Department of Environmental Medicine, University of Rochester, 575 Elmwood Avenue, Rochester, New York 14642
ABSTRACT
The article highlighted in this issue is "Silica-Induced Caspase Activation in Mouse Alveolar Macrophages is Dependent Upon Mitochondrial Integrity and Aspartic Proteolysis" by Michael Thibodeau, Charles Giardina, and Andrea K. Hubbard from the University of Connecticut.
Silica-induced lung injury and the development of silicosis is a significant occupational illness with estimates of ~5000 newly recognized cases of silicosis per year (Rosenman et al., 2003). Accumulation and deposition of respirable silica mineral containing particles within the lung produces a chronic lung disease characterized by granulomatous and fibrotic lesions. Recent work in a model of murine silicosis implicates apoptosis as a mechanism underlying the immunopathology found in silicosis (Borges et al., 2002
). This implication presents a paradox, as inflammatory responses such as that underlying silica-induced pulmonary fibrosis are not thought to arise following an apoptotic response. Indeed, a near-dogmatic characteristic of apoptosis versus necrosis is that apoptotic cells are thought to be phagocytosed by neighboring cells or professional phagocytes in order to prevent inflammation.
Recent work, including that of Thibodeau and colleagues in this issue of Toxicological Sciences, showing that silica can initiate apoptotic cell death compels us to rethink the potential role of apoptosis in the inflammatory response and immunopathology associated with silicosis. Thibodeaus work begins to identify the caspases involved and to define the biochemical pathways activated in silica-induced apoptotic cell death. Targeting specific caspases for therapeutic intervention, perhaps pharmacologically, may prove to be important in treating silica-induced lung disease.
Although silica has been documented to cause apoptotic cell death, the cellular pathways leading to caspase activation have not been extensively investigateduntil now. Thibodeau and colleagues have examined the causal role of the intrinsic apoptotic death pathway as a mechanism of silica-induced apoptosis. Using a macrophage cell culture model, the work demonstrates that -quartz silica directly impacts cell survival and induces apoptotic cell death (i.e., increases the number of cells with subdiploid DNA content). There is an obvious limitation with this in vitro model; the MH-S alveolar macrophage cell line used in the study is by its very nature continuously in cell cycle; whereas, the majority of macrophages in vivo are quiescent. This begs the question as to whether the apoptosis inducing effects of silica on MH-S cells are simply linked to cell cycle dysregulation. Although not quantified in the paper, it does not appear that the cycling cell compartment (S plus G2/M cells) is affected by silica, but there is a rapid (i.e., by 6 hours) and pronounced decrease in the frequency of G1 cells concomitant with the appearance of apoptotic events. Although not proven, the data suggest that it is highly likely that the apoptotic cells are arising from the G1 compartment. Thus, arguably the MH-S cells targeted to undergo silica-induced apoptosis are comparable to alveolar macrophagesat least with respect to their cell cycle properties. It is somewhat troubling that a caspase inhibitor prevents the appearance of subdiploid events without restoring the G1 peak, but inhibitor approaches generally speaking are wrought with difficulties. All caveats aside, the work importantly demonstrates that silica directly induces caspase-dependent apoptosis in macrophage cells. This observation raises questions concerning the mechanism whereby apoptosis is induced by silica and the potential role of apoptosis in silica-induced lung injury.
The mechanism whereby silica induces apoptosis is addressed further in the paper. In addition to the caspase inhibitor approach, the paper very nicely demonstrates that CPP32 family or caspase-3-like caspases are activated in a concentration-dependent and time-dependent fashion by silica. Both fluorogenic substrate-based enzyme activity assays and routine immunoblotting approaches are used in a series of carefully performed experiments to implicate caspase-3 activation in response to silica treatment. As predicted by the caspase inhibitor experiments, active caspase-3 cleavage products appear prior to subdiploid cells, which aids in establishing a causal role for caspase-3 in the execution of the cells. Activation of the caspase executioner is yet another piece of evidence implicating apoptosis as the mechanism underlying silica-induced cell death. Data in the paper strongly support that stimulation of the intrinsic apoptotic pathway by silica is the mechanism underlying caspase-3 activation and cell death. Again through an inhibitor approach (i.e., the selective caspase-9 inhibitor, Z-LEHD-FMK) and direct measurement of caspase-9 cleavage products by immunoblotting, the data very nicely establish a role for caspase-9 in silica-induced apoptosis and correlate the caspase-9 activation with caspase-3 activation. These are rather straightforward results and highly predictive that caspase-9 would be involved in an apoptotic pathway elicited by an agent (i.e., silica) that purportedly acts via generation of reactive oxygen species (Mossman and Churg, 1998). Nevertheless, the manuscript firmly establishes that caspase-9 is activated upstream of caspase-3, whereas caspase-8, a mediator of the extrinsic apoptotic pathway, is not. As the authors point out, this is an important difference between their work and others, where silica has been reported to initiate apoptosis via a death receptor-mediated pathway (Borges et al., 2002
).
