Impaired alveolar epithelial fluid transport is a characteristic feature in patients with ALI and has been associated with increased morbidity and mortality. Among ALI patients, we previously reported that patients with impaired AFC (<14%/h) exhibit reduced survival (62% hospital mortality versus
20% with AFC ≥ 14%/h) and a decreased median duration of unassisted ventilation (0 versus
23 days for AFC ≥ 14%/h) (2
). Thus, the mechanisms responsible for the decrease in AFC are of major clinical and biological importance.
Several mechanisms can explain the decrease in AFC in the clinical setting. First alveolar epithelial barrier function in patients with ALI may be reduced because of severe injury from mechanical and inflammatory factors that results in epithelial cell death (apoptosis and necrosis) (3
) as well as by hypoxia (30
). Injury may also be mediated by activation of proinflammatory pathways that down-regulate sodium and chloride transporters that are responsible for vectorial fluid transport across the alveolar epithelium as we and others have hypothesized in prior studies (15
). Therefore, in this study, we tested the direct effect of human ALI pulmonary edema fluid on cultured human alveolar epithelial type II cells to determine whether edema fluid, in contrast to simultaneously collected plasma, would alter net fluid transport.
The initial studies demonstrated that ALI edema fluid markedly reduced net fluid transport compared with plasma (). This was a remarkable finding that provides direct translation of our clinical studies of impaired alveolar epithelial fluid transport in patients with ALI (1
) to our laboratory model of cultured human alveolar type II cells. To study the mechanisms, we carried out further studies to evaluate the effects of ALI pulmonary edema fluid on cell death and the expression of the major known alveolar epithelial ion transporters on cultured human alveolar epithelial type II cells. The primary goal was to understand some of the mechanisms by which undiluted pulmonary edema fluid from patients with ALI reduced net fluid transport (i.e.
resolution of pulmonary edema or AFC). We collected and combined pulmonary edema samples from 19 patients with ALI. We chose sepsis as the source of ALI due to the high mortality associated with this etiology of ALI (33
) and combined the fluid due to the limited volume of pulmonary edema fluid available from each individual patient as in our prior studies (12
Total cell death was modest for human alveolar epithelial type II cells exposed to ALI pulmonary edema fluid (10 ± 1%) compared with plasma (5 ± 1%) by 24 h. The level of necrosis was elevated in type II cells exposed to ALI pulmonary edema fluid (4 ± 1 versus
1 ± 0.3%, p
< 0.01), whereas the level of apoptosis was not significantly different (). However, by confocal and transmission electron microscopy, the vast majority of the cells appeared intact, retaining features characteristic of alveolar epithelial type II cells without evidence of apoptosis or injury when exposed to either the ALI pulmonary edema fluid or plasma (Figs. and ). The modest increase in necrosis but not apoptosis among type II cells in these in vitro
studies was in general agreement with several prior studies of lung injury (29
To further determine whether the modest increase in total cell death caused by ALI pulmonary edema fluid contributed to the changes in protein permeability or net fluid clearance, we exposed human alveolar epithelial type II cells to cytomix, a mixture of the major biologically active cytokines found in ALI pulmonary edema fluid, as a surrogate for the pulmonary edema fluid itself (21
). We previously found by flow cytometry that total cell death was not significantly different between human alveolar type II cells exposed to control media or cytomix, <5%. Similar to ALI pulmonary edema fluid, cytomix decreased net fluid clearance to zero while increasing protein permeability significantly above the level of the control media (). Cytomix also increased the gene expression of the major inflammatory cytokines and decreased the expression of the major sodium transport proteins in a dose-dependent manner (). Combined with the relatively low level of cell death in these current studies, the changes in net fluid transport were not likely to be primarily explained by necrosis or apoptosis.
In an earlier study, we reported that IL-1β
and, to a lesser extent, TNFα
are the predominant cytokines responsible for the proinflammatory activity in pulmonary edema fluid in patients with ALI (12
). In this study, we confirmed that both TNFα
were markedly elevated in the ALI pulmonary edema fluid compared with simultaneously collected plasma ().
