PERSPECTIVE
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Not all of the effects listed in Table 1 are generally accepted, and in some cases the initial report has not been confirmed by subsequent studies. For instance, the defective acidification hypothesis was highly appealing in that it could explain the pleiotropic effects of CF (10). According to the hypothesis, failure of CFTR in intracellular vesicles led to decreased acidification of their contents. This would clearly affect the posttranslational processing and trafficking of many proteins. But several later studies failed to show differences in intravesicular pH in CF (11).
The key questions to the many proposed actions of CFTR are (1) whether the methods are adequate and (2) do the results have any relevance to the function of affected human epithelia in vivo. Specific methodologic problems include random variation among cell lines, use of nonpolarized cells to draw conclusions about epithelial function, overexpression of CFTR, inadequacies in the transgenic mouse model of CF, and use of nonspecific pharmacologic agents.
Many studies have compared CF and non-CF cell lines, most of which are poor models of native epithelium because they do not form tight junctions and polarize. Realizing this, many workers supplement their results on cell lines with data from primary cell cultures. These can very closely resemble the native epithelium in both their structure and function (12), and CF and non-CF cultures can be closely matched for cell number, structure, transepithelial resistance, and other parameters. However, the primary cultures used are generally very poorly characterized. At minimum, investigators should check whether their primary cultures are polarized, but even this simple assay (for transepithelial resistance) is rarely performed.
Transfection of cell lines and expression of exogenous CFTR is also commonly used. The most sophisticated application of this approach involves the "recovery" of CF cell lines by expression of wild-type CFTR. The problem, however, with expression experiments is that the expressed CFTR is usually present at levels far greater than found in native epithelia. Excess of any ion channel in the membrane might be expected to have multiple effects on function. It is also possible that CFTR trafficking pathways may become saturated and then CFTR trafficked to unusual locations. In retinal pigment epithelium, for instance, CFTR is normally trafficked to the apical membrane, but exogenously expressed CFTR appears in the basolateral (S. Miller, personal communication). Similarly, in rat Fischer thyroid cells, the relative amounts of exogenous CFTR passing to the apical and basolateral membranes depend on the level of expression (13). Furthermore, if trafficking pathways are overwhelmed with CFTR, then trafficking of proteins other than CFTR may be affected. To control for the effects of overexpression, it is usual to transfect control cells with an irrelevant reporter gene such as lacZ. Yet this is not an entirely adequate control because the reporter gene is usually not an integral membrane protein and uses trafficking pathways different from CFTR.
The CF mouse is a powerful investigational tool (14), but it must be remembered that mouse airways and their epithelia differ greatly from those of humans. Mouse airway epithelium has comparatively large numbers of Ca- activated chloride channels (15) and lacks mucous glands and goblet cells (16). In fact, ion transport by the tracheal epithelium of CF transgenic mice is indistinguishable from wild-type mice (17), and most CF mice show barely any airway pathology (14).
Another approach to test for involvement of CFTR in a particular cell function is to use blockers. However, there are no good blockers for this channel; all must be used at high concentrations and have nonspecific actions (18, 19).
At their worst, the studies above involve merely overexpressing CFTR in a nonpolarizing cell type. It is hardly surprising that so many functions have been reported as "CFTR-dependent." In fact, if the levels of CFTR expression are high enough, cell viability itself is reduced (20).
In the current edition of the American Journal of Respiratory Cell and Molecular Biology, a new role for CFTR is proposed in the article titled "Cystic Fibrosis Transmembrane Conductance Regulator-Dependent Regulation of Epithelial Inducible Nitric Oxide Synthase 2 Expression" (21).
Nitric oxide (NO) is produced by many different cell types of human airways, including the epithelium (22). NO is formed by nitric oxide synthase (NOS), of which there are several forms (23). In airway epithelia, the predominant form is iNOS (inducible NOS), otherwise known as NOS2. This enzyme can be induced by a variety of inflammatory agents, and epithelial-derived NO may influence airway disease by its antimicrobial activity and by effects on ion transport and, therefore, mucociliary clearance (24). Levels of exhaled NO are markedly elevated in asthma and viral infections of the upper respiratory tract, possibly by proinflammatory cytokines released from lymphocytes (22). Conversely, epithelium-derived NO may modulate the immune response and airway cytokine balance (23). Despite this association of NO with airway inflammation in some diseases, there are recent reports that levels of NOS in airway epithelium are reduced in both human and murine CF (24, 25), as are levels of NO in exhaled gases (26). These results have led to the proposal that NO is a defense against inflammation, and that the inability to increase NO production in CF airways predisposes them to infection (25).
