The pathophysiology of the acute respiratory distress syndrome

Pathophysiology 5 (1998) 1 – 13

Review article

The pathophysiology of the acute respiratory distress syndrome Donna L. Carden a,b,*, J. Steven Alexander b, Ronald B. George a b

a Departments of Medicine, Louisiana State Uni6ersity Medical Center, Shre6eport, Shre6eport, LA 71130, USA Molecular and Cellular Physiology, Louisiana State Uni6ersity Medical Center, Shre6eport, Shre6eport, LA 71130, USA

Accepted 3 March 1998

Abstract The pathophysiology of the acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema, decreased lung compliance and profound arterial hypoxemia. The syndrome has several apparent ‘triggers’ and involves several cell types, most notably microvascular endothelial cells and polymorphonuclear leukocytes or neutrophils. These cells interact through several classes of adhesive determinants on both the endothelial cell and neutrophil which govern leukocyte binding, transendothelial migration and the extent of injury to the lung. The lung injury elicited by leukocytes involves the release of several mediators which include oxidants and proteases, of which elastase now appears to be the most important in pulmonary injury. There are several potential targets of oxidants and proteases in the lung which include the endothelial cell membrane, glycocalyx and basement membrane as well as endothelial and epithelial junctional proteins. Destruction of these elements appears to be responsible for increased pulmonary microvascular permeability and lung edema formation and may also facilitate neutrophiltransendothelial migration. This review focuses on the structure and function of the alveolar-capillary membrane and the forces that govern leukocyte trafficking in the lungs as a background to understanding the pathophysiology of lung injury in ARDS. © 1998 Elsevier Science B.V. All rights reserved. Keywords: ARDS; Neutrophils; Alveolar–capillary membrane; Permeability

1. Introduction Acute lung injury is a syndrome of lung inflammation manifested by increased vascular permeability which is associated with several well-defined risk factors [1]. The syndrome is characterized by: (1) profound arterial hypoxemia resistant to oxygen therapy; (2) radiographically identified diffuse, pulmonary infiltrates; and (3) an absence of clinical or radiographic evidence of left heart failure (Table 1) [3]. Rather than a single and distinct disease entity, acute lung injury actually represents a progressive continuum of lung damage, the most severe form of which is the acute respiratory distress syndrome (ARDS) [4]. ARDS * Corresponding author. Tel.: +1 318 6756887; fax: + 1 318 6754221; e-mail: [email protected] 0928-4680/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0928-4680(98)00004-2

shares all of the characteristics described above for acute lung injury, except that the degree of arterial hypoxemia is more severe (Table 1). ARDS was first described by Ashbaugh and Petty in 1967 [5]. Although the incidence of ARDS has been reported to be over 150000 per year and the mortality rate estimated at 50–70%, neither the exact incidence nor the true mortality rate of ARDS are known [6]. Obtaining accurate epidemiologic data has been challenging due to controversies regarding the clinical definitions and the distinguishing characteristics of acute lung injury and ARDS. In addition, the non-specific injury response of the lungs to a variety of precipitating events means that patients with dissimilar illnesses are often grouped together, obscuring the exact time of onset, true incidence and clinical course of acute lung injury and ARDS.

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Table 1 American – European Consensus Conference definitions of acute lung injury and acute respiratory distress syndrome [1] (modified with permission from Canonico and Brigham [2])

Acute lung injury ARDS

Onset

Arterial oxygenation

Chest radiograph

Left atrial pressure

Acute

PaO2/FiO25300 mmHg with or without positive end expiratory pressure (PEEP) PaO2/FiO25200 mmHg 9 PEEP

Bilateral pulmonary inflitrates Bilateral pulmonary inflitrates

PWP518 mmHg or absence of left heart failure PWP518 mmHg or no evidence of left heart failure

