Mediation of ARDS by Leukocytes

Mediation of ARDS by Leukocytes* Clinical Evidence and Implications for Therapy Jean E. Rinaldo, M.D.

he mortality rate for AROS remains extremely T high. It is now widely believed that elucidation of 1

the pathophysiology of cell injury and aberrant repair in ARDS will be necessary to substantially influence survival rates by identifying appropriate pharmacologic therapy. In recent years, attention has been directed towards blood leukocytes as major mediators of acute lung injury in ARDS. The role of blood leukocytes as mediators of pathophysiology is of theoretical interest but more importantly suggests potential therapeutic approaches. The rationale for several proposed therapeutic agents including corticosteroids, prostaglandin E, and antioxidant therapy, are based at least in part upon the concept that leukocytes mediate acute lung injury in ARDS. Many observations in vitro and from animal models suggest that under certain defined experimental conditions, activated granulocytes are capable of injuring other cells. The mechanisms of injury include release of highly reactive oxidant species, of proteolytic enzymes, and of metabolites of arachidonic acid. This evidence has recently been summarized in detail. 2 However; despite its popularity, the hypothesis that harmful interactions between blood leukocytes and pulmonary vascular endothelial cells are causally related to ARDS is not definitively established. The purpose of the present review is to critically reexamine the hypothesis based both on the clinical studies that are currently available, and on newly emergent theoretical considerations. First, available clinical studies will be examined: despite a wealth of basic information suggesting a potential for neutrophilmediated lung injury, clinical data indicating that this potential is realized in AROS are relatively sparse. Secondly, several recent basic observations will be introduced which appear to cast some doubt on the ·From the University of Pittsburgh School of Medicine, Division of Pulmonary Medicine, Pittsburgh__ This investigation was su~ported by grants from the American Lung Association, the Health Researcn and Services Foundation, the Upjohn Pharmaceutical Company, and Presbyterian-University Hospital. Reprint requests: Dr: Rinaldo, 3550 Terrace Street, Room 440, University Of Pittsburgh, Pittsburgh 15261

590

hypothesis that blood leukocytes injure normal pulmonary endothelium severely, leading to AROS. THE "COMPLEMENT" HYPOTHESIS

It was observed in 1968 that profound granulocytopenia accompanied cellophane membrane hemodialysis. Mild hypoxemia occurred concurrently. Activation of the complement cascade by the dialysis membrane, via the alternate pathway, was implicated. In correlative experimental studies, neutrophils were found aggregated in the microvascular beds of tissues following complement activation, particularly in the lung where a large microvascular network is present. A peptide product ofactivated complement (CSA) which has chemotactic properties appeared to be the mediator of intrapulmonary neutrophil leukostasis. The etiology of dialysis-associated hypoxemia remains in dispute, but advocates of the "complement hypothesis" proposed that vasoactive mediators released by the aggregated neutrophils cause disturbances in ventilation and perfusion matching which could impair gas exchange. The limited clinical pertinence of these observations to patients undergoing hemodialysis was overshadowed by the emergence of a hypothesis that changed the direction of ARDS-related research. It was proposed that an exaggerated version of these events which accompanied hemodialysis-complement activation-intrapulmonary neutrophil aggregation and neutrophil-mediated pulmonary microvascular dysfunction-could result in AROS. 3 In support ofthis idea, it was noted that endotoxemia, soft tissue trauma, and pancreatitis have in common their ability to activate intravascular complement and their status as risk factors for AROS. The "complement hypothesis" gained credibility in 1980 when it was reported that the presence in plasma ofa leukocyte aggregant (said to be C5A) accurately predicted subsequent onset of ARDS." Among clinical studies, the study of Hammerschmidt et al" is the cornerstone of the "complement" hypothesis. It seemed at once to definitively establish the pathophysiologic basis of ARDS, and to Mediation of ARDS by Leukocytes (Jean E. Rinaldo)

identify potential victims in an early phase in which preventative therapy could be instituted. Subsequent studies attempted to conBrm this important observation, but failed to do SO.5.6 Weinberg and co-workers5 found that in patients with the sepsis syndrome, complement activation is universal, and has no prediCtive value either for the onset or the subsequent progression of ARDS. Duchateau et al6 reported similar observations suggesting nonspecificity of complement activation in several other predisposing conditions for ARDS. The reason for the discrepancy between these recent studies and the earlier series reported by Hammerschmidt and associates· is uncleat: There are several differences in study designs. More recent studies have tended to employ sensitive radioimmunologic measurements of complement components, while the earlier one assayed C5A indirectly by measuring granulocyte aggregating activity in plasma. The ARDS criteria in the recent studies were also more rigidly defined. Based on these studies, it can be concluded that intravascular complement activation presently has no proven predictive value in identifying incipient ARDS. STUDIES

