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Chlorine is a highly toxic respiratory irritant that when inhaled causes epithelial cell injury, alveolar-capillary barrier disruption, airway hyperreactivity, inflammation, and pulmonary edema. Chlorine is considered a chemical threat agent, and its release through accidental or intentional means has the potential to result in mass casualties from acute lung injury. The type 4 phosphodiesterase inhibitor rolipram was investigated as a rescue treatment for chlorine-induced lung injury. Rolipram inhibits degradation of the intracellular signaling molecule cyclic AMP. Potential beneficial effects of increased cyclic AMP levels include inhibition of pulmonary edema, inflammation, and airway hyperreactivity. Mice were exposed to chlorine (whole body exposure, 228–270 ppm for 1 h) and were treated with rolipram by intraperitoneal, intranasal, or intramuscular (either aqueous or nanoemulsion formulation) delivery starting 1 h after exposure. Rolipram administered intraperitoneally or intranasally inhibited chlorine-induced pulmonary edema. Minor or no effects were observed on lavage fluid IgM (indicative of plasma protein leakage), KC (Cxcl1, neutrophil chemoattractant), and neutrophils. All routes of administration inhibited chlorine-induced airway hyperreactivity assessed 1 day after exposure. The results of the study suggest that rolipram may be an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery.
Chlorine is a widely used industrial chemical that is highly toxic to the respiratory system. Chlorine is considered a chemical threat agent because of its respiratory toxicity, its ready availability, and its history of use in warfare. Large amounts of chlorine are produced and transported within the United States, and numerous accidental releases leading to high-level human exposures have occurred (Joyner and Durel, 1962; Jones et al., 1986; Van Sickle et al., 2009). Acute effects associated with chlorine exposure in humans include dyspnea, airway obstruction, hypoxemia, pulmonary edema, and pneumonitis (Hasan et al., 1983; Evans, 2005; Van Sickle et al., 2009). Most individuals who survive an episode of acute chlorine poisoning recover normal lung function (Jones et al., 1986), but a subset exhibits long-term consequences of exposure, including airway obstruction and airway hyperreactivity (Hasan et al., 1983; Schwartz et al., 1990; Lemiere et al., 1997; Malo et al., 2009).
Inhaled chlorine reacts with epithelial lining fluid of the respiratory tract and possibly also directly with epithelial cells to deplete antioxidant defenses and produce additional toxic products (Squadrito et al., 2010). Chlorine dissolves to produce hypochlorous acid and also reacts directly with biological molecules in epithelial lining fluid including antioxidants, proteins, amino acids, and phospholipids. Many of the products of these reactions are themselves oxidizing agents that can propagate cellular damage. Low-level chlorine exposure stimulates irritant-responsive sensory nerves (Gagnaire et al., 1994; Morris et al., 2005; Bessac et al., 2008) and results primarily in airway injury characterized by inflammation, vascular leakage, and airway hyperreactivity (McGovern et al., 2010). These effects can occur in the absence of overt histological changes in the airways (McGovern et al., 2010), suggesting subtle epithelial injury and the involvement of neuronal mechanisms (Bessac and Jordt, 2010). Exposure to higher doses of chlorine causes more severe injury of the conducting airways, including the death of large numbers of epithelial cells, and also damages alveolar epithelial cells resulting in pulmonary edema (Winternitz et al., 1920; Martin et al., 2003; Wang et al., 2004; Leustik et al., 2008; Tian et al., 2008).
Treatment for lung injury induced by inhalation of high levels of chlorine has consisted primarily of supportive care, including oxygen administration and lung-protective mechanical ventilation, often in conjunction with therapeutics (e.g. β-adrenergic agonists, corticosteroids, nebulized sodium bicarbonate) for which clinical efficacy has yet to be demonstrated (Van Sickle et al., 2009). Pharmacological agents that raise intracellular concentrations of the signaling molecule cyclic AMP (cAMP) have the potential to produce multiple beneficial effects in the injured lung. Increased cAMP levels are associated with increased alveolar fluid clearance, decreased pulmonary edema, improved pulmonary function, and inhibition of inflammation (Hoyle, 2010). Therapeutic increases in cAMP levels can be achieved by stimulating its production, e.g. by treatment with β-adrenergic agonists, or by inhibiting its degradation by treatment with phosphodiesterase (PDE) inhibitors. Type 4 PDEs are major isozymes in lung epithelial cells, airway smooth muscle cells, and immune cells (Torphy, 1998). Pharmacologic inhibitors of type 4 PDEs have been investigated as novel anti-inflammatory agents for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease (Spina, 2008). Based on these known activities, we hypothesized that the selective type 4 PDE inhibitor rolipram would be an effective rescue treatment for chlorine-induced lung injury. The goal of the present study was to evaluate the efficacy of rolipram given after exposure by multiple routes of administration in inhibiting acute lung injury caused by inhalation of chlorine gas. The study focused on acute injury and inflammation that occurs 6–48 h after exposure.