Acute

2. Causes of ARDS Although the list of disorders associated with ARDS is impressive (Table 2), Hudson et al. [7] recently identified seven clinical risk factors specifically associated with an increased incidence of ARDS [7]. ARDS is most often associated with: (1) the sepsis syndrome (42.6%) and (2) multiple emergency blood transfusions (i.e. 15 units of blood within 24 h; 40%); but (3) near-drowning; (4) pulmonary contusion; (5) aspiration of gastric contents (i.e. acid aspiration); (6) multiple fractures; and (7) drug overdose are also associated with a substantial risk for the development of ARDS. In addition to these seven major risk factors, experimental and clinical evidence indicates that ischemiareperfusion of the intestine [8 – 12] or lower extremities [13,14], acute pancreatitis [15] and thermal injury (burns) are also associated with ARDS. Despite intense experimental and clinical efforts to determine the causes of and to develop effective therapies for ARDS, the mortality rate for this disorder remains unacceptably high [7]. One difficulty in designing effective interventions is that this syndrome is not defined by a single pathology, but rather reflects the lungs general injury response to a variety of precipitating insults. However, one consistent abnormality in ARDS, regardless of the initiating process, is loss of integrity of the alveolar-capillary barrier. Loss of this barrier at the blood – air interface results in fluid and protein flooding the alveoli, with accompanying interstitial edema, arterial hypoxemia and decreased lung compliance. Hence, most of the clinical findings of ARDS can be explained by the loss of alveolar –capillary functional integrity and its sequelae. This discussion will review the structure of the alveolar – capillary membrane and the forces that govern fluid and protein exchange in the alveolar – capillary unit within the lungs as a background to understanding the pathophysiology of lung injury in ARDS.

3. Structure of the alveolar – capillary membrane The alveolar–capillary membrane is composed of an endothelial layer separated from the alveolar epithelium

by a thin interstitial space (Fig. 1). The closely apposed epithelial and endothelial barriers are both involved in the regulation of solute exchange in the lung. However, the alveolar capillary allows a net outward movement of fluid and small solutes from the vascular to the interstitial space. In contrast, the alveolar epithelium forms a highly restrictive solute exchange barrier that limits the movement of water and ions into the alveolus. While the vascular layer of the alveolus appears to be formed only by endothelial cells, the alveolar airspace is lined by at least two types of epithelial cells. Type I epithelial cells cover the majority of the alveolar lining, while type II cells cover a smaller percentage of the airspace, but actively synthesize and secrete surfactant. The type II cells also appear to regenerate damaged type I cells (Fig. 1). The interaction of these two types of epithelial cells comprises the main restrictive barrier for solute exchange in the lung.

4. Microvascular fluid and solute exchange in the lung The forces that influence the movement of fluid from the pulmonary microvasculature to the interstitial or alveolar space are classically defined by the Starling equation for filtration of fluid across semipermeable membranes [16]

Table 2 Conditions associated with an increased risk of ARDS (modified with permission from Canonico and Brigham [2]) Indirect lung injury

Direct lung injury

Sepsis syndrome Shock Severe non-thoracic trauma Multiple transfusions Multiple fractures Head injury Acute pancreatitis Eclampsia Drug overdose Heroin Aspirin Propoxyphene

Near drowning Pulmonary contusion Aspiration of gastric contents Pneumonia Viral Bacterial Mycobacterial Fungal Parasitic Toxic inhalation Carbon monoxide Oxygen (high concentrations) Corrosive chemicals

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Fig. 1. The alveolar – capillary membrane. This figure shows the anatomical structure of the alveolar – capillary membrane. The central alveolar airspace (A) is covered by type I (I) and type II alveolar epithelial cells (II), which together, cover the surface of the airspace. Within the alveolus and in contact with the alveolar epithelial cells, are alveolar macrophages (AM) which actively phagocytize bacteria and particulate matter entering the airspace. Immediately below the epithelial barrier is the basement membrane (BM), which is secreted by both endothelial and epithelial cells. Pulmonary endothelial cells (Endothelial cell), adjacent to the basement membrane, form the capillary network surrounding the airspace.

Q =Kf, c[(Pc −Pi)− s(pp −pi)]

(1)

In this equation, Q represents fluid flux across the vascular endothelium per unit time. The capillary filtration coefficient, Kf, c describes the hydraulic conductivity, or ‘ease’ with which water crosses the endothelium. Often described as a constant, the Kf, c is actually a dynamic and sensitive indicator of the integrity of the vascular endothelial barrier [17 – 19]. If Kf, c increases, the net transvascular fluid filtration increases. Pc is the hydrostatic pressure in the pulmonary capillary, Pi represents the opposing hydrostatic pressure of the interstitial space and (Pc – Pi) is the pressure gradient across the vascular wall. The osmotic reflection coefficient, s, describes the protein permeability of the endothelial wall. A s of 1 describes a membrane that is impermeable to protein. As s approaches 0, the membrane becomes freely permeable to protein, allowing unrestricted movement of fluid and protein between the vascular and interstitial space. (pp – pi) is the oncotic pressure gradient between the pulmonary vessels (pp) and the interstitium (pi). This oncotic pressure gradient normally favors the reabsorption of fluid from the interstitial space into the vasculature. Once thought to be composed of passive structural cells, the alveolar epithelium is now recognized as the primary barrier against solute movement into the alveolus. In fact, current evidence suggests that the alveolar epithelium is at least an order of magnitude less permeable to protein and small solutes than the pulmonary