OF

ARDS PATIENTS USING

BRONCHOALVEOLAR LAVAGE

The "complement" hypothesis describes intravascular events: endothelial cells lining the pulmonary microvasculature are allegedly injured by aggregated, activated, intravascular leukocytes. A major difficulty in proving or disproving this hypothesis is the inaccessibility of the luminal side of the alveolarcapillary membrane to observation and sampling. In contrast, the cellular contents of the airspace side of the alveolar-capillary barrier are easy to sample using bronchoalveolar lavage (BAL). The accessibility of the inflammatory cell populations of the airspaces to sampling, and growing familiarity with the sampling technique, have led to many recent research studies of ARDS patients using BAL. Some caution should temper enthusiasm for these studies. First, even after extensive experience, the contribution of BAL in elucidating the pathophysiology of chronic interstitial lung disease (ILD) remains in dispute. Second, unlike ILD, ARDS is frequently complicated by occult pneumonia. 7 Concurrent pulmonary infection inevitably obscures findings of BAL. Third, appropriate control patients are difficult to identify: in animal models, factors such as mechanical ventilation and oxygen toxicity alter BAL findings per se. Therefore, normal control subjects are not sufficient if abnormalities observed in BAL fluid from ARDS patients are to be attributed to ARDS rather than to respiratory support. Finally, it is difficult to determine to what extent inflammatory cells recovered from airspaces reflect inflammatory events on the luminal side of small

vessels of the lung, where injury is presumably initiated. With these qualifications in mind, it is appropriate to examine available BAL data derived from ARDS patients. Lee et al8 published the first such study. Although the report has been faulted because only/' tracheal washings were performed in some cases, and normal control subjects were used for comparison, it clearly showed elevated recovery of lung neutrophUs from ARDS patients compared with normal patients. Neutrophil elastase, a proteolytic product of neutrophils, was also present, and collagenase9 has since been identified as well. Recently, Parsons et alto have confirmed an abnormal recovery of neutrophils, and isolated chemotactic factors for neutrophils in lavage supernatants from ARDS patients. Abnormalities in soluble antiproteases recovered by BAL from ARDS patients have also been observed. 11 The antiproteases appear to have been rendered inactive by oxidation of methionyl groups which are essential for antiproteolytic activity. Based on these findings, it has been proposed that airspace neutrophils both release proteolytic enzymes and inactivate by oxidation those endogenous antiproteases which might normally exert a protective effect. The studies cited seem to demonstrate unequivocally that neutrophils and their secretory products are present in abnormal numbers in lavageable airsp~ces of ARDS patients. They also suggest potenthese observations tial mechanisms ofinjury. Howeve~ do not prove that the neutrophils are causing injury. It may be plausibly suggested that the bronchoalveolar neutrophils are participating in the phagocytosis of cellular debris or in the process of repair. Several recent studies have tried to establish a causal link between airspace neutrophils and the pathogenesis of ARDS by using statistical methods to relate the recovery of neutrophils in BAL to quantifiable pathophysiologic abnormalities of ARDS such as hypoxemia or protein permeability. Weiland et allJ found a significant correlation between the percentage and absolute numbers of neutrophils recovered by BAL and both the severity of hypoxemia and the concentration of airspace protein in BAL fluid supernatants. The latter measurement was employed as an indicator ofalveolarcapillary permeability. However, Hyers et alJ3 reported that patients in well-defined groups predisposed to ARDS show an influx of neuQuphils into airspaces regardless of whether there was concurrent clinical evidence of ARDS. Thus, recovery of neutrophils by lavage may be (like complement activation) a nonspecific Bnding in predisposed patients rather than being associated with ARDS itsel£ In summary, BAL studies, have not yet forged a conclusive link between neutrophils and pathogenesis of acute lung injury in man. CHEST I 89 I 4 I APRIL, 1986

591

STUDIES OF ARDS IN LEUKOPENIC PATIENTS, AND STUDIES USING BLOOD LEUKOCITES

illness were less able to mount an appropriate leukocytosis in response to sepsis or stress.

