Perspective Mechanisms of Cell Injury and Death in Hyperoxia Role of Cytokines and Bcl-2 Family Proteins Constance Barazzone and Carl W. White Departments of Pediatrics and Pathology, Centre Médical Universitaire, Geneva, Switzerland; and Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado
Prevailing concepts of pulmonary oxygen toxicity pathogenesis and hyperoxic tolerance have evolved over the past two decades. The “free radical” theory stating that lung cells poison themselves by producing an endogenous excess of reactive oxygen species (ROS) within various organelles had garnered considerable experimental support in the 1980s (1). The possible role of inflammation in compounding or propagating injury during sublethal hyperoxic exposure was also acknowledged. Subsequently, considerable evidence was obtained to support an association between increasing lung antioxidant enzyme activities and acquired tolerance to hyperoxia. Studies using pharmacologic supplementation and genetically altered mice have provided additional support for the importance of antioxidant enzymes, particularly the mitochondrial MnSOD. Nonetheless, certain inevitable intramitochondrial events, such as inactivation of aconitase with concomitant liberation of ferrous iron from the active site in the matrix and an associated decline in respiration (2), cannot be prevented by MnSOD induction via cytokines or genetic manipulation (3).
Cytokines and Hyperoxic Tolerance Our understanding of mechanisms of tolerance was enhanced when it was found that cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1, when administered together intravenously (4) or individually by the intratracheal route (5, 6), induce tolerance. These treatments were also associated with the induction of lung MnSOD. However, these cytokines can induce other cytokines, notably IL-6 and numerous other proteins. Hence the mechanisms of cytokine-induced protection have been widely explored but not fully understood (Table 1). The study by Ward and colleagues (7) in this issue of the Red Journal has directed our attention to a possible role for IL-6 and acute phase proteins such as tissue inhibitor of metalloproteinases in the protective response. It also was noted that the protective anti-apoptotic protein Bcl-2 was induced in response to transgenic IL-6 expression, raising
(Received in original form March 11, 2000) Address correspondence to: Carl W. White, M.D., 1400 Jackson Street, Room J101, Denver, CO 80206. E-mail:
[email protected] Abbreviations: interleukin, IL; keratinocyte growth factor, KGF; reactive oxygen species, ROS; tumor necrosis factor, TNF. Am. J. Respir. Cell Mol. Biol. Vol. 22, pp. 517–519, 2000 Internet address: www.atsjournals.org
the issue of a potentially important role of apoptosis in this animal model for lethal hyperoxic lung injury. This discussion should be prefaced by saying that significant cell dysfunction undoubtedly precedes cell death in these models and that appropriate early interventions might prevent such dysfunction and subsequent death. Among such possibly effective, therapeutic interventions would be the induction of protective responses, such as the acute phase response, which, among other effects, can modulate protease–antiprotease balance and also the homeostasis of metals such as iron, which in turn can modulate free radical–dependent injury. The current article, however, suggests that direct modulation of cell death processes could also influence outcome; that is, specific changes in proteins of the Bcl-2 family and/or other anti-apoptic growth or survival factors could also be sufficient to afford protection (7).
Apoptosis and Hyperoxia Exposure Epithelial cells are crucial in maintaining the integrity of the alveolar-capillary barrier, and their damage causes leakage at this site. In animal models, death of endothelial and epithelial cells seems to be an essential feature of endstage oxygen-induced alveolar damage. To date, two distinct mechanisms of cell death have been recognized: necrosis and apoptosis. Necrosis is associated with the disruption of the cell membrane, resulting in a loss of cytoplasm, and, finally, a random nuclear degradation. Apoptosis occurs in a cell with intact plasma membrane, and there, DNA degradation is the result of the activation of specific endonucleases (8). Apoptosis and necrosis were commonly conceived as different and mutually exclusive processes (8). This statement, which may be true in vitro, has not been convincingly demonstrated in vivo. There is evidence that the intracellular events leading to apoptosis and necrosis can occur sequentially. In the initial step, there is a collapse of mitochondrial membrane potential and loss of cellular glutathione. At this stage, pharmacologic intervention in the form of caspase inhibitors can prevent cell death. In a second step, there is disruption of the plasma membrane (necrosis) (9). The possibility remains that apoptosis and necrosis might occur simultaneously in an exclusive fashion within one cell population or sequentially in the same cell (necroptosis). Alveolar cell death is difficult to assess in vivo mainly because the distinction between alveolar type I cells, endothelial cells, and interstitial cells is very difficult by light microscopy. Even by electron microscopy, when cell injury
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TABLE 1
Cytokine regulation during hyperoxia Cytokine
Induction
TNF
↑
IL-1
↑
Effect on Lung Injury
Protective* Detrimental† (⫾)
Protective* IL-6
↑↑
IL-8*
↑
IL-11 IGF, IGFR, IGR-BP PDGF-
Reference(s)
(4) (5) (26) (6) (27) (26) (4)
? Overexpression protective Detrimental
(28) (29) (7) (30)
ND
Overexpression protective
(19)
↑↑
?
↑
?
(31–34)
Figure 1. Simplified overview of apoptosis.
(35)
G-CSF
(↑)
Detrimental
VEGF
↓↓
?