endothelium and hence is the main regulator of fluid and solute exchange across the alveolar–capillary membrane [18,20]. The restrictive properties of the epithelial barrier are thought to depend on the homotypic association of several specialized proteins found in the tight and adherens junctions [21–23]. The alveolar epithelium is more than a passive barrier in that it plays an active role in the reabsorption of fluid which enters the alveolus [24] (Fig. 2). Several lines of evidence demonstrate the importance of sodium transport as the primary mechanism driving fluid reabsorption from the airspaces (Fig. 2). Amiloride-sensitive Na + channels appear to be the principal entry pathway for sodium at the apical membrane of alveolar epithelial cells [25] after which sodium is actively removed from the basolateral cell surface by the Na + –K + ATPase [26,27]. Chloride and water follow the transport of sodium through paracellular or transcellular pathways to maintain electrochemical and osmotic neutrality across the alveolar epithelial membrane. In addition to transcellular and paracellular pathways, water pores [28] termed aquaporins, contribute to the alveolar fluid homeostasis. While the major site of fluid exchange in the lung is in the microcirculation of the alveolar vessels [29], some fluid also leaks from the small arterioles and venules at the junctions of alveolar walls. However, several factors normally prevent the fluid filtered out of the vasculature from entering the alveolar airspace. For example, the oncotic pressure gradient (pp –pi) normally favors

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Fig. 2. Ion and water transport through the alveolar epitheilal barrier. Water and ions are transported between the alveolar airspace and the capillary lumen by convective and osmotic forces created by the active transport of sodium (Na + ) at the basolateral surface of type I alveolar epitheilal cells (left). The energy dependent pumping of sodium (Na + ) from the type 1 cells to the interstitial space draws Na + into the apical cell surface through amiloride-sensitive sodium channels. Water and chloride (Cl − ) are convectively drawn into the cell by specialized channels, termed ‘aquaporins’.

the vascular reabsorption of fluid from the interstitium. In addition, lymphatic drainage increases as a compensatory response to increased transvascular fluid filtration. As a final defense against alveolar edema, the epithelial barrier actively participates in the reabsorption of fluid that accumulates within the airspace. In ARDS, injury to the endothelial barrier, epithelial barrier or both results in loss of the restrictive properties of the alveolar–capillary membrane and as a consequence, interstitial edema and airspace flooding occur. ARDS precipitated by a systemic inflammatory insult (i.e. the sepsis syndrome, multiple transfusions, ischemia-reperfusion disorders) may initially injure the endothelial barrier with resultant increases in Kf, c and decreases in s. These changes greatly increase the transvascular movement of fluid and protein into the interstitium and diminish the oncotic pressure gradient driving fluid reabsorption. Initially, the interstitial fluid load is compensated for by increased lymphatic drainage but as lung injury and the inflammatory process continues, compensatory mechanisms become overwhelmed, and the airspaces are flooded with edema fluid. Other causes of ARDS such as gastric acid aspiration, drowning, and some forms of necrotizing pneumonia are precipitated by direct injury to the alveolar epithelial barrier, again resulting in the translocation of interstitial fluid and solute into the airspace [30]. Al-

though initially confined to the endothelial or epithelial barrier, the integrity of the entire alveolar–capillary membrane may become compromised as lung inflammation progress due to the damaging effects of stimulated inflammatory macrophages and neutrophils [31].