A third group of clinical studies has tried to draw a causal link between neutrophils and ARDS based on blood leukocytes. Blood leukocytes can be quantified and studied with relative ease. Also, interactions between blood leukocytes and pulmonary endothelial cells can perhaps be more reasonably inferred than with leukocytes lavaged from airspaces. In animal models, blood neutropenia induced prospectively by chemotherapeutic agents markedly ablates pathophysiologic manifestations of experimental "ARDS."2 It seems reasonable to infer that if ARDS in humans resembles these animal models, patients with a low number ofcirculating blood leukocytes should be relatively "immune" to ARDS. Therefore, considerable recent interest has been focused on the occurrence of ARDS in neutropenic patients. Present data suggest that even profound leukopenia does not preclude the development of ARDS. Patients who have undergone total marrow irradiation to render them candidates for bone marrow transplantation have been reported to have developed ARDS despite apparent bone marrow aplasia. 14 Patients with drug-induced neutropenia appear to have developed ARDS as well. 15 It is possible that leukopenia imparts partial protection, though not complete immunity. One retrospective uncontrolled series observed that in leukopenic patients with ARDS,15 resolution of leukopenia induced by chemotherapy was associated with deterioration ofpulmonary gas exchange. Clinical studies ofthis sort are severely limited by the inability to prove that patients with reduced numbers of leukocytes circulating in blood are truly leukopenic: significant numbers of leukocytes may be present in tissue despite profound blood leukopenia. If activated blood leukocytes in the lung cause ARDS, it WOQld be expected that the state ofactivation of blood leukocytes in ARDS patients would be abnormally high, and that the number ofleukocytes circulating might be at least transiently diminished as they are sequestered in the lung. Both observations have been reported. Zimmerman et al17 observed a higher state of activation ofblood leukocytes in ARDS patients than in critically ill control patients without ARDS. Additional studies confirming this observation are needed. Thommasen et al16 found that in patients with several common dispositions for ARDS, a fall in the number of circulating neutrophils to 4,200 or less had excellent predictive value (SO percent sensitivity, 87 percent specificity) for the subsequent onset of ARDS. It was inferred that the neutropenia in these patients reflected leukocyte sequestration in the lung, but this was not proven by histology or other means. A plausible alternative might be that patients who developed ARDS were those who by virtue of other underlying

PR
592

ENDOTHELIAL CYlUfOXICITY

IN VIVO

Concurrent with the burgeoning number of clinical studies of leukocytes in ARDS, interest of basic investigators in cell-cell interactions between leukocytes and endothelial cells has grown. Recently, the premise that activated neutrophils cause severe injury to endothelial cells in vivo by releasing oxygen radicals and other mediators has underJ,tone closer scrutiny. A major cornerstone to the "complement hypothesis" was the observation of Sacks et al lS that activated leukocytes kill endothelial cells in vitro by generation of oxygen radicals. However, several recent observations have raised the possibility that cytotoxic damage induced by this mechanism is limited by modulating mechanisms in vivo. First, recent studies suggest that biologic surfaces such as endothelial cell surface membranes may interact with activated neutrophils in ways that limit endothelial damage: the presence of an endothelial monolayer was recently shown to markedly inhibit the respiratory burst of activated neutrophils induced by chemotactic agents. 19 Secondly, erythrocytes have been found to be potent scavengers of superoxide anion." Even minute amounts of glutathione, present in erythrocytes, appear to efficiently scavenge oxygen radicals and prevent neutrophil-mediated cytotoxicity. Presumably, this protective mechanism is available within the vascular lumen where erythrocytes are present. Ultrastructural studies have shown that chemotactically driven migration of neutrophils into sheep pulmonary artery endothelial explants occurs without damage to the endothelium. 21 Finally, using the most sensitive biochemical measures of acute lung injury available, Ward et al,22 have demonstrated that oxygen radical-mediated acute lung injury caused by phagocytes following intravascular activation ofthe complement system is evanescent and self-limited. These recent observations support the contention that there are potent protective mechanisms operating in vioo which down-regulate potential neutrophil-induced endothelial cell injury in the lung. Based on similar considerations, it has recently been proposed that interactions between granulocytes and endothelial cells in the lung are not injurious to normal pulmonary endothelium. 23 CONCLUSIONS Neutrophils were shown to be capable of killing other cells in vitro, and seemed to contribute to pulmonary dysfunction in several alleged animal models of ARDS. 2 A single clinical study indicated that evidence of intravascular complement activation predicted the onset of ARDS in man. 4 Based on these observations, the notion that activated circulating Mediation of ARDS by Leukocytes (Jean E. Rinaldo)

