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Effect of Inflammatory Stimuli on Airway Ion Transport

Laboratory of Molecular Genetics, Istituto Giannina Gaslini, Genoa, Italy

Correspondence and requests for reprints should be addressed to Luis J. V. Galietta, Ph.D., Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Largo Gerolamo Gaslini, 5, 16148 Genoa, Italy. E-mail: galietta{at}unige.it

	ABSTRACT

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 
The airway epithelium controls the chemical and physical properties of airway surface fluid and consequently mucociliary clearance. The treatment for 24–48 hours of human bronchial epithelial cells with interferon- or interleukin-4 leads to marked changes in transepithelial ion transport properties. Both cytokines downregulate the activity of the epithelial Na⁺ channel and, at the same time, upregulate Ca²⁺-dependent Cl^- secretion. Interleukin-4 also increases the expression and function of the cystic fibrosis transmembrane conductance regulator Cl^- channel. These results suggest that some inflammatory stimuli may change the balance between fluid absorption and secretion to favor hydration of the airway surface and consequently mucus clearance.

Key Words: airway epithelium • interferon- • interleukin-4 • ion transport • mucociliary clearance

The airway surface is covered by a fluid, the airway surface liquid (ASL), interposed between the mucous layer and the epithelium. The ASL has chemical and physical properties that are essential in mucociliary clearance and in the defense against pathogens. Furthermore, the ASL contains proteins, secreted by different cell types, that may have pro-/antiinflammatory or bactericidal functions. The airway epithelium controls the thickness and ion composition of ASL through the activity of ion channels and transporters (1). A significant fraction of ASL is generated in the lung periphery and moved toward the mouth via the action of cilia. Therefore, absorption is essential to prevent the airways from being overwhelmed as fluid moves from the peripheral airways to the trachea. The amiloride-sensitive epithelial Na⁺ channel (ENaC) is essential in this process. Indeed, knockout mice with abolished ENaC function die at birth because of excess fluid in the airways (2). ASL is also controlled by activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl^- channel of the apical membrane of epithelial cells. Besides being responsible for Cl^- secretion, CFTR also regulates ENaC activity. Indeed, impairment of CFTR function in cystic fibrosis leads to Na⁺ and fluid hyperabsorption and consequently ASL depletion (3, 4). These studies suggest that the ASL volume is controlled by a delicate balance between absorption and secretion. This mechanism is important to ensure proper mucus hydration, cilia beating, and mucus clearance. We hypothesized that ion transport properties of the airway epithelium may change as a way to respond to injuring agents and proinflammatory conditions. During normal ventilation about 20,000 L of air per day pass in and out of the respiratory system. Air contains particulate material, such as pollen, ash, mineral dust, mold spores, bacteria, and viruses, all of which are capable of damaging the lungs either directly or by predisposing the lung to infection. The airways and the lung must be able to handle this load, repair any damage to cells resulting from exposure to these materials, and clear inhaled infectious particles.

To elucidate the role of proinflammatory stimuli in airway ion transport we tested the effect of cytokines on polarized preparations of human bronchial epithelial cells.

	EFFECT OF IFN- ON TRANSEPITHELIAL ION TRANPORT

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 
Previous studies had shown that IFN- and tumor necrosis factor- (TNF-) may affect expression of the CFTR gene and therefore the extent of Cl^- secretion in intestinal cells (5, 6). We asked whether such cytokines were also able to change CFTR expression in cultured human airway epithelial cells. Short-circuit current measurements of polarized bronchial epithelia revealed that IFN- decreased CFTR-dependent Cl^- secretion by more than 60% (7) at 48 hours of treatment. The same treatment markedly reduced the amount of ENaC-dependent short-circuit current. On the other hand, similar long-term treatment with TNF- did not change CFTR or ENaC activity. It has been reported that ENaC is inhibited by nitric oxide (8). Therefore, we considered whether the IFN- effects were mediated by upregulation of the inducible form of the nitric oxide synthetase, an enzyme that is induced by inflammatory cytokines. Several lines of evidence ruled out this hypothesis (7). First, nitrite levels in cell supernatants and direct measurements of nitric oxide synthetase activity in cell lysates were not increased by IFN-. Second, ENaC- and CFTR-dependent ion transport were not affected by treatment with nitric oxide donors. Finally, the effects of IFN- could not be prevented by preincubation with nitric oxide synthetase inhibitors.