(37) (38)
KGF
⫽
Protective†
(20) (21)
(Barazzone et al., unpublished data) (36)
Definition of abbreviations: tumor necrosis factor, TNF; interleukin, IL; platelet-derived growth factor, PDGF; insulin-like growth factor, IGF; granulocyte-stimulating stem factor, G-CSF; vascular endothelial growth factor, VEGF; keratinocyte growth factor, KGF. *Protective when given before. † After the establishment of the injury.
is present the distinction between these cell types is difficult. Moreover, the study of cell death during lung injury is complex because the destructive process and the reparative phase can occur simultaneously. However, several recent studies have focused on the importance of apoptosis in oxygen toxicity. To date, researchers have demonstrated that this injury is associated with features of both cell necrosis and apoptosis (10, 11). Apoptosis can be triggered by different pathways that are responsible for caspase activation. These include activation of members of the TNF superfamily (TNF receptor, Fas, CD40) located on the plasma membrane—some of which contain a death domain. Additional pathways to apoptosis include withdrawal of growth factors and/or direct insults to the plasma membrane (starvation, stress, oxidative stress), which involve different intracellular proteins and enzymes (Figure 1). Exposure to hyperoxia may be similar to oxidative stress in vitro (12); it increases levels of ROS, which, among their deleterious effects, can provoke DNA strand scission, lipid peroxidation of cellular membranes, and activation of various genes whose products are involved in inflammation and cell death (1, 13). Mounting evidence suggests that ROS and oxidative stress are important players in the chain reaction leading to cell death. A variety of key cellular events focus on mitochondria, including the release of caspase activators, changes in transmembrane potential, altered oxidation-reduction status,
and participation of Bcl-2 family proteins, which may be associated with the mitochondria. Although inhibition of caspases does not completely inhibit cell death caused by certain agents, anti-apoptotic proteins such as Bcl-2 and Bcl-XL can maintain cell survival in the face of such agents (14). Because mitochondria are major generators of ROS and can harbor proteins of the Bcl-2 family, they probably play an important role in the control of processes which culminate in cell death as a result of hyperoxia (15, 16). Three interrelated mitochondrial mechanisms are probably responsible for the control of cell death or life: (1) the disruption of electron transport and mitochondrial release of cytochrome c, resulting in loss of its function and decreased ATP production, (2) the release of caspaseactivating proteins, such as members of Bcl-2 family and some procaspases, and (3) alteration of the cellular redox state. However, the fine regulation of caspases by the Bcl2 family is not completely understood, except that it depends on the cell type and on cytochrome c availability (14). The Bcl-2 family comprises both pro- and anti-apoptotic members, and these can act upstream of several caspases and of mitochondrial dysfunction (17). During exposure to hyperoxia, we have shown that Bax and Bcl-X messenger RNA were strongly upregulated within the lung, raising the question of whether these molecules are protective or deleterious in this model (10). Molecules such as Bax (death agonist) and Bcl-2 (death antagonist) act in competition, and their relative abundance and dimerization can determine cell death or cell survival. The study featured in this issue, showing that IL-6 overexpression in transgenic mice can modify the basal level of Bcl-2 protein without changing the level of Bax protein, suggests that the balance between these molecules is important in vivo (7). The importance of apoptosis in determining the outcome of alveolar injury has also been demonstrated by administration of agents involved in programmed cell death, such as anti-CD40 L monoclonal antibody, or by overexpressing IL-11 in Clara cells (18, 19). These two studies underline not only the potential importance of the pathway leading to apoptosis, but also the type of cells that should be protected from apoptosis. Indeed, p53 knockout mice
Perspective
or Fas null mice show no protection during exposure to hyperoxia (10). Keratinocyte growth factor (KGF) has been shown to protect against alveolar damage during exposure to hyperoxia (20, 21). However, the mechanisms of KGF protection are not fully understood. In cultured epithelial cells, KGF can increase DNA repair (22). During exposure to hyperoxia, diminished apoptosis could not be detected in vivo or in vitro after KGF treatment (20, 23). A very surprising finding in the studies of both Waxman and associates and Ward and coworkers is the potential importance of Clara cells in maintaining alveolar-capillary membrane integrity. It would be interesting to both determine whether increased expression of IL-6 or IL-11 in CCL10 can modify Bcl-2 only in these cells and to understand the precise role of Clara cells in maintaining the alveolar-capillary barrier. It also would be of interest to know whether Clara cell–specific expression allows distal delivery of IL-6 and/or alters Bcl-2 expression in adjacent cells, such as epithelial type I and II cells and endothelial cells. Another possibility would be that bronchiolar epithelium in mice, which consists mainly of Clara cells, could participate in the genesis of alveolar epithelium during the differentiation of Clara cells (24). Although Clara cells are a progenitor of bronchiolar epithelium, no evidence supports a role for them in the genesis of more distal respiratory epithelium. It remains possible that other effects on Clara cell function in this model could affect outcome in hyperoxic lung injury in mice (25). Acknowledgments: C.B. is supported by the Swiss National Science Foundation grant 32-56949.99 and the Wolfermann-Nägele Foundation. C.W.W. is supported by the National Heart, Lung, and Blood Institute grants HL-52732, HL56263, HL-57144, and HL-30068. The authors thank David H. W. Riches for helpful comments and Stephanie Park for preparing the manuscript.
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