, significant thickening of the submucosa with fibroblastic tissue was seen at day 21 (see later, Fig. 6b) and increased progressively thereafter. At 28 days after SM exposure, IHC staining suggested that the lungs from rats exposed to SM had increased expression of the fibrosis marker CTGF , the myofibroblast marker α-smooth muscle actin (α-SMA), and infiltration of CD8+ cells (Fig. 4). At 39 days post exposure, hematoxylin and eosin (H&E staining) (Fig. 5a–c), trichrome staining (Fig. 5d), and IHC staining for α-SMA (Fig. 5e) of adjacent lung sections suggested that SM promotes chronic lung inflammation, collagen deposition, and myofibroblast proliferation, respectively. These results indicate that SM inhalation induces a delayed profibrotic response in the lung. In addition, SM induces emigration of CD8+ cells in the lung; the role of these cells in lung fibrosis is not clear.
TGF-β is the major profibrotic cytokine that is produced primarily by T cells and macrophages . To determine the role of TGF-β in SM-induced chronic lung injury and development of pulmonary fibrosis, control and SM-exposed rat lungs were stained for TGF-β+ cells by IHC at 3 days and 28 days post SM exposure. While at 3 days, staining of TGF-β was not significant (not shown), whereas at 28 d post SM exposure, lungs showed a strong presence of TGF-β+ cells (Fig. 6). These results suggest that SM inhalation causes delayed expression of TGF-β in the lung.
SM-induced lung fibrosis is progressive and appears late after SM inhalation, suggesting an “autoimmune-like” etiology. Many autoimmune and fibrotic lung diseases are associated with the presence of Th17 cells. To determine whether SM promotes the accumulation of IL-17+ cells in the lung, lung sections from vehicle control and SM-exposed rats (28 days post SM/air inhalation); and vehicle control and SM-exposed monkeys (60 days post SM/air inhalation) were evaluated for the presence of IL-17+ cells by IHC staining. As shown in Fig. 7, SM inhalation exposure induced the migration of IL-17+ cells into the rat lung (Fig. 7a and 7b) and monkeys (Fig. 7c and 7d) at 28 days and 60 days, respectively. Interestingly, in monkeys, IL17+ cells were not seen in significant numbers prior to 30 days post SM exposure, where only a few IL-17+ cells were seen in the lung (not shown); however, a strong increase in the presence of these cells was observed at day 60 post SM exposure. Moreover, the infiltration of IL-17+ cells was observed only in the areas of lung inflammation/fibrosis. These results suggest that IL-17+ may be intimately associated with SM-induced chronic lung inflammation and fibrosis.
The surviving SM victims of the Iran-Iraq war continue to suffer from chronic respiratory complication of SM exposure . In humans, respiratory exposure to mustard agents (sulfur and nitrogen mustards) produces acute lung injury followed by chronic progressive lung fibrosis [6, 7, 26, 29, 30]. Moreover, the SM-exposed patients show persistent signs of immune dysfunction and accumulation of CD8+ cells in the lung [10–13]. Thus, sublethal exposures to mustard agents may induce two distinct but partially overlapping phases of lung injury: the acute phase involving production of proinflammatory factors and the chronic phase characterized by immunological alterations leading to progressive lung fibrosis.
To better define the early and late events in SM-induced lung injury in this study, we exposed rats and monkeys to sublethal doses of SM. In the rat model, within 3 days after SM exposure, mRNA expression of the prototypic inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α, IL-17) and chemokines (CCL2, CCL3, CCL11 and CXCL1) was significantly higher in the lung. This was accompanied with higher protein content of proinflammatory cytokines (IL-1β and TNF-α) in the BALF from SM-exposed animals. Histopathology of lung tissues indicated increased numbers of neutrophils, eosinophils, and lymphocytes in the lungs of SM-exposed rats. It is conceivable that increased leukocytic infiltration resulted from the increased expression of proinflammatory cytokines and chemokines. Cytokine chemoattractants such as CCL2, CCL3, and CXCL1 are pivotal in the development of inflammatory responses. CCL2 (MCP-1) is a small secreted protein that has chemoattractant activity for not only monocytes, but also memory T cells, natural killer cells, and perhaps dendritic cells resulting in their recruitment to the sites of tissue injury and inflammation [31–33]. CCL2 is also implicated in chronic inflammatory/autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, glomerulonephritis  and pulmonary fibrosis . CCL3 (MIP-1α) is produced by a variety of cells including monocyte/macrophages, mast cells, epithelial cells, and lymphocytes. It has been shown as a neutrophil chemoattractant in allergen and infection associated lung inflammations [36, 37]. Interestingly, CCL3-mediated neutrophilic immigration in the lung may be mediated by IL-1β and TNF-α [36, 38]–both the proinflammatory cytokines were upregulated in the SM lung. CXCL1 (Gro-α) acts like IL-8 and is a strong chemoattractant for neutrophils in experimental models of human inflammatory diseases [39, 40]. Eotaxin is an established chemoattractant for eosinophils in the lung , and it is likely that increased levels of eotaxin contributes to the eosinophilic infiltration seen in SM-exposed lungs. In addition to these cytokines/chemokines, SM caused moderate increases (2–3 fold) in the expression of IL-4 and IL-5 (not shown); interestingly; however, IL-13 expression in the SM lung did not increase significantly. While IL-5 promotes eosinophilic responses, IL4 and IL-13 are fibrogenic in several fibrotic conditions . Moreover, in rats at 3 days post SM exposure, the expression of TGF-β or IL-13 had not increased significantly. These results suggest that early response in the lung is mostly inflammatory and not profibrotic.
