The discovery of mechanisms that regulate salt and water transport by the alveolar and distal airway epithelium of the lung has generated new insights into the regulation of lung fluid balance under both normal and pathological conditions. There is convincing evidence that active sodium and chloride transporters are expressed in the distal lung epithelium and are responsible for the ability of the lung to remove alveolar fluid at the time of birth as well as in the mature lung when pathological conditions lead to the development of pulmonary edema. Currently, the best described molecular transporters are the epithelial sodium channel, the cystic fibrosis transmembrane conductance regulator, Na+-K+-ATPase, and several aquaporin water channels. Both catecholamine-dependent and -independent mechanisms can upregulate isosmolar fluid transport across the distal lung epithelium. Experimental and clinical studies have made it possible to examine the role of these transporters in the resolution of pulmonary edema.

SummaryReversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A routine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalburain and challenged with aerosolized antigen. No airway hyperreactivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4 + T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperreactivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti-IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperreactivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.

Substantial progress has been made in understanding the role of the distal airway and alveolar epithelial barriers in regulating lung fluid balance. Molecular, cellular, and whole animal studies have demonstrated that reabsorption of fluid from the distal air spaces of the lung is driven by active sodium transport. Several different in vivo, in situ, and isolated lung preparations have been used to study the mechanisms that regulate fluid transport in the normal and injured lung. Catecholamine-dependent and -independent regulatory mechanisms have been identified that modulate fluid transport, probably by acting on apical sodium channel uptake or the activity of the Na, K-ATPase pumps. Recently, a family of molecular water channels (aquaporins) has been identified that are small (approximately 30 kDa) integral membrane proteins expressed widely in fluid-transporting epithelia and endothelia. At present, four different water channels have been identified in trachea and lung. Measurements of osmotic water permeability in in situ perfused lung and isolated perfused airways suggest a significant contribution of these molecular water channels to measured water permeability. However, further studies are required to determine the role of these water channels in normal pulmonary physiology and disease. Recent studies have provided new insights into the role of the alveolar epithelial barrier in clinical and experimental acute lung injury. Unlike the lung endothelium, the alveolar epithelium is resistant to several clinically relevant types of injury, including endotoxemia and bacteremia as well as aspiration of hyperosmolar solutions. In addition, even when the alveolar barrier has been injured, its capacity to transport edema fluid from the distal air spaces of the lung recovers rapidly. Future studies need to integrate new insights into the molecular mechanisms of alveolar epithelial sodium and water transport with functional studies in the normal and injured lung.

Acid aspiration lung injury may be mediated primarily by neutrophils recruited to the lung by acid-induced cytokines. We hypothesized that a major acid-induced cytokine was

Exogenous administration of fi-adrenergic agonists has previously been reported to increase lung liquid clearance by stimulation of active sodium transport across the alveolar epithelium. We hypothetized for this study that endogenous release of epinephrine in septic shock would stimulate liquid clearance from the airspaces in rats. Liquid clearance from the air spaces was measured by the concentration of protein over 4 h in a test solution of 5% albumin instilled into one lung. Bacteremic rats developed severe systemic hypotension and metabolic acidosis that was associated with a 100-fold rise in plasma epinephrine levels. There was a 100% increase in liquid clearance from the airspaces of the lung in the bacteremic compared with control rats. To determine the mechanisms responsible for this accelerated lung liquid clearance, amiloride (10'-M), a sodium transport inhibitor, was added to the air spaces. Amiloride prevented the increase in liquid clearance from the airspaces, indicating that this effect depended on increased uptake of sodium across the lung epithelium. The addition of propranolol (10-4 or iO' M) to the instillate also prevented the acceleration in alveolar liquid clearance in the bacteremic rats. We conclude that the release of endogenous catecholamines associated with septic shock markedly stimulates fluid clearance from the distal airspaces of the lung by a 8-adrenergic mediated stimulation of active sodium transport across the epithelial barrier. This data provides evidence for a previously unrecognized mechanism that can protect against or hasten the resolution of alveolar edema in pathological conditions, such as septic shock, that are associated with the endogenous release of catecholamines. (J. Clin. Invest. 1994. 94:663-671.)

Because beta-adrenergic agonist therapy may be useful clinically as a treatment to hasten the resolution of alveolar edema, this study was designed to examine the dose-dependent effects of beta-adrenergic agonist therapy on alveolar epithelial fluid clearance. The studies were done by instilling an isosmolar 5% albumin solution into the distal air spaces of both ex vivo rat and ex vivo human lungs that were inflated with 8 to 10 cm H2O with 100% oxygen and placed in a 37 degrees C humid incubator. Alveolar fluid clearance was measured by the progressive increase in concentration of protein over 1 or 4 h. Salmeterol, a new long-acting lipophilic agent, was more potent than terbutaline in stimulating alveolar fluid clearance from the ex vivo human lung. Therefore, salmeterol was used for these studies. The results indicated that: (1) basal, unstimulated alveolar fluid clearance in rat lungs was significantly faster than in human lungs (24 +/- 4% over 4 h in rat lungs compared with 11 +/- 2% over 4 h in human lungs, p < 0.05); (2) comparison of equivalent doses of beta-adrenergic stimulation indicated that stimulated clearance rates were also faster in rat lungs than in human lungs; (3) very low doses of salmeterol were effective in ex vivo rat lungs (10(-8) M); and (4) relatively low doses were effective in the ex vivo human lungs (10(-6) M) as a treatment for increasing alveolar fluid clearance. In summary, there are significant differences in the basal and stimulated rates of alveolar epithelial fluid clearance in rat and human lungs, although the ex vivo human studies may have underestimated maximal alveolar fluid clearance in the intact human lung. The human lung responds well to relatively low doses of beta-adrenergic agonist therapy, a finding with potentially important clinical implications for hastening the resolution of alveolar edema.

Transition from placental to pulmonary oxygenation at birth depends on a rapid removal of fetal lung fluid from the developing alveoli. Alveolar fluid clearance was examined in ventilated, anesthetized developing guinea pigs of the ages newborn, 2-d-old, 5-d-old, 30-d-old, and 60-d-old (adult). An isosmolar 5% albumin solution was instilled into the lungs of the guinea pigs; the guinea pigs were then studied for 1 h. Alveolar fluid clearance was measured from the increase in alveolar protein concentration as water was reabsorbed. Newborn guinea pigs had a very high alveolar fluid clearance rate that declined rapidly within the first 5 postnatal days towards adult levels. The high alveolar fluid clearance at birth was apparently mediated by the beta-adrenergic system as demonstrated by the elevated plasma epinephrine levels and the increased sensitivity to inhibition by the beta-adrenergic antagonist propranolol immediately after birth. Surprisingly, exogenous addition of epinephrine was not able to stimulate alveolar fluid clearance in the newborn lung, but exogenous epinephrine stimulation increased over time to adult levels. The elevated alveolar fluid clearance at birth was associated with a significantly greater amiloride sensitivity in the newborn guinea pig lung. Northern blot analysis of distal lung tissue as well as isolated alveolar epithelial type II cells showed and confirmed higher levels of the alpha-subunit of the epithelial sodium channel mRNA in the newborn lung that rapidly tapered off toward adult levels. In conclusion, these data demonstrate the importance of the beta-adrenergic system and amiloride-sensitive sodium transporting pathways for clearance of fetal lung fluid at birth.

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.