Besides CFTR, which is regulated by cAMP, other Cl^- channels are also involved in epithelial anion secretion. These channels, termed Ca²⁺-dependent Cl^- channels (CaCC), are activated by an increase in cytosolic free Ca²⁺ concentration. Interestingly, we found that IFN- treatment markedly upregulated the response to apical application of uridine triphosphate (UTP) (7), a nucleotide that interacts with purinergic receptors, thus eliciting an intracellular Ca²⁺ spike and CaCC activation. We performed a series of experiments to try to elucidate the mechanisms underlying CaCC upregulation by IFN-. This cytokine could act at different levels in the signaling cascade downstream of the purinergic receptors. It could upregulate the receptors themselves or increase the ability of cells to mobilize intracellular Ca²⁺. Alternatively, it could indirectly affect anion secretion by upregulating basolateral K⁺ channels, which are required to provide the driving force for apical Cl^- secretion. Finally, IFN- could directly increase CaCC gene expression. Our findings suggest that the latter is the simplest explanation. Indeed, stimulation with ionomycin, an ionophore that directly increases intracellular Ca²⁺ without the intervention of plasma membrane receptors, still resulted in a higher response in cells treated with IFN- (7). Furthermore, we measured intracellular free Ca²⁺ concentration with the Fura-2 fluorescent probe. We found that UTP, as expected, elicited a cytosolic free Ca²⁺ increase characterized by a peak in the first seconds of stimulation followed by a slow decay to resting levels (7). This kind of response was unaltered in cells treated with IFN-. Such experiments demonstrated that upregulation of Ca²⁺-dependent Cl^- secretion by IFN- is not due to changes in intracellular Ca²⁺ homeostasis. Finally, to investigate the involvement of basolateral channels, we permeabilized the basolateral membrane with amphotericin B, an ionophore that largely increases the cation permeability of the membrane to which it is applied. Under this condition, the effect of basolateral K⁺ channels and other transporters on the Cl^- driving force is abolished. Using permeabilized epithelia, we found that Cl^- transport induced by UTP was still greater in IFN-–treated cells with respect to control (7), thus indicating that the effect was not mediated by changes in basolateral channel activity but rather to a more direct upregulation of CaCC activity/expression. Although we have not tested other physiological agonists besides UTP, our results suggest that IFN- may upregulate the response to other Ca²⁺-elevating agents (e.g., bradykinin and histamine).

Interestingly, the transepithelial resistance was markedly increased by IFN- treatment (7). This finding seemed to rule out any toxic effect of this cytokine on epithelial integrity.

To evaluate the net effect of IFN- on transepithelial ion transport, we have measured fluid transport in polarized bronchial epithelia over a 24-hour interval (7). Under resting conditions, these cells removed fluid from the apical side at a rate of about 15 µl · cm^–2 · 24 h^–1. Incubation with IFN- markedly reduced fluid absorption to a level close to that measured in the presence of amiloride. This finding, which is in agreement with the inhibition of ENaC activity detected in short-circuit experiments, suggests that IFN- may favor hydration of the airway surface.

	IL-4 FAVORS CHLORIDE ION SECRETION

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 
Our results with IFN- led us to test other cytokines and in particular IL-4, which plays an important role in lung pathology. IL-4 is involved in the helper T cell Type 2 immune response and is increased in the airways of subjects with asthma (9, 10). The role of IL-4 in asthma pathogenesis is also demonstrated by the finding that mice without critical elements in the IL-4 signaling pathway lack features of asthma, like airway hyperresponsiveness and mucus hypersecretion (11, 12). We incubated bronchial epithelial cells with IL-4 and measured ion transport properties. As shown in Figure 1A, ENaC activity was markedly inhibited by this cytokine (13). The effect was quite fast, already significant at 6 hours and maximal at 24 hours. In contrast with IFN-, IL-4 induced a striking increase in CFTR activity (13). Indeed, the current induced by cAMP was more than doubled by treatment for at least 24 hours with this cytokine. To confirm that this effect was indeed involving CFTR, we studied bronchial cells from patients with cystic fibrosis. The current induced by cAMP was small even after treatment with IL-4, thus excluding the intervention of another type of cAMP-dependent channel.

