In this article, we examine the role that NK-1 receptor plays in airway epithelial injury and proliferation during repair, and whether it modulates neutrophil emigration into airways after acute ozone inhalation. First, we demonstrate that the NK-1 receptor antagonist, SR140333, did not significantly affect the rapid, shallow breathing induced by an acute ozone exposure in Wistar rats. Second, we show that treatment with the NK-1 receptor antagonist significantly attenuated epithelial cell death in terminal bronchioles, but not in proximal airways, suggesting an airway generation–specific response in ozone-induced cell death. Third, we show that treatment with the NK-1 receptor antagonist significantly attenuated epithelial cell proliferation after acute ozone exposure in both proximal and distal airways. Fourth, the NK-1 receptor antagonist did not influence the emigration of neutrophils into airways after ozone injury. Supplementing these observations, we show that: (1) airway epithelial cells expressing NK-1 receptors are distributed down the entire length of the airway; (2) ozone-induced epithelial cell death in the terminal bronchioles occurs via a nonapoptotic mechanism; (3) the NK-1 receptor antagonist, SR140333, does not attenuate cell injury by acting as an antioxidant; and (4) ethidium-positive cells in the terminal bronchioles of ozone-exposed rats are positive for the nuclear orphan receptor, Nur77. Taken together, these data indicate that substance P acting via NK-1 receptors is an important mediator in orchestrating airway epithelial cell death and proliferation after acute ozone exposure.
We found airway generational differences in the amount of epithelial cell death and proliferation in vehicle-treated rats exposed to 1.0 ppm ozone for 8 hours followed by an 8-hour postexposure period. The highest density of dead cells was found in the terminal bronchioles (). Similar site-specific differences in ozone-induced injury and repair have been previously reported (33
). It has been proposed that this distribution of ozone-induced lesions is the result of numerous factors, including the distribution of ozone uptake at different airway sites, and the sensitivity of different cell types to ozone-induced injury (33
). Previous work in our laboratory has shown that the pattern of distribution of ozone-induced airway injury is in part dependent upon the development of rapid, shallow breathing during exposure, which tends to protect the large conducting airways, but produces a more even distribution of injury in terminal bronchioles (31
). Subsequently, Alfaro and colleagues (32
) demonstrated that rapid, shallow breathing results in a redistribution of ozone uptake, in part explaining the findings of Schelegle and coworkers. In the current study, we found that the selective NK-1 receptor antagonist SR140333, did not significantly affect ozone-induced rapid shallow breathing in a limited subsample of rats. Therefore, the observed decreases in site-specific, ozone-induced epithelial injury and proliferation were due to the direct effect of the antagonists, and not due to a change in the distribution of ozone uptake.
Although never directly studied, it has been assumed that ozone-induced cell death of airway epithelial cells is the direct result of oxidant damage induced by ozone and/or its reaction products. Our data show that treatment with SR140333 significantly reduced the ethidium labeling in the terminal bronchioles after ozone inhalation, indicating that NK-1 receptors play some role in mediating ozone-induced cell death. Several papers have found that NK-1 receptors contribute to cellular injury and death in various animal and cell culture models of tissue injury. SR140333 reduced the epithelial ulceration and severity of colitis and infarct volume after cerebral ischemia in rats (35
). Castro-Obregón and colleagues (37
) demonstrated, in a neuronal cell line overexpressing the NK-1 receptor, that substance P induces a nonapoptotic programmed cell death. They showed that this nonapoptotic cell death involves the recruitment of a mitogen-activated protein kinase phosphorylation cascade by the scaffold protein, arrestin 2, leading to the phosphorylation of the orphan nuclear receptor, Nur77. More recently, Lucattelli and colleagues (25
) have shown that bleomycin-induced cell death of type I pneumocytes in mice is dependent upon a similar NK-1 receptor–dependent pathway. It is possible that a similar NK-1 receptor–dependent mechanism contributes to the ozone-induced cell death observed in the terminal bronchioles in our rats. We assessed the amount of apoptosis using immunohistochemistry for active caspase 3, a key enzyme involved in the apoptotic pathway and an established marker of apoptotic cells in tissue. Terminal bronchioles in rats exposed to ozone and treated with both the NK-1 receptor antagonist and vehicle showed no significant number of apoptotic cells. Additionally we demonstrate that the ethidium-positive cells colocalize with Nur77 in ozone-exposed rats not treated with SR140333. Taken together, our results suggest that substance P, acting through the NK-1 receptor and Nur77, may contribute to epithelial cell death through a non–caspase 3–mediated mechanism.
Other mechanisms for this epithelial protective effect may exist. In this study, we investigated the possibility that SR 140333 could have direct antioxidant effects, thus protecting the epithelium from ozone-induced injury. We found no evidence of antioxidant effects when HBE cells were treated with 400 and 800 mM H2O2 as an oxidant in the presence of a range of SR140333 levels, including those used in rats in this study.