Experiments involving animals were approved by the University of Louisville Institutional Animal Care and Use Committee and were conducted in accordance with the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). FVB/N mice were purchased from the Jackson Laboratory (Bar Harbor, ME) at 8 weeks of age and were used within 2–4 weeks. Mice were exposed to inhaled chlorine using a whole body exposure system as described (Tian et al., 2008; Hoyle et al., 2010b). The standard exposure period was 1 h with an additional 10 min of ventilation prior to removal of mice from the chamber. Continuous monitoring of chamber chlorine concentration was performed using an X-Stream 2 gas analyzer (Rosemount Analytical, Solon, OH). Total exposure dose was determined by integrative sampling using a modified version of an ASTM method for airborne chlorine (Rando and Hammad, 1990; Hoyle et al., 2010b). Mice were exposed to chlorine doses of 228–270 ppm-h; these levels of exposure produce significant acute lung injury, including epithelial cell death, inflammation, pulmonary edema, and airway hyperreactivity, while producing minimal mortality within the first two days of exposure (Tian et al., 2008).
Rolipram (from Tocris Bioscience, Ellisville, MO) was dissolved in methanol and diluted with phosphate buffered saline for intraperitoneal (i.p.), intranasal (i.n.), or aqueous intramuscular (i.m.) administration to mice. Rolipram doses for systemic and i.n. delivery were determined in initial dose ranging studies (not shown). Treatment of unexposed mice with these doses of rolipram by any of the routes of administration had no effect on lung weight or airway hyperreactivity (not shown). Vehicle solutions consisted of methanol diluted in phosphate buffered saline. A nanoemulsion formulation for i.m. injection was prepared by dissolving rolipram in a mixture of soybean oil and lecithin and then emulsifying by sonication with the aqueous phase. The final concentrations of inactive components in the nanoemulsion were 30% soybean oil, 5% lecithin, 1% methyl paraben, 1% propyl paraben, 1% Pluronic® F68, 2.5% glycerol, 5% polyethylene glycol 400, remainder water. A vehicle nanoemulsion was prepared by omitting rolipram from the mixture. In all cases, rolipram was administered starting 1 h after the end of the chlorine exposure. Mice were sedated by inhalation of 2–3% isoflurane for i.n. and i.m. delivery of rolipram.
Pulmonary edema was assessed 6 h after chlorine exposure, either by weighing the left lung or by measuring extravascular lung water in the whole lung. For weighing the lung, mice were anesthetized with tribromoethanol (375 mg/kg i.p.) and were exsanguinated by cutting the abdominal aorta. The chest cavity was opened, suture was placed around the left main bronchus (to allow inflation of the right lung with fixative for other studies), and the left lung was removed for weighing. Left lung weight was normalized to mouse body weight. Extravascular lung water was measured as described previously (Su et al., 2005). Hemoglobin measurements in blood and lung homogenates for the extravascular lung water procedure were measured using commercially available reagents according to the supplier’s instructions (Arbor Assays, Ann Arbor, MI).
Collection of lung lavage fluid and differential cell counts were performed as described (Tian et al., 2008). KC (Cxcl-1) was measured by ELISA using reagents from R&D Systems (Minneapolis, MN). IgM was measured by ELISA using reagents from Bethyl Laboratories (Montgomery, TX).
Rolipram or vehicle was administered i.p., i.n., or i.m. 1 h and 10–12 h after the end of chlorine exposure. The day after exposure, rolipram was administered to each mouse 2 h before pulmonary function testing. Pulmonary function and airway reactivity to methacholine were measured by forced oscillation using a FlexiVent system (SCIREQ, Montreal, Quebec, Canada). Mice were anesthetized with tribromoethanol (400 mg/kg i.p.) and a tracheal cannula was inserted and connected to a ventilator and pressure transducers. Mice were placed on a warming plate, attached to EKG leads, and mechanically ventilated with a tidal volume of 6 ml/kg at 150 breaths/min. Mice were administered pancuronium bromide (0.8 mg/kg i.p.) to inhibit endogenous breathing effort. Baseline measurements of respiratory system resistance and compliance were collected, as well as lung mechanics parameters calculated from fitting lung impedance data to the constant-phase model (Hantos et al., 1992; Tomioka et al., 2002). Following baseline respiratory measurements, mice were administered increasing doses of aerosolized methacholine (generated from solutions of 1.6, 3.1, 6.3, and 12.5 mg/ml) to measure airway reactivity. Methacholine was aerosolized for 10 sec from an Aeroneb nebulizer that delivered 0.15 ml/min, and respiratory parameters were repeatedly collected for a total of 15 measurements of each parameter. For each methacholine dose, the average of the 15 measurements was calculated.