As mentioned above, activation of caspase-9 is typically the result of stimulation of the intrinsic or mitochondrial pathway of apoptotic cell death a characteristic of which is a decrease in the inner mitochondrial transmembrane potential, m. Treatment of MH-S macrophages with silica elicits measurable and marked changes in
m, and partial inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A, a known inhibitor of the MPT pore, prevents activation of caspase-9 and caspase-3 in response to silica treatment. Again, it would have been surprising had this not been the case. Nevertheless, these data further underscore that its the intrinsic caspase-9 dependent pathway that is activated by silica in these cells.
The final piece of data in the manuscript by Thibodeau et al. raises the interesting possibility that silica-induced injury to the endolysosomal compartment leads to mitochondrial damage, and as such is a mechanism contributing to the activation of the intrinsic apoptotic pathway. Unlike the rest of the manuscript where multiple approaches were taken to establish the signaling pathway triggered by silica treatment, the potential role of lysosomal proteases in activating m, caspase-9 and caspase-3 is approached more cursorily. That is, there are no direct measures of silica on lysosomal proteases (e.g., cathepsins) or lysosomal injury; the implication of the lysosomal pathway relies soley on the use of purported lysosomal protease inhibitors. Nevertheless, as a first approach the data suggest that cathepsin D is involved in activating the MPT and subsequent downstream activation of caspase-9 and caspase-3. Recent work by Reiners et al. indicates that activation of the intrinsic apoptotic pathway following lysosomal photodamage involves conversion of Bid to tBid (Reiners et al., 2002
), a proapoptotic member of the Bcl-2 supergene family. The lysosomal protease(s) responsible for Bid cleavage in this model is not known, but it does not appear to be cathepsins B, D, or L. Essentially, a question raised by the findings of Thibodeau et al. is what is the mechanism linking lysosomal injury to the intrinsic apoptotic pathwayit may or may not involve cathepsins. At face value, it likely does not involve cleavage of Bid, as the cytochrome c release and downstream activation of caspase-9 that is triggered by tBid occurs in the absence of a loss of
m, and cannot be suppressed by cyclosporin A or other inhibitors of the MPT.
Thibodeau and colleagues include a diagram in their paper showing a proposed mechanism of silica-induced caspase activation and apoptosis. Data in their paper strongly support that the intrinsic, caspase-9-mediated, mitochondrial apoptotic pathway is activated following silica treatment. These findings raise important questions as to the mechanism(s) whereby silica produces mitochondrial injury and/or lysosomal injury in the first place. Perhaps the oxidative burst following phagocytosis and intracellular compartmentalization of silica particles in the endolysosomes leads to the generation of reactive oxygen species that activate lysosomal proteases and subsequently the intrinsic program. With such a scenario in mind, the diagram proposed by Thibodeau and colleagues should perhaps be weighted toward endolysosomal injury as the apoptotic initiator. Given the framework established by the current work, additional studies aimed at determining the mechanism of signal initiation are warranted.
Ultimately, one must consider the biological significance of silica-induced apoptosis in the context of silica-induced lung injury, which is characterized by pulmonary inflammation and inflammatory lesions. On the surface, it might be expected that apoptosis would act to counter an inflammatory process, and in fact, some have proposed that silica-induced apoptosis occurs during the resolution of the inflammatory response (Shen et al., 2001). Others have implicated silica-induced apoptosis more causally in the inflammatory response occuring in the lungs of silica-treated mice (Borges et al., 2002
). The present work by Thibodeau et al. is limited in addressing the consequences of silica-induced apoptosis to lung inflammationits an in vitro model aimed at examining the intracellular mechanisms of silica action. Despite this limitation, given the primary role of alveolar macrophages as a first line of defense against respirable particles including silica, its tempting to speculate that silica-induced apoptosis of the alveolar macrophages could potentially favor a proinflammatory state. That is, given the role of alveolar macrophages in removing other injured cells, their deletion may be detrimental to the anti-inflammatory response.
NOTES
1 To whom correspondence should be addressed: Fax: 585-256-2591. E-mail: michael_mccabe{at}urmc.rochester.edu.
REFERENCES
Borges, V. M., Lopes, M. F., Falcao, H., Leite-Junior, J. H., Rocco, P. R., Davidson, W. F., Linden, R., Zin, W. A., and DosReis, G. A. (2002). Apoptosis underlies immunopathogenic mechanisms in acute silicosis. Am. J. Respir. Cell Mol. Biol. 27, 7884.
Mossman, B. T. and Churg, A. (1998). Mechanisms in the pathogenesis of asbestosis and silicosis. Am. J. Respir. Crit Care Med. 157, 16661680.[ISI][Medline]
Reiners, J. J., Jr., Caruso, J. A., Mathieu, P., Chelladurai, B., Yin, X. M., and Kessel, D. (2002). Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage. Cell Death. Differ. 9, 934944.[CrossRef][ISI][Medline]
Rosenman, K. D., Reilly, M. J., and Henneberger, P. K. (2003). Estimating the total number of newly-recognized silicosis cases in the United States. Am. J. Ind. Med. 44, 141147.[CrossRef][ISI][Medline]
Shen, H. M., Zhang, Z., Zhang, Q. F., and Ong, C. N. (2001). Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages. Am. J. Physiol Lung Cell Mol. Physiol 280, L10L17.