on Alveolar Epithelial Barrier Function
M.A. Matthay and J.-W. Lee
Introduction
There has been considerable progress in understanding how the alveolar epithe- lium regulates fluid balance under normal and pathologic conditions [1]. There is a growing understanding of the important role of the alveolar epithelium in regulating inflammatory responses as well as in responding to several pathologic stimuli. Progress has been made possible because of the availability of excellent an- imal models for in vivo studies as well as several in vitro models including studies of cultured human alveolar epithelial type II cells. This chapter will focus on how four different inflammatory molecules (tumor necrosis factor [TNF]- α , leukotriene D
4(LTD
4), interleukin [IL]-1 β , and transforming growth factor [TGF]- β ) can ex-
ert differential effects on the barrier and fluid transport capacity of the alveolar epithelium with a particular focus on their relevance to acute lung injury (ALI).
Normal Alveolar Epithelial Barrier
The normal alveolar epithelial barrier is very tight, resisting the passive movement of even small molecules and solutes such as electrolytes. Thus, the alveolar epithe- lium can be viewed as mostly impermeable to macromolecules such as proteins including albumin and immunoglobulins. Tight junctional proteins maintain this tight barrier between alveolar type I and type II epithelial cells [1].
In addition to these tight barrier properties, the alveolar epithelium has spe-
cialized functions that facilitate gas exchange. First, the alveolar epithelial type II
cell is the source of surface active material, which is necessary for the maintenance
of normal alveolar stability in the gas filled lung. Secondly, alveolar epithelial
type II cells, as well as alveolar epithelial type I cells, have the capacity to re-
move excess alveolar fluid by vectorial ion transport. Sodium is taken up by apical
ion channels and extruded actively by the basolateral Na/K-ATPase [1]. Chlo-
ride follows by unknown pathways under normal conditions and via the cystic
fibrosis transmembrane conductance regulator (CFTR) under cAMP stimulated
conditions [2, 3]. Water follows the mini-osmotic gradient produced by vectorial
transport of sodium and chloride into the interstitium and results in isomolar
alveolar fluid clearance. Both catecholamine-dependent and catecholamine inde-
pendent mechanisms can upregulate alveolar fluid clearance [2,4]. The best studied
mechanisms are cAMP-dependent fluid clearance [1], which also has relevance at the time of birth when endogenous catecholamines upregulate alveolar fluid clear- ance [5]. Endogenous release of catecholamines has the capacity to upregulate alveolar fluid clearance under pathologic conditions of severe shock when there is a large increase in plasma catecholamine levels [6]. Delivery of a beta 2-adrenergic agonist to the distal airspaces of the lung markedly increases the rate of alveolar fluid clearance in several species, including the human lung [1].
The Alveolar Epithelial Barrier to Pathologic Stimuli
Prior studies from our research group indicated that the alveolar epithelial bar- rier is remarkably resistant to injury. In these early studies, we instilled either autologus plasma or autologus serum into the distal airspaces of sheep. We found that large numbers of neutrophils and monocytes were chemoattracted to the alveoli, presumably secondary to release of chemotactic molecules from alveolar macrophages. The large numbers of inflammatory cells did not result in a change in alveolar epithelial permeability to protein [7]. Furthermore, fluid transport mechanisms were intact with a normal rate of alveolar fluid clearance.
A subsequent study in human volunteers was carried out to further assess the response of the alveolar epithelial to inflammatory stimuli [8]. Leukotriene B
4(LTB
4was instilled into the distal airspaces of human volunteers with a fiberoptic bron- choscope. The volunteers were lavaged subsequently at 4 and 24 hours in the LTB
4- instilled right middle lobe as well as in the contralateral control saline-instilled lingula. The results showed that large numbers of neutrophils were attracted to the alveoli, similar to the numbers of neutrophils lavaged from patients with acute respiratory distress syndrome (ARDS), but there was no increase in permeability to protein across the alveolar epithelium (Table 1). These results indicated that the epithelial barrier was resistant to injury and could permit the passage of inflam- matory response cells, neutrophils and monocytes, to chemotactic stimuli in the airspaces without injuring the alveolar epithelium.