Data are presented as group means ± standard error of the mean (SEM). Effects of treatment on airway reactivity to methacholine were analyzed by repeated measures analysis of variance (ANOVA). Effects of exposure condition/treatment on other parameters were analyzed using one-way ANOVA with Fisher’s Protected Least Significant Difference test. Differences were considered to be statistically significant at the p<0.05 level.
The effects of rolipram treatment on multiple aspects of chlorine-induced lung injury in FBV/N mice were investigated. In initial experiments, the effects of rolipram on lung weight were measured as an indicator of pulmonary edema. Chlorine exposure increased lung weight, and both i.p. and i.n. rolipram treatment inhibited the increase in weight (Fig. 1). In subsequent experiments, extravascular lung water was measured as a more specific indicator of pulmonary edema. In agreement with the lung weight analysis, i.p. administration of rolipram to chlorine-exposed mice inhibited the chlorine-induced increase in extravascular lung water (Fig. 2A). I.m. injection of rolipram was tested as route of administration that would be amenable for use as a rescue treatment in a mass casualty scenario. Injection of an aqueous solution of rolipram i.m. 1 h after exposure had no effect on extravascular lung water assessed 6 h after exposure (Fig. 2B). Likewise, a nanoemulsion rolipram formulation for i.m. injection failed to inhibit chlorine-induced pulmonary edema (Fig. 2C).
IgM concentration in lung lavage fluid was measured as an indicator of plasma protein leakage into the lung. Chlorine exposure increased lavage fluid protein concentration 6 h after exposure, and rolipram treatment did not inhibit this effect (Fig. 3).
We showed previously in chlorine-exposed mice that the neutrophil chemoattractant KC (Cxcl1) was increased in lung lavage fluid 6 h after exposure, whereas the appearance of neutrophils in lavage fluid is delayed, with higher numbers observed at 48 h compared with 6 or 24 h after exposure (Tian et al., 2008). We therefore assessed the effect of rolipram treatment on chlorine-induced inflammatory processes by measuring lavage fluid neutrophils 48 h after exposure and lavage fluid KC 6 h after exposure. As expected, chlorine exposure produced neutrophilic inflammation (Fig. 4). Rolipram administered by the aqueous i.m. route produced up to 36% inhibition of lavage fluid neutrophils but did not have any effect following administration by other routes. Chlorine inhalation caused significant increases in lavage fluid KC levels, and these increases were not inhibited by rolipram treatment (Fig. 5). In fact, the highest dose of rolipram resulted in significantly higher amounts of KC for three of the four routes of administration tested.
Chlorine inhalation induces airway hyperreactivity to inhaled methacholine measured 1 day after exposure (Hoyle et al., 2010a; Song et al., 2011). Rolipram may potentially inhibit airway hyperreactivity both by relaxation of airway smooth muscle and by inhibition of pulmonary edema (Hoyle, 2010). To test this experimentally, mice were exposed to chlorine and treated with rolipram i.p., i.n., or i.m. three times post-exposure prior to lung function measurements on the day after exposure. All four of the delivery methods tested resulted in inhibition of airway hyperreactivity observed in response to methacholine challenge (Fig. 6). I.m. treatment with the nanoemulsion formulation appeared to produce the greatest inhibition of airway hyperreactivity (Fig. 6D) and returned the lung resistance measurements in chlorine-exposed mice to normal levels (Hoyle, 2010). The responses of other respiratory parameters to i.m. injection of rolipram nanoemulsion are shown in Fig. 7. Rolipram caused significant reversal of chlorine-induced changes in resistance (Fig. 7A), compliance (Fig. 7B), tissue damping (G, Fig. 7D), and eta [ratio of tissue damping (G)/tissue elastance (H), Fig. 7F], but not Rn (Newtonian, or central airway, resistance, Fig. 7C).