Another potential protective effect of SR140333 may be related to its ability to block substance P–induced vasodilation and microvascular permeability (38
). Acute ozone inhalation in humans has been shown to result in the elevation in BAL fluid of several macromolecules normally restricted to the vascular compartment that may contribute to cellular injury and necrosis, including complement 3a (39
). Interestingly, Park and colleagues (40
) have recently demonstrated that either depleting and/or antagonizing complement in mice significantly reduces ozone-induced airway hyperresponsiveness. Several studies have also shown that NK-1 antagonists (including SR140333) blocks airway epithelial permeability after ozone exposure and airway hyperresponsiveness in other models, implicating a role of substance P in the induction of airway hyperresponsiveness as well (41
). Blocking microvascular permeability could limit the influx of mediators to the site of ozone-induced oxidant stress, reducing the subsequent epithelial cell death. Countering this possibility is the observation of Sertl and coworkers (43
) that substance P–induced vascular permeability is limited to the large conducting airways of the rat.
Interestingly, we found a significant decrease in epithelial necrosis in the terminal bronchioles, but not in central or proximal conducting airways, of rats treated with SR140333 and exposed to ozone compared with vehicle-treated and ozone-exposed rats. This unique observation could be due to several possibilities. First, it could indicate that NK-1 receptors have a differential effect on ozone-induced injury at the different airway generations studied. This may, in part, be the result of the different cell types that are located within the proximal airways and terminal bronchioles. The epithelial populations of rat proximal bronchi are ciliated, serous, basal, and mucous cells, whereas the terminal bronchioles contain ciliated and Clara cells, and distal terminal bronchiole and proximal alveolar ducts contain type 1 and 2 pneumocytes. Although our results demonstrate that NK-1 receptors are present throughout these airways, there could be functional differences in the receptors or other mediators, which may modulate the receptors' effect on epithelial injury at these sites. Second, the delivery of SR140333 via the vasculature to the surface epithelium may differ between the airway sites studied. Third, the ethidium label is not cumulative, and we cannot rule out the possibility that epithelial injury occurs at different airway generations at different time points after ozone exposure.
SR140333 significantly attenuated ozone-induced proliferation of epithelial cells at all the sites studied. This effect is similar to the effect that substance P has on modulating the migration and proliferation of cells after the mechanical injury of tracheal epithelium (21
). Substance P stimulates proliferation and migration of guinea pig tracheal epithelial cells (19
), and works synergistically with insulin-like growth factor-1 to stimulate corneal epithelial wound healing (20
). Vesely and colleagues found less BrdU incorporation in terminal bronchiolar epithelium of capsaicin-treated rats exposed to filtered air and ozone, suggesting that neuropeptides released by C fibers may modulate basal and reparative airway epithelial cell proliferation (27
). Our observations, that the NK-1 antagonist, SR140333, decreases airway cell proliferation after ozone exposure, confirms the previous findings of Vesely and colleagues (27
), and extends them to the large conducting airways examined in this study. This implies that the release of substance P from lung C fibers acts to orchestrate the proliferation of basal cells, nonciliated Clara cells, and type II pneumocytes to replace the ciliated cells and type I pneumocytes lost during and after the acute inhalation of ozone. This illustrates the need to better understand the precise mechanisms that lead to the activation of lung C fibers during acute ozone exposure. This is especially true in the terminal bronchioles, where substance P release appears to contribute to ozone-induced cell death, as well as to cell proliferation. Specifically, we propose that oxidant-stressed ciliated cells and type I pneumocytes release one or more mediators that activate lung C fibers to release substance P, and that this substance P activates cell death and proliferation pathways in the terminal bronchiolar epithelium and cell proliferation pathways in the proximal conducting airway epithelium that is in proportion to the ozone-induced oxidant stress.
Our results indicate that the NK-1 receptor antagonist did not significantly affect neutrophil emigration into airways after ozone exposure. There was no significant difference in the number of neutrophils in BAL or in terminal bronchioles of rats treated with SR140333 compared with vehicle-treated rats exposed to ozone. These observations are consistent with our previous observations in rats treated neonatally with capsaicin to ablate C fiber afferents (26
). Taken together, these results suggest that substance P is not a critical mediator for neutrophil emigration into airways after ozone injury in the rat.
Within the literature, there is some discrepancy as to the importance of substance P in neutrophil function and recruitment in different experimental models. In vitro
studies show that substance P primes and activates human neutrophils for superoxide, H2
, and nitric oxide production (23
). In humans, substance P also appears to be important in neutrophil chemotaxis, mediating neutrophil binding to epithelial and endothelial cells and inducing release of cytokines, such as IL-8 (45
). NK-1 receptor knockout mice have shown the importance of substance P in mediating neutrophil accumulation in pancreatitis and inflamed skin (46
In contrast, other papers have not substantiated the importance of substance P in neutrophil recruitment. Similar to our results with SR140333, neutrophil recruitment was not affected in a model of thermal injury in rats (48
). Roch-Arveiller and colleagues examined the response of rat neutrophils to substance P and found concentration-dependent chemotaxis only at high concentrations (10−6
In conclusion, these observations illustrate the pivotal role that the activation of C fibers, with the subsequent release of substance P, plays in the complex cascade of events that are initiated by the acute inhalation of ozone. This role is not limited to reflex responses, such as rapid shallow breathing, but includes the modulation of cellular injury and subsequent proliferation of epithelial cells during repair. Further studies need to be conducted to examine the direct and/or indirect mechanisms that contribute to this modulation of cellular injury and repair by NK-1 receptors.