To further understand the pathogenesis of oxidant-induced lung injury syndromes, studies were undertaken to define the pathways that regulate the toxic respiratory effects of supraphysiologic concentrations of oxygen. These studies demonstrate that A1 is induced in lungs from WT mice during hyperoxia, where it feeds back to control the severity of this pathologic pulmonary response. They also demonstrate that A1 is induced by IL-11 and VEGF and that this induction is a key event in the pathogenesis of the cytoprotective effects that these cytokines have in HALI. Mechanistic insights were also obtained, since these studies demonstrate that the protective effects of A1 are associated with decreased hyperoxia-induced induction and activation of key caspases and components of the extrinsic/death receptor and intrinsic/mitochondrial cell death pathways, and with the A1-dependent induction of the antiapoptosis proteins Bcl-2 and Bcl-xl. Lastly, they demonstrate that A1 is induced by hyperoxia and IL-11 in vitro and that the overexpression of A1 inhibits the oxidant-induced necrosis and apoptosis of epithelial cells in culture. When viewed in combination, these studies demonstrate that A1 is an endogenous and cytokine-induced cytoprotective molecule that plays a critical role in the regulation of the apoptotic and necrotic responses that are induced by hyperoxia in the lung.
The ability of 100% O2
to cause acute lung injury with endothelial and epithelial cell injury, noncardiac pulmonary edema, and eventually death is well documented in rodent and other modeling systems (2
). For reasons that are not clear, however, inbred mice can survive for as little as 3 days or as long as 6 days in 100% O2
). The mechanisms that account for the delay in the appearance of HALI and for the noted animal-to-animal variation are poorly understood. This lack of understanding may be the result of an inadequate appreciation of the factors that control oxidant-induced injury in the lung. The present studies and studies from our laboratory and others (2
) demonstrate that 100% O2
causes a TUNEL+
cell death response with features of apoptosis and necrosis in vivo. They also address the complex nature of this cell death response by demonstrating that hyperoxia can induce apoptosis, necrosis, or a mixture of the 2 in vitro (2
). Importantly, A1 was a potent inhibitor of all of these in vivo and in vitro responses. The demonstration that A1 is a critical regulator of pulmonary apoptosis is in accord with prior studies that demonstrate that A1 inhibits the apoptosis induced by a variety of stimuli in a variety of cells and tissues (14
). In contrast, to our knowledge, these are the first studies to demonstrate that A1 is an important regulator of oxidant-induced necrosis. These findings are not without precedent, however, because it has recently been appreciated that pathways that were previously thought to be unique to apoptosis (caspase-8 and Bid activation) also play key roles in cellular necrosis (15
IL-11 is a multifunctional IL-6–type cytokine that is produced by epithelial cells, fibroblasts, eosinophils, and other cells in response to a number of stimuli, including IL-1, TGF-β1
, and respiratory virus (33
). Early studies of IL-11 focused on its stimulatory roles in megakaryocytopoiesis and thrombopoiesis and its ability to activate osteoclasts and induce bone resorption. It has subsequently been appreciated that IL-11 has remarkable mucosal protective effects in the setting of chemotherapy and radiation therapy that may result from its ability to inhibit macrophage production of IL-1, TNF, and IL-12 and inhibit the activation of NF-κB (33
). Previous studies from our laboratory demonstrated that IL-11 also confers cytoprotection in HALI (6
). This protective effect could not be accounted for by antioxidant alterations. Instead, IL-11 appeared to be a potent inhibitor of the hyperoxia-induced cell death response (6
). The present studies add to our knowledge of the mechanism of this protective response by demonstrating that A1 is a critical mediator of this cytoprotection. They also demonstrate that this protective response is not IL-11–specific, since VEGF, a known regulator of apoptosis (22
), also conferred cytoprotection via a mechanism that is, at least partially, A1-dependent. Transgenic GM-CSF has recently been shown to confer protection in HALI (30
). It is tempting to speculate that A1 contributes to this response as well, because A1 was originally described by Prystowsky and colleagues as a gene in bone marrow that was potently stimulated by GM-CSF (43
). In combination, these observations suggest that A1 induction is a commonly employed pathway that cytokines and, potentially, other stimuli use to control oxidant-induced injuries. It is important to point out, however, that A1-independent pathways also appear to be operative, since IL-11 and VEGF transgenic mice with null mutations of A1
still lived for an extended interval in 100% O2
when compared with WT controls. Additional experimentation will be required to define the A1-independent mechanisms that are operative in this setting.