The purpose of the present investigation was to determine if it is the absence of functional CFTR that causes decreases in NOS in CF airway epithelia. The first set of experiments used a nonpolarizing airway epithelial cell line (27), stably transfected with the R-domain of CFTR, a maneuver that blocks CFTR chloride channel function while leaving CFTR messenger RNA (mRNA) levels unaltered (28). Cells expressing the R-domain showed a dramatic reduction in mRNA for NOS2 compared with mock-transfected control cells. Reflecting the change in NOS2 mRNA, lipopolysaccharides did not increase NO production in cells expressing R-domain, but increased it four-fold over baseline in the control cells. Puzzlingly, the authors neglected to give the values for baseline NO production; one hopes it was higher in the mock-transfected cells. The major criticism of the above experiments is that cell lines stably expressing a foreign protein will experience different selection pressures than nontransfected cells, and with successive passages these cell lines may come to differ from the parent cell line in many ways only distantly related to the presence or absence of functional CFTR.
Transgenic mice were used in the second set of experiments. In addition to wild-type mice, the authors used CF knockout mice and CF knockout mice expressing human CFTR hooked to an intestinal promoter (FABP). Thus, there were three different types of mice. One had no CFTR in either the nasal or ileal epithelia. A second had functioning human CFTR in the ileum, but not in the nasal epithelium. The third type had murine CFTR in both tissues. To test for CFTR in the nasal epithelium of these mice, the nasal surface was superfused with chloride-free medium containing forskolin (an agent that elevates cAMP). If CFTR is present, it is opened by the elevation in cAMP, and the chloride gradient from cytosol to lumen across the apical membrane results in the potential difference across the nasal epithelium becoming more lumen- negative. In wild-type mice, the combination of forskolin and chloride-free solution caused the airway lumen to become more negative by 14 mV. By contrast, the same maneuver shifted the nasal potential difference by 5 to 6 mV in the opposite (i.e., positive) direction in both kinds of CF mice. Ussing chamber studies showed CFTR activity in the ilea of wild-type and FABP-hCFTR mice, but not in the knock-out mouse. Immunocytochemical staining of nasal and ileal epithelia for NOS2 showed a strict dependence on the presence of CFTR. NOS2 was found in the intestine of the FABP-hCFTR mouse and in both the nose and ileum of wild-type, but not in the nose of the FABP-hCFTR mouse or either tissue of CF knockout mice. The results from this study are summarized in Table 2.
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Steagall and associates' results support their hypothesis that the expression of NOS2 depends on the presence of functional CFTR. But how surprising is this? There are really only three basic possibilities as to how CF could induce a change in function: lack of functional CFTR, presence of mutant CFTR, or some secondary consequence of the disease's pathology. The possibility that the decrease in NOS in native epithelium is secondary to pathology is made unlikely by earlier findings that show that NOS2 expression is reduced in both mouse and human airways; the mouse shows trivial airway inflammation and pathology. The authors further exclude this possibility by showing that NOS2 is reduced by the presence of functional CFTR in cultured cell lines. The possibility that the CF-related effects on NOS expression were due to the presence of mutant CFTR is also easy to exclude: the mice used in these studies and earlier immunocytochemical work were null mice devoid of any murine CFTR.
The conclusion that functional CFTR enhances NOS expression was to be expected from the previous cytochemical studies. Nevertheless, the present work helps reinforce the important conclusion that NO release by airway epithelium is reduced in CF. The resulting reduction in antimicrobial capability may contribute to the characteristic colonization of airways by bacteria in this disease (29). Also, reduced NO levels may change ion transport across airway epithelium (24). This, in turn, could alter the volume and composition of the airway surface liquid in such a way as to promote further bacterial colonization.
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Footnotes |
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Address correspondence to: J. H. Widdicombe, D. Phil., University of California San Francisco, Cardiovascular Research Institute, 3rd and Parnassus Avenues, Box 0130, San Francisco, CA 94143-0130.
(Received in original form October 5, 1999).
Abbreviations: cyclic adenosine monophosphate, cAMP; cystic fibrosis, CF; cyclic fibrosis transmembrane conductance regulator, CFTR; inducible nitric oxide synthase, iNOS (or NOS2); fatty acid binding protein, FABP; nitric oxide, NO.| |
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