5. The alveolar epithelial barrier As previously stated, type I alveolar epithelial cells constitute most of the surface of the alveolar airspace. Type I cells are characterized by extremely thin, widely spread, cytoplasmic extensions. These structural modifications minimize the diffusion distance for gas exchange at the air–blood interface. Conversely, type II alveolar epithelial cells are cuboidal in shape, have numerous microvilli on their apical surfaces and contain lamellar bodies that store pulmonary surfactant (Fig. 1). The alveolar epithelium is not only more restrictive to solute exchange than the pulmonary endothelium, it is also more resistant to injury than the vascular barrier [32]. However, the alveolar epithelial barrier can become damaged under certain circumstances such as gastric acid aspiration or necrotizing pneumonia, resulting in barrier disruption and permeability edema. Intraalveolar instillation of Pseudomonas aeruginosa, a bacterium that causes a necrotizing pneumonitis associ-

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Fig. 3. Adhesion molecules on the alveolar capillary membrane. Several classes of adhesion molecules participate in the rolling, adhesion, diapedesis and alveolar trafficking of leukocytes in addition to altering the barrier function of the alveolar capillary membrane in ARDS. Initially, leukocytes roll on activated endothelial cells by forming a loose bond between neutrophil sialyl LewisA, fucosylated sialyl LewisX, PSGL-1 and endothelial cell P-selectin. The subsequent, firm adhesive contact between neutrophil CD11b/CD18 and endothelial cell ICAM-1 facilitates leukocyte migration across the endothelium into the interstitial space. During migration, neutrophils pass across endothelial and epithelial junctions containing cadherins and occludin. These membrane proteins may be transiently or permanently altered in ARDS. Finally, alveolar epithelium ICAM-1 may facilitate leukocyte trafficking and adhesion in the alveolar airspace.

ated with the development of ARDS, induces a loss of alveolar epithelial barrier function [32]. In addition to the damage elicited by specific bacteria or bacterial products, there is also evidence that oxidants [33] and proteases [34] derived from inflammatory leukocytes directly injure the alveolar epithelial barrier. Adhesive determinants expressed on the alveolar epithelium may contribute to this injury by facilitating the intra-alveolar migration of leukocytes followed by macrophageepithelial [35] or neutrophil-epithelial interaction (Fig. 3). In support of this concept, it has been reported that airway epithelial cells express intercellular adhesion molecule 1 (ICAM-1), an adhesive glycoprotein critical to leukocyte-endothelial cell adhesion, in response to hyperoxia [36] as well as the inflammatory cytokines IL-1 and TNF [36,37]. The alveolar epithelium can function in the presence of moderate lung injury [38] and rapidly recovers function following pulmonary damage [32]. For example, alveolar epithelial liquid clearance is preserved in the presence of hyperoxia-elicited lung injury [38]. In addition, the epithelial barrier recovers sufficiently 24 h after intra-alveolar instillation of P. aeruginosa, to remove a significant portion of accumulated alveolar fluid [32]. The alveolar epithelium may actually attenuate the intravascular and intra-alveolar procoagulant effects observed in ARDS [39]. By expressing urokinase-type

plasminogen activator and plasminogen activator inhibitor-1 [40], type II alveolar epithelial cells appear to diminish the deposition of fibrin and pulmonary thrombo-emboli that result from activation of the coagulation system in ARDS [39]. The preservation of alveolar epithelial barrier integrity appears to have a significant prognostic value in ARDS [41], in that patients who maintain a functional epithelial barrier have a better chance of survival than those who exhibit a loss of epithelial barrier integrity.

6. Pulmonary endothelial barrier Several types of inflammatory cells are activated in ARDS including neutrophils, macrophages, monocytes and platelets. The products released by these cells promote the inflammatory response and elicit endothelial cell dysfunction, including the hallmark of ARDS, enhanced vascular permeability. The resulting increase in transvascular fluid and solute flux in the lung rapidly causes decreased lung compliance and impaired oxygenation. Most of the exchange of fluid and solute across the vascular endothelium occurs at endothelial cell–cell junctions [42,43] (Fig. 3). The structural components of these junctions consist of occludin, a major component

