© 2003 American Thoracic Society
Cell Suffering and Its Prevention in LungDepartment of Cellular Biology and Anatomy and Institute for Molecular Medicine and Genetics Medical College of Georgia Augusta, Georgia Many organs in the human body generate and/or are exposed to considerable levels of mechanical stress. Medical interventions, such as mechanical ventilation of the lung, can extend such stress levels beyond what is physiological. What is the nature of the damage inflicted by mechanical stress and how can this damage, including sublethal damage to cells, be detected? Frank damage resulting in disruption of normal tissue architecture can, obviously, be assessed by conventional histologic means. More subtle cell level injuries, are also induced by mechanical stress both within and beyond the physiologic range (1). Most pertinent to this editorial are cell plasma membrane disruptions. Detection of the full extent of this type of cell injury requires that a probe for membrane integrity be employed. This is because many cells can repair or reseal plasma membrane disruptions and thereby survive them, leaving no obvious trace in conventional histology of the injury. The probe chosen for detecting "cell wounding" must be membrane impermeant, readily detected microscopically, and, practically speaking, should be as small (to maximize entry through a disruption) and as inexpensive as possible. Entry of the probe through a disruption, and its subsequent trapping in cytosol after membrane resealing, then marks a cell as wounded. In this issue of AJRCCM (pp. 1057–1063), Gajic and colleagues (2) describe the use of propidium iodide as a cell wound marker. Its small size and the fact that it fluoresces most strongly when bound to components of the cell nucleus make it a nearly ideal cell-wounding probe and therefore a welcome addition to our experimental arsenal. Using this wounding probe, Gajic and coworkers (2) show that certain clinically relevant conditions of mechanical ventilation of the lung can dramatically increase the incidence of temporary, survivable plasma membrane disruption in cells of the alveolar epithelium. A striking array of fluorescent nuclei (probably of alveolar Type I cells) set against a dark background appear in the alveolar epithelium after ventilation at high VT and zero end-expiratory pressure. The clarity of these images is probably attributable to prodium iodide's tendency to fluoresce strongly when it binds an intracellular component (in the nucleus) and not in the extracellular background. These images are demonstrated to be accurately representative by rigorous (blinded) quantification of the number of wounded cells as a function of ventilation conditions. Were these propidium iodide–labeled cells dead or alive? By exposing the lung to propidium iodide after ventilation and quantitating the number labeled under this condition, it could be shown that the disruptions occurring during ventilation were predominantly not lethal: the number of propidium iodide–positive cells labeled by probe addition after was strikingly decreased compared with probe addition during ventilation. Dead cells are incapable of maintaining plasma membrane barrier function and so would be detected equally effectively whether the prodium iodide is given during or after the mechanical insult.
What is the biological or clinical relevance of these observations? Speculation only is possible at this point. Cell wounding could initiate strain-related remodeling of the blood–gas barrier. Indeed, such remodeling, as revealed by conventional histologic analysis and changes in lung weight, is demonstrated by Gajic and coworkers (2) to correlate well with a quantitative index of cell wounding. Plasma membrane disruption can be viewed as a mode of mechotransduction. For example, gene expression, including that for proinflammatory mediators such as nuclear factor What design elements protect cells from damage due to these stresses? There are, of course, organ-level adaptations, such as the lumenal folding characteristic of many hollow organs that cyclically fill and empty. Unfolding prevents undue stress during the filling phase. But, as is becoming clear from work such as that being done by Hubmayr's group, there are also important cell and molecular level adaptations. Lung epithelial cells are not merely passive recipients of stretch-induced injury. They apparently can sense the imposition of stress and react by increasing their surface area. They could thereby reduce the level of tension produced by subsequent stretch insults. Thus, Hubmayer's group previously showed that when an epithelial cell line, cultured in vitro on an elastic substratum and irreversibly labeled in the surface plasma membrane with the fluorescent lipid, BIODIPY-FL, are stretched, this probe becomes diluted in the surface membrane (5). The most obvious explanation of this stretch-induced dilution was that an internal source of membrane, lacking the fluorescent probe, was being added to the plasma membrane by exocytotic fusion events. In fact, stretch-induced exocytosis in lung epithelial cells could be demonstrated using another lipdic dye, FM 1-43, widely employed for documenting synaptic exocytotic events. Nonspecific methods of blocking this stretch-induced exocytosis resulted in a higher level of plasma membrane disruption for a given level of stretch, consistent with the idea that the new membrane addition was protective. Unanswered questions include the nature of the signal for exocytosis. This signal appears not to be sublethal plasma membrane disruption, which is documented to induce exocytosis (6), because only a minority of the cells were wounded under conditions in which a majority increased their surface areas. This stretch-induced increase in lung epithelial cell surface area is reminiscent of a perhaps better-characterized response of the bladder umbrella epithelial cell to stretch. The surface plasma membrane of these cells is sharply folded: plaques, composed of densely packed hexagonal arrays of the protein uroplakin alternate with hinge regions (more typical of plasma membrane structure). These folds disappear when the umbrella cell is stretched by bladder filling. Moreover, additional plaques are harbored just below the apical surface in a specialized vesicular compartment, the discoidal vesicles. When the umbrella cell is stretched for a prolonged interval, excytotic fusion of the discoidal vesicles with the apical plasma membrane increase its surface area, protecting it, as is hypothesized for the alveolar epithelial cell, from stretch-induced damage (7). Does the lung epithelial cell possess similar specializations? A recent article suggests that many cell types contain a previously unrecognized organelle, the "enlargesome" (8). This organelle is exocytosed on an increase in intracellular calcium elevation, including that which occurs on cell wounding. Its proposed function is the addition of new membranes to the cell surface, as is required for resealing and perhaps for protective responses, such as those exhibited by the lung epithelial cell. Perhaps there are a plethora of cell adaptations waiting to be discovered that either participate in the process of plasma membrane repair or prevent its occurrence, and the lung epithelial cell appears to be an excellent choice for further study. REFERENCES
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B, is known to be potently induced in the wounded cell itself, probably as a consequence of the signal generated by entry of calcium through a disruption (