View larger version (22K): [in this window] [in a new window]   Figure 1. Epithelial sodium channel (ENaC) downregulation by proinflammatory stimuli. (A) Representative short-circuit current recordings showing the effect of amiloride application on human bronchial bronchial cells treated with and without interleukin-4 (IL-4). The amiloride-sensitive current, that is, the extent of ENaC activity, is decreased by IL-4. (B) Determination of ENaC subunit expression by semiquantitative RT-PCR (taken with permission from Reference 13; Copyright 2002. The American Association of Immunologists, Inc.). The messenger RNA (mRNA) levels are normalized with respect to control. Each value is the mean of three or four determinations. The asterisks indicate a significant (p < 0.05) decrease with respect to control. IL-4, but not IFN-, decreases the subunit and, to a lesser extent, the ß subunit.

Interestingly, the effects of IL-4 on ENaC, CFTR, and CaCC could be mimicked by stimulating the bronchial epithelial cells with IL-13 (13), a cytokine that largely overlaps the biological functions of IL-4 because they both share a plasma membrane receptor, the chain of the interleukin 4 receptor (IL-4R) (12, 14). By using a specific antagonist, we confirmed that IL-4 was indeed changing the ion transport by acting on the IL-4R (13).

Other authors have also found that IL-13 and IL-4 induce a hypersecretory ion transport state in human bronchial epithelial cells (15). In particular, these authors have also concluded that the upregulation of Ca²⁺-dependent Cl^- secretion by these cytokines is not due to changes in basolateral K⁺ conductance or Ca²⁺ homeostasis but probably to a more direct effect on CaCC activity.

	DETERMINATION OF ENaC AND CFTR EXPRESSION

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 
We investigated the molecular basis of the changes elicited by inflammatory cytokines on airway ion transport. Semiquantitative reverse transcription-polymerase chain reaction was performed to assess the expression of the different ENaC proteins (Figure 1B). This channel is formed by at least three subunits, , ß, and , encoded by separate genes (16). We found that IFN- did not change the messenger RNA (mRNA) levels of the three subunits despite the decrease in ENaC activity measured in short-circuit current experiments and the inhibition of fluid absorption. It is therefore possible that IFN- downregulates Na⁺ absorption by a different, possibly posttranslational, mechanism. Indeed, ENaC activity is controlled in different ways such as ubiquitination by Nedd4 (17), carboxylmethylation (18), and proteolysis (19, 20).

In contrast to IFN-, IL-4 had a profound effect on ENaC mRNA levels (13). In particular, ENaC expression was almost abolished by IL-4 treatment. ßENaC mRNA levels were partially reduced whereas ENaC expression was unaltered. The selective effect on the subunit is in agreement with the finding that treatment with maximal concentrations of IL-4 does not inhibit ENaC activity completely (13). Indeed, it has been shown that the ENaC subunits and ß alone may form a functional Na⁺ channel although with reduced plasma membrane activity (16).

We have also measured CFTR expression by quantitative reverse transcription-polymerase chain reaction. In agreement with functional data obtained from short-circuit current experiments, we found that CFTR mRNA levels were downregulated and upregulated by IFN- and IL-4, respectively (13). These findings indicate that such cytokines can modulate CFTR expression either by changing the rate of gene transcription or by affecting mRNA stability. We have also included TNF- in this analysis. Surprisingly, this inflammatory cytokine caused a dramatic 10-fold increase in CFTR mRNA, which contrasts with the lack of effect at the functional level (13). Such a discrepancy had already been observed in our previous study (7), in which TNF- had no effect on CFTR currents but increased strikingly the levels of CFTR protein. This phenomenon cannot be explained at the moment. We can speculate that the increase in CFTR expression caused by TNF- is not followed by increased activity either because the excess protein is not delivered to the plasma membrane or because the protein is in part not functional. It is possible that TNF-, besides upregulating CFTR expression, is also eliciting posttranscriptional (e.g., alternative splicing) or posttranslational mechanisms that limit CFTR activity.