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of tight junctions, [44] and cadherins, [22,45] which form the adherens junction. There is considerable indirect support for the concept that in ARDS, increased transvascular leak of fluid and protein stems from endothelial cell activation [46,47] and from defects in the integrity of the endothelial cell – cell occludin and adherens junctions. We and others have recently demonstrated that destruction and dysregulation of these elements contributes to the development of permeability edema [48]. For example, we observed proteolysis of endothelial cell junctions by activated neutrophils, and a similar proteolysis in endothelial cells exposed to neutrophil elastase. The proteolysis of endothelial junctions appears to release cadherins from the endothelial surface into a soluble pool. The observation that serum from patients with ARDS have increased circulating levels of vascular endothelial cadherin supports the concept that proteolysis of junctional proteins contributes to the pathogenesis of this syndrome [48]. In addition, to changes in junctional structure and integrity, inflamed endothelial cells express adhesive determinants which facilitate leukocyte – endothelial cell interaction (Fig. 3). For example, on stimulation with several types of pro-inflammatory stimuli, endothelial cells acutely (within 15 min), and chronically (over the course of several hours), up-regulate P-selectin which can be rapidly mobilized from pre-formed pools or synthesized after endothelial stimulation [49]. Conversely, ICAM-1, is constitutively expressed on the apical endothelial cell surface, and can be up-regulated four –five-fold by stimulation with oxidants, cytokines [50] and vasoactive stimuli. E-selectin is not expressed on non-activated endothelial cells, but is expressed over the course of several hours in response to challenge with inflammatory stimuli. P-selectin, originally described as GMP-140 [51], is a 140 kD lectin-like protein which mediates the rolling behavior of leukocytes in inflamed venules [52]. P-selectin is stored as a pre-formed pool within endothelial cells and platelets, and is mobilized within 5 min to the cell surface by fusion of either alpha granules (in the case of platelets) [51], or Weibel – Palade bodies (in endothelial cells) [53]. Once mobilized to the cell surface, P-selectin interacts with sialyl LewisA and fucosylated sialyl LewisX, sulfated glycans and P-selectin glycoprotein ligand-1 (PSGL-1) on the leukocyte cell membrane [54–56] which ‘tethers’ the leukocyte to the endothelial surface and reduces the leukocyte’s rolling velocity. Decreased speed and enhanced rolling of the leukocyte on the endothelial surface permits more stable, leukocyte–endothelial cell adhesive interactions. Consequently, P-selectin is thought to contribute to the development of acute lung injury following systemic activation of circulating leukocytes [9,57]. P-selectin can also be up-regulated through the activation of tran-

scription/translation to increase surface expression, although this process requires several hours to take place. After being mobilized to the endothelial surface, P-selectin has two possible fates: it can be recycled back to the cytoplasm, or it may be shed into the pool of soluble circulating P-selectin [58–60]. While it is not clear if circulating forms of P-selectin play significant physiological roles in inflammation, our studies indicate that it may function to reduce subsequent leukocyte binding to the endothelium [61]. On platelets, mobilized P-selectin may also contribute to the formation of platelet-neutrophil microemboli that could play a significant role in ARDS [62]. E-selectin is another lectin-like endothelial specific adhesive protein which is expressed on the endothelial cell surface in response to inflammatory stimuli [63]. As described above, E-selectin is not expressed on non-activated endothelial cells, but is expressed over the course of several hours after cytokine stimulation. It exists as a single chain 130 kD protein which like P-selectin, participates in the rolling and slowing of leukocytes within inflamed venules. The fact that E-selectin is released from the endothelial cell surface in sepsis [64,65] suggests that soluble E-selectin could modulate the progression of acute lung injury in this disorder. The counter-receptors for E-selectin on the leukocyte are sialyl LewisA and fucosylated sialyl LewisX [54–56]. ICAM-1 is a member of the immunoglobulin gene superfamily [63]. Numerous reports demonstrate that ICAM-1 mediates the firm adhesion of leukocytes to the endothelium and arrest of leukocytes from the rolling phase by binding to CD11b/CD18 on the surface of primed or activated neutrophils [66]. ICAM-1 is constitutively expressed at functional levels on the surface of virtually all endothelial cells, but its expression is increased several fold by induced protein synthesis. ICAM-1 is increased on the endothelial cell surface by exposure to lipopolysaccharide (LPS) [67], cytokines, [50] oxidants and anoxia-reoxygenation [68–70]. ICAM-1-dependent neutrophil-endothelial cell firm adhesion may prime and/or activate neutrophils for maximal degranulation [71,72], thereby promoting the disruption of adjacent epithelial or endothelial barriers [73].