Under physiologic conditions, tissue homeostasis is preserved by the tight control of apoptosis and necrosis. This is accomplished by the continuous integration of pro– and anti–cell death signals (44
). Although cell death can be triggered by a vast array of stimuli and mediated via an increasingly complex series of pathways, the vast majority of signals engage the cell death machinery at the level of the cell membrane or at the level of the mitochondria. The membrane (“extrinsic”) pathway triggers surface “death receptors” such as Fas, which binds FasL, and TNF receptor I, which binds TNF and lymphotoxin, and it activates caspase-8. Other stimuli use mitochondrial dysfunction to signal death responses. In this “intrinsic” response, BH3-domain-only family members such as Bid are activated to tBid and interact with Bax-type proteins (Bax, Bak, Bok) to form or interact with mitochondrial pores, release cytochrome c
, activate caspase-9, and induce cell death (13
). Fas system alterations and mitochondria-dependent cell death pathway activation have been noted in hyperoxia (15
). Thus, to further understand the mechanism(s) of hyperoxia-induced cell death and the effects of A1, we characterized major features of the intrinsic and extrinsic pathways in mice with WT and null A1
loci. These studies demonstrate that hyperoxia induces and activates caspase-3, -8, and -9 and key components in the extrinsic/death receptor and intrinsic/mitochondrial cell death pathways. They also demonstrate that A1 inhibits the caspase, death receptor, and mitochondrial activation events that were seen. Interestingly, they also demonstrate that hyperoxia and IL-11 induce the prototypic antiapoptotic proteins Bcl-2 and Bcl-xl, and that these inductive events are mediated via an A1-dependent pathway. It is not possible, in these complex whole-lung systems, to define the critical sites of A1 production or the relative importance of each of its many effects on the cell death machinery. However, when these studies are combined with prior studies in lymphocytes and other systems that demonstrate that A1 can inhibit Bid cleavage, sequester tBid, and inhibit the activation of caspase-3 and -8 (14
), it is clear that endogenous A1 is a multifunctional regulator of oxidant-induced apoptotic and necrotic responses in the lung. These studies also demonstrate an additional level of complexity in the Bcl-2 gene family. Specifically, in addition to differences in their function and cytokine inducibility and their need to form homo- and heterodimers to mediate their effects (14
), they interact with one another in a Bcl-2 gene cascade that plays an important role in their stimulation at sites of tissue injury.
Oxidative injury is a key element in the pathogenesis of a wide variety of diseases and disorders. This is nicely illustrated in the lung, where hyperoxia leads to acute and chronic injuries such as bronchopulmonary dysplasia in the newborn and adult respiratory distress syndrome in adults. Oxidant injury also plays a major role in the pathogenesis of interstitial lung diseases, asthma, and chronic obstructive pulmonary disease and can worsen the pathologic effects of pulmonary infections (29
). Our studies demonstrate, for the first time to our knowledge, that A1 is induced by hyperoxia and by protective cytokines and that, after this induction, it is a key endogenous regulator of oxidative injury in the lung. They also highlight the multifaceted mechanism of the protection conferred by A1, with impressive inhibitory effects on caspases and the extrinsic/death receptor and intrinsic/mitochondrial cell death pathways. These observations have a number of important implications. First, they suggest that therapies that increase the expression and/or effector functions of A1 can be therapeutically useful in the treatment of these diseases. They also suggest that genetic polymorphisms or environmental exposures that alter the production or effector functions of A1 can have major effects on an individual’s ability to tolerate an oxidative load and thus could contribute to the patient variability that is seen in the severity and natural history of these disorders.