	HYPOTHESES AND PERSPECTIVES

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 
Various reports indicate now that airway ion transport is affected by proinflammatory stimuli (7, 13, 15, 21–23). Although these studies have been performed only in vitro, it seems reasonable to assume that similar phenomena may occur in vivo. However, the actual response to inflammatory stimuli in the airways is certainly complicated by the simultaneous presence of various types of cytokines and other soluble mediators that could have synergistic or antagonistic effects. Signaling arising from cell–cell interactions (e.g., contact between activated leukocytes and epithelial cells) may add another level of complexity to the mechanisms that regulate ion transport.

Several stimuli seem to induce a reduction in ENaC activity, thus inhibiting fluid absorption (7, 13, 15). This response could be beneficial to increase the fluidity of the mucus that is hypersecreted under pathologic conditions and to clear pathogens and particulate material from the airway surface (Figure 2). For instance, IL-4/IL-13 elicit goblet cell metaplasia and mucus hypersecretion in the airways (12, 24, 25). The accompanying increase in ASL volume resulting from ENaC inhibition would be required to provide adequate hydration of mucous layer.

View larger version (49K): [in this window] [in a new window]   Figure 2. Hypersecretory state induced by inflammatory stimuli in airway epithelium. The schematic summarizes the effects of cytokines on net ion and fluid transport. The apical membrane contains Na+ channels (ENaC), cAMP-dependent Cl- channels (CFTR), and Ca2+-activated Cl- channels (CaCC). The downregulation of ENaC and the upregulation of CFTR and CaCC may favor anion secretion with respect to Na+ absorption. This may in turn increase fluid secretion and hydration of the mucus layer (ML) overlying the airway surface liquid (ASL). The relative thickness of the two layers is not drawn to scale.

The depth and composition of the airway surface liquid (ASL) depend also on secretion from the submucosal glands. It would be interesting to assess whether bronchial gland fluid secretion is also modulated by inflammatory conditions. It is reasonable to speculate that the presence of pathogens on the airway surface may elicit a hypersecretory state in the glands by the release of cytokines and other soluble mediators from the surface epithelium and leukocytes. In this respect, it has been reported that CFTR expression is upregulated by interleukin-1ß (IL-1ß) in Calu-3 (21), an epithelial cell line that is representative of submucosal gland cells. This kind of response could be useful to increase fluid secretion to flush and deliver to the airway surface mucus and bactericidal molecules (e.g., lactoferrin, lysozyme, immunoglobulins, and ß-defensins) that are produced by glands under pathologic conditions.

These findings also stress the importance that the CFTR may have under inflammatory conditions in individuals who are not affected by cystic fibrosis. We can speculate that the downregulation of CFTR expression (which may happen in response to IFN- or other unknown stimuli) or inadequate CFTR upregulation may cause a transient and local defect in Cl^- secretion resembling the cystic fibrosis phenotype. At present there is no clear evidence that genetic alterations in CFTR function, besides being responsible for cystic fibrosis, are also a factor of risk for more frequent lung pathologies (26). However, it is possible that drugs able to increase CFTR activity may be beneficial to ameliorate the mucociliary clearance in chronic obstructive pulmonary disease and other related disorders (27).

(Received in original form June 19, 2003; accepted in final form October 6, 2003)

	REFERENCES

TOP ABSTRACT EFFECT OF IFN-{gamma} ON... IL-4 FAVORS CHLORIDE ION... DETERMINATION OF ENaC AND... HYPOTHESES AND PERSPECTIVES REFERENCES

 

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