7. The role of neutrophils in ARDS

7.1. Neutrophil retention 6ersus neutrophil mediated injury Several investigators have reported that even minor surgical manipulations elicit lung neutrophil retention that is not necessarily associated with increased vascular permeability. Further, alterations in neutrophil stiff-

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Fig. 4. Micrograph of intestinal ischemia-reperfusion elicited lung inflammation stained for ICAM-1 (blue), neutrophils (pink) and fluorosceinlabed albumin to delinate the vascular space (green). Systemic inflammation induces marked lung leukocyte retention, neutrophil-endothelial cell interaction and fluid and protein extravasation. Image courtesy of D. Carden, V. Specian and R. Specian.

ness can lead to the physiological retention of neutrophils in the pulmonary microvasculature [74–76] without causing injury. The concept that leukocyte stiffness contributes to lung neutrophil retention is supported by evidence in humans in which a correlation has been reported between lung neutrophil sequestration in vivo and neutrophil deformability in vitro [77]. Neutrophil deformability is particularly relevant to neutrophil retention in the microvasculature of the lung for several reasons. Neutrophils are normally much less deformable than red blood cells and the mean diameter of neutrophils (7–8 mm) is larger than the mean diameter of pulmonary capillaries (5 – 5.5 mm) [78]. Furthermore, the intravascular pressures in the pulmonary capillaries are 1/10 those in the systemic circulation and the flow within the microvasculature of the lung is pulsatile with periods of flow interspersed with intervals of no-flow [79]. Thus, the effect of hydrodynamic dispersal forces and neutrophil deformability are important determinants of neutrophil retention in the pulmonary microcirculation [79]. In addition, exposure

of neutrophils to inflammatory stimuli results in a further decrease in their deformability due to reorganization of the neutrophil cytoskeleton and net filamentous actin (F-actin) assembly [76]. We have recently reported that complement activation is an important inflammatory stimulus which occurs as a consequence of systemic inflammation and elicits a prolonged increase in neutrophil stiffness [12]. These changes diminish the ability of leukocytes to deform during capillary transit [76,80] and cause neutrophil retention within capillary sized pores in vitro and in the pulmonary microvasculature in vivo[11,12,76]. While changes in neutrophil stiffness can clearly affect lung neutrophil trafficking independent of injury, it is generally accepted that most forms of ARDS involve lung neutrophil entrapment and activation as well as neutrophil-mediated pulmonary injury [30,81]. For example, we and others have demonstrated that blocking mAbs directed against neutrophil CD11/CD18 or endothelial P-selectin or ICAM-1 prevent the lung injury induced by systemic inflammation secondary to intesti-

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nal ischemia-reperfusion [9,11,82]. Under these conditions, the presence of sequestered, activated leukocytes appears to be responsible for the majority of complications associated with ARDS. The dramatic lung neutrophil accumulation and interstitial edema formation characteristic of systemic inflammation initiated lung injury is apparent in Fig. 4. This image represents an inflamed alveolus subjected to intestinal ischemia-reperfusion which is stained for ICAM-1 (blue), neutrophils (pink) and the vascular space (green). The lung pathology produced by activated neutrophils appears to be mediated by several factors including neutrophilderived oxidants, proteases and other granule constituents, e.g. peptide defensins and cationic proteins. We will consider the evidence for each of these classes of agents in acute lung injury.

8. The role of neutrophil-generated products in lung injury

8.1. Oxidants Neutrophil derived oxidants play a highly significant role in the bactericidal function of the leukocyte [83]. Inappropriate and/or excessive activation of neutrophils often leads to stress and injury of normal tissue through several mechanisms. Some oxidants, e.g. hydroxyl, can directly modify proteins, e.g. surfactant and a-1 antiprotease inhibitor, and lead to changes in the endothelial and epithelial cells environment. Oxidation of cytoplasmic constituents can lead to DNA and mitochondrial damage, as well as injury to several types of biological membranes including the cell and nuclear envelopes, golgi and endoplasmic reticulum compartments. Oxidants are known to deplete endothelial ATP by dissipating ionic gradients in the mitochondria and are thought to uncouple several steps in oxidative phosphorylation. These effects on cell metabolism have acute effects through energy depletion, and may also lead to apoptosis by the induction of unique oxidant sensitive transcription factors within cells, e.g. NFkB [84]. Oxidants like peroxide and hydroxyl radical also lead to alterations in several important second message systems inside cells, including cell calcium, protein kinase C and tyrosine kinase. Because of these pleiotropic effects, it is very difficult to relate the individual effects of oxidants to particular aspects of lung pathology. However, it is clear that oxidants do play an important role in ARDS, based on many studies which demonstrate that antioxidants and oxidant scavengers effectively attenuate neutrophil mediated lung injury [10,14,85].