In cultured human alveolar epithelial type II cells, ALI pulmonary edema fluid markedly increased the gene expression of all the major inflammatory cytokines and chemokines compared with plasma (). The changes in gene copy numbers suggested that human alveolar epithelial type II cells were capable of propagating an inflammatory response once stimulated in an autocrine manner. In sharp contrast, ALI pulmonary edema fluid decreased the gene expression of all the major sodium and chloride transport proteins () that are essential for net fluid transport or AFC. By Western blot analysis (), the protein levels of α
ENaC, CFTR, and α
1Na,K-ATPase decreased significantly. The decrease in protein levels of the major sodium and chloride transport proteins involved in AFC corroborates previous studies of the effect of individual cytokines (TNFα
), and TGFβ
)) on the mRNA and protein levels of α
ENaC and CFTR and fluid transport.
In contrast to the other transport proteins, the gene copy number of β
1Na,K-ATPase increased by 3% (p
= not significant), and the protein levels increased by 58% on exposure to ALI pulmonary edema fluid compared with plasma (). The β
1 subunit, expressed in significantly lower amounts than the α
1 subunit, is the regulatory and rate-limiting element in the assembly of the functional αβ
Na,K-ATPase enzyme complex whose expression is controlled at the transcriptional, posttranscriptional, and translational levels (32
). In a state where Na,K-ATPase enzyme activity is decreased such as during exposure of LLC-PK1 cells, a pig kidney cell line, to low extracellular potassium, Lescale-Matys et al.
) found that the gene expression of β
1 subunit is increased compared with the α
1 subunit, leading to an actual increase in the αβ
active complex delivered to the membrane. The decrease in the sodium transport proteins, α
ENaC and α
1Na,K-ATPase, as well as the likely consequent changes in the gradient and membrane potential due to ALI pulmonary edema fluid in these studies may stimulate an environment in which Na,K-ATPase enzyme activity is decreased, leading to an increase in β
1Na,K-ATPase protein expression.
In parallel with the reduced gene and protein expression of the key alveolar epithelial transport proteins, net alveolar type II fluid transport was significantly decreased () whereas paracellular protein permeability was increased () for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with plasma. Interestingly net fluid transport for human type II cells exposed to plasma from ALI patients was similar to that of human type II cells stimulated with forskolin and IBMX, suggesting that plasma from ALI patients can activate cAMP perhaps due to a modest increase in epinephrine levels as reported previously (39
). The lack of improvement in net fluid transport for cells treated with forskolin and IBMX following treatment with ALI pulmonary edema fluid () is consistent with our finding that CFTR mRNA and protein levels were significantly decreased following ALI pulmonary edema exposure; the CFTR protein is important in mediating the cAMP up-regulation of AFC especially in β
-adrenergic receptor-driven alveolar Na+
Despite the changes in the sodium and chloride transport proteins reported previously with individual cytokine exposure (15
), pretreatment of the human alveolar type II cells with inhibitors of TNFα
or with IL-1RA prior to exposure to ALI pulmonary edema fluid had no impact on net fluid transport or paracellular protein permeability perhaps because neutralization of individual cytokines has limited value in the context of the actual ALI edema fluid that contains several proinflammatory cytokines. Because of a limitation of the quantity of edema fluid available for study, we could not test the potential additive effect of combining different neutralization strategies such as IL-1RA plus an anti-TGFβ
Because neutralization of individual cytokines did not have any effect on reversing fluid transport, we tested inhibitors of intracellular signaling pathways that we and other investigators have found to be linked to vectorial sodium transport in prior studies of alveolar epithelial type II cells (18
). Although inhibition of the ERK1/2 MAPK pathway had no effect, pretreatment of ALI edema fluid with SB202190, a p38 MAPK inhibitor (10 μm
), improved net fluid transport (). Pretreatment with SB202190 also attenuated the increase in paracellular protein permeability from 28% for cells treated with ALI pulmonary edema fluid alone to 11% for cells pretreated prior to exposure to ALI pulmonary edema, although this difference did not quite reach statistical significance (p
= 0.06; ).