Apoptosis is a tightly regulated mechanism for cell death that eliminates unwanted, damaged, and infected cells. Apoptosis is believed to have contributed to the lung injury in Iranian victims of SM exposure . Increased LDH activity (cell death) was observed in guinea pig lungs  and tracheal epithelial cells of pigs exposed to SM inhalation . If the cell injury/death involves an apoptotic process, the nuclear condensation of apoptotic cells can be visualized by TUNEL staining . Our results indicated significant damage to the epithelial cells and the presence of TUNEL-positive cells in SM-exposed but not control lungs 3 days after SM inhalation. Malaviya et al.  also reported an early activation (6 h after SM inhalation) of an apoptotic response (TUNEL-positive cells and caspase 3/9 activation) in the lungs. Increasing evidence suggests that xenobiotic-induced apoptotic cell death in the lung is associated with lung fibrosis, and blocking apoptosis ameliorates fibrotic response in in vitro and in vivo models of lung injury [47–49]. On the other hand, fibroblasts obtained from established lung fibrotic disease may become resistant to apoptosis and contribute to the progressive fibrotic process [50–52]. Together these results suggest that SM inhalation induces a potent inflammatory response in the lungs that is associated with apoptotic cell death, which may initiate the fibrotic response. Early interventions might be useful in inhibiting the extensive fibrotic response seen at later stages of SM exposure.
The major cause of morbidity and mortality among human SM victims is lung fibrosis; however, the mechanism of lung fibrosis is not clearly understood. It is generally accepted that neutrophils and eosinophils are the abundant cell type during the early stages of lung injury. The cytokines and chemokines released at the site of injury stimulate angiogenesis and activate T cells that are the major producers of the profibrotic cytokines IL-4/IL-13 and TGF-β [28, 42]. These cytokines, particularly TGF-β, activate macrophages and fibroblasts, and the activated fibroblasts may transform into α-SMA-expressing myofibroblasts . Apart from TGF-β, IL-13 and IL-4 have been identified as key fibrogenic cytokines in many fibrotic conditions [42, 54, 55]. However, our results show that a single sublethal dose of SM did not induce significant expression of IL-13 at any time after SM exposure. There was only a moderate transitory increase in IL-4, which, in addition to being an archetypal Th2 cytokine, promotes TGF-β production from eosinophils . Moreover, increased TGF-β expression was detected only at later times (2 wk after SM inhalation), and the increased fibrotic response as seen by trichrome staining and augmented expression of CTGF and α-SMA was detected several weeks after SM exposure. Coincident with lung fibrosis, was the accumulation of CD8+ and IL-17+ cells in the lung. The presence of CD8+ cells has been reported in other fibrotic lung diseases, including COPD [57–59], idiopathic pulmonary fibrosis [60–62], and bronchiolitis obliterans . Interestingly, increased numbers of CD8+ cells are present in the BAL of SM-exposed patients who show signs of bronchiolitis obliterans [13, 64]. We also observed the presence of CD8+ cells in skin sections of dermally SM-exposed guinea pigs . Thus, CD8+ cells seem to be associated with a number of fibrotic diseases including SM-exposed humans and animals; however, whether CD8+ cells perpetuate the injury as cytotoxic cells or play a direct role in lung fibrosis is not clear.
Based on cytokine/transcription factor expression, CD4+ T cells have been divided into three subpopulations: Th1 (IFN-γ+/T-bet+), Th2 (IL4+/STAT3+), and recently added Th17 (IL-17+/ROR-γt) . IL-17+ cells have been implicated in a number of autoimmune and experimental fibrotic lung diseases, including cystic fibrosis , sarcoidosis , systemic sclerosis-related pulmonary fibrosis , and bleomycin-induced lung fibrosis . IL-1β, IL-6, and TGF-β are critically important for differentiation of Th17 cells, production of IL-17, and lung fibrosis [69, 70]. Our results indicate that SM significantly increases the expression of IL-17, IL-6, and TGF-β; however, the expression of TGF-β was delayed and preceded the development of frank lung fibrosis. Because, IL-17 is also produced by neutrophils, it is likely that early expression of IL-17 was associated with the infiltration of neutrophils in the lungs of SM-exposed animals. Most of these studies were carried out in rats; however, in a limited number of monkeys, SM caused delayed lung fibrosis that was associated with the presence of IL-17+ cells in the lung. Moreover, in monkeys, SM caused a patchy inflammatory response, and IL-17+ cells were abundantly present only in inflamed areas of the lung. Thus, IL-17+ cells are clearly associated with lung inflammation. We also have preliminary evidence suggesting that SM can induce similar responses in C57BL/6 mice, including the increased presence of CD4+ IL-17+ T cells and cellular and molecular imprints of lung fibrosis (Mishra et al., unpublished observation). Thus, our results indicate that IL-17 may be involved in SM-induced lung fibrosis across the mammalian species and a potential target for therapeutic interventions.
This work was supported by the CounterACT Program, the National Institutes of Health Office of the Director, and the National Institute of Neurological Diseases and Stroke (grant number 5U54NS058185). The authors would like to thank Drs. Tom March and Julie Hutt for pathological assessment of the slides
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