8.2. Proteases In addition to oxidants, neutrophils release several proteolytic enzymes which have recently been shown to participate in ARDS pathology. These proteases include elastase, collagenase, gelatinase and several other related enzymes. They may not contribute to the leukocyte bactericidal functions to a great extent, but rather may function in helping neutrophils extravasate and to assemble within tissues during chemotaxis [86–88]. The excessive release of these enzymes, which normally act to degrade the basement membrane in leukocyte motility, leads to extensive tissue destruction in ARDS. Further, neutrophil-derived oxidants appear to potentiate these proteases since many of the endogenous inhibitors of these proteases are inactivated by oxidants. While proteases also participate in ARDS pathology, the full spectrum of pulmonary pathologies associated with ARDS cannot be entirely attributed to the release of these proteases. Nevertheless, several recent studies, including those in our laboratory, have demonstrated the potential prophylactic and therapeutic efficacy of antiproteases [48]. More recently, endothelial and perhaps epithelial junction proteins have been identified as important potential targets for neutrophil derived proteases. It has been observed that endothelial cadherins, components of the adherens junctions, and occludin, components of the tight junction appear to be significantly degraded by leukoproteases. Since these junctions constitute the main barrier to exchange between the vascular, interstitial and alveolar compartments in the lung, their destruction by proteases would be expected to result in profound interstitial and alveolar edema. Whether the interstitial and alveolar edema in ARDS is a result of proteolysis of cadherins and occludin is currently a topic of intense investigation.

9. The role of alveolar macrophages in ARDS Alveolar macrophages constitute at least 90% of resident leukocytes in the uninjured lung. These inflammatory cells are responsible for the phagocytosis and destruction of particles which are small enough to escape the upper airway mucociliary defense mechanism and reach the distal airway. In the process of phagocytosis and alveolar clearance, macrophages release proteases, metalloproteases and oxidant species, potentially damaging adjacent endothelium and/or epithelium. In addition to directly producing lung injury, macrophages release the early response Th-1 cytokines IL-1 and TNF, which elicit acute lung injury in animal models of systemic inflammation [89,90] and effectively amplify the inflammatory response [91]. The macrophage-derived cytokines, IL-1 and TNF, as well as the bacterial cell wall component, lipo-

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polysaccharide, elicit endothelial cell activation and increased endothelial adhesion molecule (ICAM-1) expression [69,92,93]. The interaction of the alveolar macrophage b2 integrin, CD11/CD18 [94] with ICAM1 facilitates the close apposition of macrophages with endothelial or epithelial cell targets [35] and may promote the release of macrophage-derived oxidants, proteases and metalloproteases in close proximity to these permeability barriers. Finally, by producing cytokines which promote neutrophil chemotaxis, and inducing the up-regulation of cellular adhesion molecules, alveolar macrophages maintain and amplify ongoing lung neutrophil recruitment and injury. The net result of this un-regulated stimulation of the alveolar macrophages is lung injury as well as amplification of the inflammatory cascade.

10. Therapeutic interventions and clinical trials

10.1. Interference with the proposed mediators of sepsis syndrome Since ARDS is most often associated with sepsis [7] it was initially hoped that interference with proposed mediators of the sepsis syndrome (lipopolysaccharide, IL-1, TNF) would decrease the incidence and improve the survival of patients with ARDS [3]. However, in prospective clinical trials, blocking these mediators has not reduced the overall mortality rate of ARDS [95– 97]. Ketoconazole, an inhibitor of thromboxane and leukotriene biosynthesis, mediators which have also been implicated in the systemic inflammatory response, showed early promise in preventing ARDS in at-risk patients [98] but subsequent clinical trials failed to demonstrate a survival benefit with the drug. This may indicate that after the inflammatory cascade and acute lung injury have been initiated, it is difficult to reverse the developing pattern of lung pathology.

10.2. Interference with the inflammatory response Interference with the inflammatory response utilizing corticosteroids [99], the nonsteroidal anti-inflammatory agent, ibuprofen [100] or prostaglandin E1 (alprostadil) [101], a vasodilator that attenuates the inflammatory response and diminishes platelet aggregation, also failed to produce any remarkable survival benefit in large ARDS trials. In vitro, the vasodilator, nitric oxide, inhibits the inflammatory response in part by interfering with endothelial or leukocyte adhesion molecule expression and neutrophil-endothelial cell interaction [47,62,102]. Rossaint et al. reported that inhaled nitric oxide reduces both pulmonary artery pressures and intra-pulmonary shunting in severe ARDS [103]. Still, preliminary survival results of a prospective clinical