The results of the present study indicate that the PDE 4 inhibitor rolipram inhibits chlorine-induced lung injury and may represent a potential rescue treatment for chlorine inhalation. Rolipram administered 1 h after exposure inhibited chlorine-induced pulmonary edema and airway hyperreactivity. I.p. and i.n. routes of administration were effective in inhibiting pulmonary edema, and i.p., i.n., and i.m. routes produced inhibition of airway hyperreactivity. No inhibitory effect of rolipram on plasma protein leakage or inflammatory parameters (neutrophils and KC in lavage fluid) was observed.
Rolipram inhibited chlorine-induced pulmonary edema, and this is effect is consistent with the known mechanism of action of PDE inhibitors. Chlorine inhalation injures lung epithelial and endothelial cells leading to fluid leakage and pulmonary edema. The effects of PDE inhibitors on pulmonary edema are thought to be related to the known stimulation of alveolar fluid clearance by cAMP (Matthay et al., 2005). Consistent with this, Song et al. demonstrated that i.n. administration of the long-acting β agonist R-formoterol to mice reversed chlorine-induced impairment of alveolar fluid transport, although the effects on pulmonary edema were not reported (Song et al., 2011). In the present study, rolipram did not reduce the concentration of IgM in lavage fluid, which was an indication that it did not inhibit plasma protein leakage. This result would be expected if rolipram stimulated alveolar fluid clearance but did not promote repair of the disrupted epithelial/endothelial barrier.
Phosphodiesterase inhibitors are known to produce a spectrum of anti-inflammatory effects in lung injury models (Souness et al., 2000), although the underlying molecular mechanisms are not well characterized. In some models, PDE inhibitors selectively decrease the production of a subset of inflammatory mediators (Herbert et al., 2008). In the present study, anti-inflammatory effects of rolipram following chlorine exposure were assessed by measuring neutrophils and KC in lavage fluid. The highest dose of rolipram resulted in increased lavage fluid KC for three of the four delivery methods tested. However, rolipram treatment had either no effect or a minor inhibitory effect on lavage fluid neutrophils. It therefore appears that the observed increases in chemokine expression are not sufficient to affect neutrophilic inflammation. Previous studies have shown that PDE 4 inhibitors can increase cytokine expression, including KC, in some cell types (McCluskie et al., 2006; Hertz et al., 2009). Therefore the effects of PDE 4 inhibitors on inflammation may depend on the context of the specific injury or proinflammatory stimulus and on the specific cell types involved. Because an inhibitory effect of rolipram on chlorine-induced neutrophilic inflammation was not observed, it is possible that increased efficacy may be obtained by combined treatment with rolipram and an anti-inflammatory agent.
Our results indicated that rolipram treatment did not affect baseline lung mechanics in chlorine-exposed mice, but did inhibit methacholine-induced increases in respiratory system resistance. Chlorine inhalation alters baseline lung function and causes airway hyperreactivity to methacholine in mice (Martin et al., 2003; Hoyle et al., 2010a; Song et al., 2011). Agents such as PDE inhibitors that raise intracellular cAMP levels can produce bronchodilation through a direct relaxant effect on airway smooth muscle. cAMP signaling pathways appear to have a minor effect on basal airway tone, but can be targeted with β-agonists or PDE inhibitors to counteract bronchoconstriction or airway hyperreactivity that occur in pathological states (Deshpande and Penn, 2006). This was similar to published findings with R-formoterol, which inhibited airway hyperreactivity to inhaled methacholine in chlorine-exposed mice (Song et al., 2011). Chlorine inhalation causes extensive injury to the epithelium of the central airways (Tian et al., 2008; Song et al., 2011). One potential mechanism of airway hyperreactivity to inhaled methacholine in this model would be the enhanced availability of aerosolized methacholine to the airway smooth muscle. Other injury models involving increases in airway epithelial permeability exhibited this phenomenon with an associated increase in Rn (Bates et al., 2006; Allen et al., 2009). In contrast, our analysis of lung mechanics revealed that Rn was not increased by the doses of methacholine we used and that rolipram treatment, which had a profound effect on Rrs, did not inhibit Rn. The results suggest that rolipram was not inhibiting resistance by relaxing the large airways; rather, the data (e.g. the significant inhibition of tissue damping) are consistent with effects on the lung periphery, which could potentially occur through the inhibitory effects of rolipram on small airways or on pulmonary edema.