In addition to these functional effects, pretreatment with a p38 MAPK inhibitor prior to ALI pulmonary edema fluid exposure partially restored the gene copy number of the inflammatory cytokines/chemokines and sodium/chloride transport proteins closer to those of the plasma control (). In addition, Western blot analysis showed that αENaC, α1Na,K-ATPase, and β1Na,K-ATPase protein levels of human alveolar type II cells pretreated with SB202190 prior to ALI pulmonary edema fluid were partially restored to the levels of type II cells exposed to the plasma ().
Despite the restoration of the gene copy number of CFTR with SB202190 pretreatment, CFTR protein levels remained depressed following exposure to SB202190 and ALI pulmonary edema fluid. Regulation of CFTR-mediated chloride transport in the alveolar epithelium may occur at multiple levels: phosphorylation leading to channel opening, trafficking of CFTR to the apical membrane, CFTR endocytosis and degradation, and CFTR transcription (41
). In this study, ALI pulmonary edema fluid decreased the gene expression and protein levels of CFTR compared with plasma. Our results were similar to previous studies that demonstrated that inflammatory cytokines (42
) or mediators such as nitric oxide (NO) (43
) are capable of suppressing CFTR cAMP-stimulated activity. In human colonic epithelial cells in culture, Howe et al.
) demonstrated that TGFβ
suppresses CFTR gene expression, intracellular protein levels, and apical membrane channel number. They also found that the inhibition of cAMP-mediated Cl-
secretion by TGFβ
is reversed with p38 MAPK inhibition not c-Jun NH2
-terminal kinase (JNK) or ERK1/2 inhibition (42
). In our current study, pretreatment with a p38 MAPK antagonist prior to ALI pulmonary edema fluid exposure fully restored the gene copy number of CFTR, but protein levels remained depressed. In the future, to understand the role of CFTR in net fluid transport on exposure to ALI pulmonary edema fluid, we may need to perform 1) biotinylation studies to determine the changes in the number of CFTRs at the apical membrane and 2) short circuit studies to determine any changes in CFTR activity. The persistent decrease in CFTR protein levels correlated with the lack of improvement in net fluid transport with cAMP agonist treatment following ALI pulmonary edema fluid exposure.
There are other possible mechanisms that could contribute to the decrease in alveolar epithelial fluid transport that we measured. Although we primarily studied the role of the inflammatory cytokines in this study, ALI pulmonary edema fluid contains other biologically active substances such as proteolytic enzymes, lipids, and reactive oxygen species, particularly the NO metabolite peroxynitrite, which can affect not only fluid clearance but other alveolar epithelial type II cell activity such as surfactant homeostasis. In a similar model in the rat, O’Brodovich and co-workers (45
) found that endotoxin-stimulated alveolar macrophages or the corresponding supernatant decreases ENaC mRNA levels and activity as well as amiloride-sensitive sodium channel activity among distal lung epithelial cells. The inhibitory effect is prevented by the addition of a nitric-oxide synthase inhibitor or an antioxidant. The role of NO or reactive oxygen species in the activity of the ALI pulmonary edema fluid is complex and will require further extensive analyses. Interestingly we found that human ALI pulmonary edema significantly increased the level of NO induced from human alveolar epithelial type II cells following 24 h of exposure ().
Another potential contributing mechanism includes the trafficking of ENaC, CFTR, or Na,K-ATPase channels to the apical or basolateral membranes, although the failure of cAMP agonists to increase alveolar fluid clearance makes this possibility less likely (31
). Because we studied type II cells alone, we cannot comment on what effects the edema fluid might have on alveolar epithelial type I cells. Recent work has shown that alveolar epithelial type I cells contain functional sodium and chloride transport proteins, which are essential for AFC (40
). We also did not measure the electrophysiologic characteristics of these human type II cells, although these studies were beyond the scope of this work (49
In conclusion, ALI pulmonary edema reduced the gene expression of major ion transporters in human alveolar type II cells. There was a substantial decrease in all of the major sodium and chloride transport gene and protein levels. These changes were associated with a decrease in net vectorial fluid transport as well as an increase in paracellular protein permeability across cultured human type II cell monolayers. Inhibition of p38 MAPK phosphorylation partially reversed the effect of the ALI pulmonary edema fluid on protein levels, net fluid transport, and paracellular permeability. These results demonstrate that ALI pulmonary edema fluid contains soluble factors that are capable of adversely affecting the resolution of pulmonary edema.