A subsequent experimental study in our laboratory demonstrated that instilla- tion of Escherichia coli endotoxin into the distal airspaces of the sheep lung resulted in a large influx of neutrophils at both 4 hours and 24 hours in anesthetized as
Table 1. Bronchoalveolar lavage fluid cells and proteins after LTB4instillation in human lungs [8]
Variable NaCl LTB4 P value
Total Cells (106) 6.8± 1.0 26.4± 5.0 0.002
Neutrophils (%) 12.2± 4.6 55.7± 6.0 0.001
Macrophages (%) 82.7± 5.9 40.5± 6.1 0.001
Total Protein (mg) 15.4± 4.8 23.4± 3.5 NS
The data are the mean ± SE of data from 11 human subjects.
well as unanesthetized sheep. Again, similar to the studies with LTB
4, there was no change in alveolar epithelial permeability to protein in either the alveolar to the interstitial direction or from the vascular compartment to the airspaces. Also, the normal rate of alveolar fluid clearance was well preserved in these sheep studies over 24 hours [9].
When live bacteria were instilled into the airspaces, specifically Pseudomonas aeruginosa, there was evidence of a modest bidirectional increase in alveolar ep- ithelial permeability. This finding was evident at both 4 and 24 hours in studies in sheep. Further, the rate of alveolar fluid clearance was diminished by the presence of bacteria and the alteration in epithelial barrier permeability. Nevertheless, there still was measurable net alveolar fluid clearance, although the rate of clearance was reduced [9]. Follow-up studies demonstrated that several products of Pseu- domonas were responsible for the decrease in fluid clearance and the increase in lung epithelial permeability.
Several other studies in experimental animals using clinically relevant models of acute lung injury (ALI) demonstrated, as expected, that the alveolar epithelial barrier can be injured and that alveolar fluid clearance is reduced. For example, acid-induced lung injury, as a model of aspiration in humans, resulted in a reduc- tion in alveolar fluid clearance proportionate to the degree of alveolar epithelial injury. In one study in rabbits, we found that an anti-IL-8 monoclonal antibody reduced acid-induced lung injury by reducing neutrophil mediated injury [10].
Also, in a more recent study, treatment with a beta-2 adrenergic agonist upregu- lated alveolar fluid clearance and decreased lung endothelial permeability in rats with acid-induced lung injury [11].
Pro-inflammatory Molecules and Alveolar Epithelial Fluid Transport
We and other investigators have measured several pro-inflammatory mediators in the airspaces of patients with ALI as well as in animal models [12, 13]. The acute pro-inflammatory response is an important part of innate immunity that regulates neutrophil and monocyte influx designed to neutralize a variety of in- fectious agents and microbial products. The effects of some of these inflammatory molecules on alveolar epithelial function have resulted in several interesting effects.
For example, we discovered several years ago that TNF- α can markedly upreg- ulate the rate of alveolar fluid clearance in rats with Pseudomonas pneumonia [14].
Another group of investigators confirmed this finding with a different model of ischemia-reperfusion and shock in rats [15]. Finally, additional studies by our group demonstrated that TNF- α has the capacity to upregulate sodium-dependent transport in both human type II cells as well as in the rat lung [16, 17]. There is also some evidence that prolonged exposure to TNF- α in vitro can have a depres- sant effect on gene expression and ion transport in the alveolar epithelium [18].
Thus, the presence of a pro-inflammatory molecule may upregulate alveolar fluid
clearance, perhaps an adaptive response that is useful for the alveolar epithelium
to minimize the quantity of excess fluid in the airspaces of the acutely injured and inflamed lung.
Another group of investigators discovered that LTD
4, a pro-inflammatory molecule, has the capacity to upregulate alveolar epithelial sodium and fluid trans- port [19]. Previous studies from our research group demonstrated that markedly elevated levels of LTD
4are found in patients with ALI [20]. At that time we thought that the effects of LTD
4were simply to increase vasoconstriction and broncocon- striction as part of the inflammatory response in the lung. However, this recent work indicates that LTD
4can upregulate alveolar fluid clearance through increased activity and membrane localization of the Na/K-ATPase. The effect is mediated through the CysLT receptor 2, which was identified in both A549 cells and rat alveolar epithelial type II cells. Thus, both TNF- α and LTD
4have the capacity to upregulate alveolar fluid clearance at the same time that they are enhancing the inflammatory responses in the airspaces of the lung.
There are other inflammatory molecules that we have studied that have the opposite effect on alveolar epithelial fluid transport. The best studied and perhaps the most relevant are IL-1 β and TGF- β 1. Both of these cytokines are important in the pathogenesis of ALI and interestingly both of them appear to have deleterious effects on alveolar epithelial barrier function and the capacity of the epithelium to reabsorb edema fluid.