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trial using inhaled nitric oxide in ARDS remain disappointing [104]

10.3. Interference with products of acti6ated neutrophils Neutrophil-derived oxidants have been implicated as key mediators of the lung injury elicited by systemic inflammation, and it might be reasonably predicted that antioxidants would prove beneficial in ARDS. Unfortunately, acetylcysteine, an oxidant scavenger and precursor of the cellular antioxidant, glutathione, has no appreciable benefit in terms of restoring gas exchange or improving survival in ARDS [3,105]. Neutrophil elastase appears to mediate at lease part of the lung injury in ARDS [14,86] as evidenced by the fact that elastase inhibitors attenuate neutrophil-mediated lung injury in experimental models of ARDS. The protease inhibitors may prevent lung damage by preventing the proteolysis of cell–cell junctional proteins which maintain monolayer integrity and which become disrupted after a systemic inflammatory insult. A phase II trial of ONO-5046, a synthetic elastase inhibitor in systemic inflammation, is currently underway.

10.4. Replacement of pulmonary surfactant Patients with ARDS have dysfunctional surfactant, a problem that may contribute to airway instability, increased airway pressures and increased shunt. Although it was initially anticipated that addition of exogenous surfactant would be beneficial in reversing these adverse pulmonary effects of ARDS, a large clinical trial was terminated because of a survival benefit could not be demonstrated in patients given a synthetic surfactant [106].

10.5. No6el therapeutic inter6entions Extracorporeal membrane oxygenation for patients with severe ARDS was evaluated in a prospective, clinical trial but the overall mortality rate in this study was not different than that in patients treated with conventional ventilation [107]. Other oxygenation approaches like liquid fluorocarbons have recently been used clinically [108] in cases of severe hypoxemic respiratory failure to promote oxygenation and prevent distal airway collapse but the effect of these agents on the mortality of ARDS is not known.

11. Future considerations Hopefully, the future still holds promise for therapies which will effectively treat acute lung injury and ARDS. It is important to note however, that although many treatments are effective in experimental models,

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they may not improve the clinical outcome of ARDS for several reasons. One key difference between experimental and clinical approaches is the timing of the administration of interventions. In experimental models, therapies can be given early, often before the onset of symptoms, and will actually interfere with ‘downstream’ events which promote or amplify the evolving lung pathology. However, in ARDS clinical trials, the lung injury of recruited individuals may have advanced well beyond the initiation stage. These individuals may not benefit greatly from therapeutic strategies given after lung inflammation and injury are fully developed. This issue also underscores the importance of rapidly identifying patients who are at risk for developing ARDS so that potentially beneficial interventions can be administered when the agent has the greatest chance of being effective. Another key difference between experimental and clinical models of ARDS is the clarity with which the initating insult is defined. Experimental approaches generally utilize a well-defined, precisely timed, insult which may affect only the epithelial or endothelial permeability barrier. Therapeutic strategies administered systemically or intra-alveolarly may significantly attenuate lung injury if the site of endothelial or epithelial injury is well-defined or confined. On the other hand, clinical trials may group patients with dissimilar precipitating illnesses, in which the initiating triggers of the inflammatory cascade, or the site of epithelial or endothelial barrier disruption, is very different. Therapeutic strategies which do not take these differences into account are unlikely to be effective in all cases. Improvements in experimental models of ARDS may reproduce more accurately the clinically salient features of acute lung injury and ARDS. The fact that the morbidity and mortality remain high in ARDS despite significant advances in our understanding of the mediators, timing and mechanisms of the syndrome, demands continued study. Clearly, there are numerous initiators and mediators which contribute to the pathophysiology of ARDS. Several studies in this field have focused on the use of a single agent to improve the clinical outcome of this disorder. While this is a scientifically logical method for carrying out therapeutic studies, it appears that success in ARDS treatment must aggressively block several simultaneous features of the disease which may involve multiple treatments. Accurately defining the initial site of barrier disruption may also dictate the route of administration of therapeutic interventions. If the timing or route of administration of an intervention are important, several clinical scenarios representing different stages of lung injury will need to be identified in order to generate effective therapies. It is therefore probably unwise and overly optimistic to expect that a single agent would significantly alter the course of

ARDS.

Acknowledgements This work was supported by a grant from the National Institute of Health (NIDDK 2 PO1 DK 43785)

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