In the present study mice were used to model chlorine injury to the lung and to investigate the ability of rolipram to ameliorate aspects of the injury. Because of the functional and anatomical differences between the mouse and human respiratory tracts, caution is necessarily required when extrapolating to effects in humans. In response to irritants such as chlorine, mice exhibit pronounced concentration-dependent changes in breathing patterns which are mediated by irritant-responsive sensory nerves. This effect, in concert with the differences in anatomy, makes it difficult to determine a dose in humans that would be equivalent to that used in this study in mice. In a disaster scenario involving large-scale chlorine release, humans will experience a variety of exposures with respect to concentration and time. Because of this, it is more important to model the spectrum of injuries that is typical following high-level exposure rather than targeting an exact human exposure dose. The general aspects of lung injury that we assessed in the mouse model, including pulmonary edema, inflammation, and impaired lung function, are typical of those documented in humans after large-scale chlorine release (Van Sickle et al., 2009).
Both β-agonists and PDE inhibitors have been used therapeutically for treatment of lung diseases (Hoyle, 2010). In theory, both types of compounds can raise cAMP levels and provide beneficial effects for treating lung injury. In practice, differential effects of the two classes of drugs have been observed. β-agonists have a long history of use as bronchodilators in asthma patients, and have also shown to be effective as inhibitors of acute lung injury in animal models (McAuley et al., 2004; Wang et al., 2004; Litvan et al., 2006; Song et al., 2011). In clinical trials, β-agonist treatment showed efficacy in an initial trial for treatment of acute lung injury/adult respiratory distress syndrome (Perkins et al., 2006), but had no effect in a subsequent larger trial (Matthay et al., 2009). As the majority of patients in such clinical trials develop lung injury associated with sepsis, the results do not rule out the possibility that β-agonists may be effective in treating acute lung injury of other etiologies such as acid aspiration or inhalation of irritant chemicals such as chlorine. A disadvantage of treatment with β-agonists is that these agents lose effectiveness with continued treatment as a result of receptor desensitization or compensatory increases in PDE activity (Johnson, 2006). Therefore patients already receiving β-agonist therapy, e.g. for asthma, may be refractory to treatment of acute lung injury with these same agents. In contrast, PDE inhibitors raise cAMP levels, and no compensatory mechanisms that limit cAMP accumulation with chronic PDE treatment have been identified. Oral administration of PDE 4 inhibitors has been clinically tested for the treatment of COPD. Such inhibitors appear to have clinical efficacy, but their use is limited by side effects including gastrointestinal irritation and nausea (Rennard et al., 2008; Calverley et al., 2009; Giembycz and Field, 2010). When considering the use of PDE 4 inhibitors as countermeasures against chlorine-induced lung injury, such adverse effects may be better tolerated or minimized in light of the limited time the drug will be taken, the potentially life-threatening nature of the illness, and administration by a route other than oral.
Ideal characteristics of an agent to be used as a countermeasure against chlorine-induced lung injury include efficacy against multiple aspects of injury, the ability to be administered quickly to large numbers of individuals, and efficacy when given as a rescue treatment subsequent to the exposure. Our experiments compared systemic and local delivery of rolipram and showed that both were effective in inhibiting pulmonary edema and airway hyperreactivity. Direct delivery to the respiratory tract has the advantage that locally high levels of drug can be targeted to the organ of interest. A disadvantage is that fluid leakage into the lungs caused by chlorine injury may interfere with the drug reaching the alveolar epithelium. In addition, delivery to unconscious victims may be difficult by this route. For countermeasure use in a mass casualty situation, a method of delivery that can be accomplished quickly by personnel with limited medical training is preferred. For treatment of human casualties, systemic delivery by intramuscular administration is likely a preferred route that combines speed and simplicity. The present study shows proof-of-principle for systemically delivered rolipram as an inhibitor of chlorine-induced lung injury. Future studies can be targeted toward developing formulations optimal for this route of delivery. An effective countermeasure must also provide therapeutic benefit when administered after exposure once lung injury has already begun to develop. In the present study, we selected a 1 h interval between the end of the chlorine exposure (2 h after the start of the exposure), and rolipram treatment was able to inhibit multiple aspects of lung injury when given at this time. The choice of the timing of treatment was based on the knowledge that lung injury, e.g. as evidenced by hypoxia, is present by 1 h after chlorine exposure (Gunnarsson et al., 1998; Batchinsky et al., 2006; Leustik et al., 2008) and on the fact that this would be a reasonable time frame during which many patients could be reached by first responders in a disaster scenario. Overall, the results of the study indicated that rolipram is an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery.
This work was supported by the National Institutes of Health CounterACT Program through the Office of the Director and the National Institute of Environmental Health Sciences (Award U01 ES015673 to G.W.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Environmental Health Sciences or the National Institutes of Health.
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