IL-1 β is one of the most biologically active cytokines in pulmonary edema and bronchoalveolar lavage (BAL) fluids of patients with ALI [21,22]. There is evidence that IL-1 β increases microvascular lung epithelial permeability based on both in vitro and in vivo models of ALI. IL-1 β also enhances alveolar epithelial repair by increasing cell spreading [23] and fibroblast proliferation [22]. In recent studies, we found that IL-1 β decreases expression of the epithelial sodium channel α -subunit
in alveolar epithelial cells via a p38 mitogen activated protein kinase (MAPK)- dependent signaling pathway. IL-1 β significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat alveolar type II cell monolayers. IL-1 β also decreased both basal and dexamethasone- induced epithelial sodium channel α -subunit ( α ENaC) mRMA levels and total and cell surface protein expression. The inhibitory effect of IL-1 β on α ENaC expression was mediated by the activation of p38-MAPK in both rat and human alveolar type II cells [24]. These results provide evidence that IL-1 β may play an important role in reducing the resolution of alveolar edema in the acutely injured lung.
Another important cytokine with pro-inflammatory properties is TGF- β 1. Re-
cent work from our research group using mouse studies with both bleomycin and
endotoxin-induced lung injury indicated that TGF- β 1 is an important early me-
diator of lung injury by increasing permeability across the lung endothelium and
epithelium [25]. In more recent studies, we determined that TGF- β 1 significantly
reduces the amiloride-sensitive fraction of sodium uptake and fluid transport
across monolayers of both rat and human alveolar type II cells. TGF- β 1 also signif-
icantly decreased α ENaC mRNA and protein expression and inhibited expression
of a luciferase reporter downstream of the α ENaC promoter in lung epithelial cells.
The inhibitory effect was mediated by activation of the MAPK, ERK1/2 [26]. Also, TGF- β 1 inhibited the amiloride-sensitive alveolar fluid transport in an in vivo rat model at a dose that was not associated with a change in epithelial protein perme- ability. These results, therefore, indicate that TGF- β 1 can decrease the capacity of the alveolar epithelium to remove excess fluid from the distal airspace the lung.
Gene Expression of Inflammatory and Transport Molecules in Human Alveolar Type II Cells
In order to explore the specific capacity of the alveolar epithelium to regulate the production of pro-inflammatory and ion transport genes, we have carried out a series of studies in cultured monolayers of human alveolar type II cells.
Several experimental preparations have been used including the use of cytomix, a combination of IL-1 β , TNF- α , and interferon (IFN) γ as well as authentic hu- man pulmonary edema fluid from patients with ALI. In these studies, the cytomix preparation and the human edema fluid induce a marked increase in gene expres- sion for several pro-inflammatory genes while at the same time inducing a marked decrease in gene expression for ion transport molecules as well as molecules that regulate epithelial cell permeability.
Conclusions
In summary, there is convincing evidence that alveolar fluid clearance and the
resolution of alveolar edema is driven by active vectorial ion transport (sodium
and chloride) across the alveolar epithelium of the lung. Mortality in patients with
ALI is significantly higher in the presence of impaired alveolar fluid clearance
(Fig. 1) [27]. Several catecholamine dependent and independent mechanisms can
markedly upregulate alveolar fluid clearance. Interestingly, evidence accumulated
in the last 10 years indicates that several pro-inflammatory molecules that have
a role in the pathogenesis of ALI by increasing lung vascular permeability can
also play an important role in the capacity of the alveolar epithelium to modulate
lung fluid balance. Specifically, TNF- α and LTD
4can upregulate alveolar fluid
clearance at the same time that they have pro-inflammatory effects in the nearby
lung parenchyma. On the other hand, IL-1 β and TGF β have now been demonstrated
to decrease alveolar epithelial fluid transport, thus probably contributing to the
magnitude of ALI by diminishing the resolution of alveolar edema as well as
enhancing the formation of lung edema. There is much to be learned about the
differential effects of pro-inflammatory genes and their protein projects on lung
fluid balance in the setting of ALI.
Fig. 1. These data demonstrates that submaximal or impaired alveolar fluid clearance in patients with acute lung injury is associated with a higher mortality when compared to patients with maximal alveolar fluid clearance. From [26] with permission
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