blood leukocytes injure the pulmonary vascular endothelium leading to ARDS gained widespread credibility. While this hypothesis has theoretic merit, existing clinical studies have failed to prove it definitively. Recent studies indicate that complement activation is a nonspecific finding in groups predisposed to ARDS, which does not predict whether or not ARDS will develop. Also, ARDS appears to occur in profoundly neutropenic patients. Bronchoalveolar lavage studies of ARDS patients indicate that neutrophils, their secretory products, and chemotactic factors for neutrophils are elevated in airspaces of ARDS patients compared to normal patients. However, whether these neutrophils are markers or mediators of injury or a response to injury is unclear. Many BAL studies have not excluded the possibility that superimposed bronchopulmonary infection or use ofmodalities of respiratory support influenced their findings, and the specificity of BAL neutrophils for ARDS itsel( rather than for conditions which predispose to ARDS, is in dispute. Finally, emerging laboratory investigation in the area of cell-cell interactions between leukocytes and endothelial cells have provided new evidence that endogenous mechanisms protect pulmonary endothelial cells from neutrophil-mediated cytotoxicity in vivo. Anti-inflammatory therapies for ARDS such as corticosteroids have been proposed based on the premise that suppression of leukocyte activation will be of therapeutic value. However, in light of current uncertainty concerning the pathogehic tole of the neutrophil in ARDS, and especially in light of a reported increased rate of infectious complications associated with corticosteroid therapy in ARDS patients,1M controlled prospective trials are urgently indicated before their use can be advocated. Meanwhile, the role of leukocytes in the pathogenesis of ARDS remains a focus of intense research interest. REFERENCES

1 Fowler A, Hamman R, Zerbe G, Benson K, Hyers 1: Adult respiratory distress syndrome: a prognosiS after onset. Am Rev Respir Dis 1985; 132:472-78 2 Tate RM, Repine J. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 128:552-59 3 Jacobs H, Craddock ~ Hammerschmidt D, Moldow C. Complement-induced granulocyte aggregation: An unsuspected mechanism of disease. N Eng} J Med 1980; 302:789-94 4 Hammerschmidt DE, Weaver LJ, Hudson LD, Craddock PR, Jacob HS. Association of complement activation and elevated plasma C5A with adult respiratory distress syndrome: pathophysiologic relevance and possible prognostic value. Lancet 1980; I(April-June):947-49 5 Weinberg ~ Matthay M, Webster R, Roskos K, Goldstein I, Murray J. Biologically active products of complement and acute lung injury in patients with the sepsis syndrome. Am Rev Respir Dis 1984; 130:791-96

6 Duchateau J, Haas M, Schreyen H, Radoux L, Sprangers I, Noel F, et aI. Complement activation in patients at risk of developing the adult respiratory distress syndrome. Am Rev Respir Dis

1984; 130:1058-64

7 Bell R, Coalson J, Smith J, Johanson W. Multiple organ system failure and infection in adult respiratory distress syndrome. Ann Intern Med 1983; 99:293-98 8 Lee CI: Fein AM, Lippman M, Holtzman H, Kimbel ~ Weinbaum G. Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory distress syndrome. N Eng} J Med 1981; 304:192-96 9 Christner ~ Fein A, Goldberg S, et al. Collagenase in the lower respiratory tract of patients with adult respiratory distress syndrome. Am Rev Respir Dis 1985; 131:690-95 10 Parsons ~ Fowler A, Hyers '£ Henson ~ Chemotactic activity in bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome. Am Rev Respir Dis 1985; 132:490-93 11 Cochrane CG, Spragg R, Ravak SO. Pathogenesis of the adult respiratory distress syndrome. J Coo Invest 1983; 71:754-61 12 Weiland JE, Davis WB, Dorinsky PM, Gadek JE. The pathogenesis of ARDS: lung neutrophils predict the severity of both pulmonary edema and gas exchange abnormalities (abstract). Am Rev Respir Dis 1984; 129:112 13 Hyers 1: Fowler A, Stephenson A, Dettenmeier ~ Fisher R, Webster R. The appearance of neutrophils and metabolites or arachadonic acid in bronchial fluid of patients at risk for ARDS. Am Rev Respir Dis 1985; 131(suppl):l35a 14 Braude S, Apperley J, Krausz 1: Goldman J, Royston D. Adult respiratory distress syndrome after allogenic bone-marrow transplantation: evidence for a neutrophil-independent mechanism. Lancet 1985; IOune):I239-41 15 Rinaldo J, Borovetz H. Deterioration of oxygenation and abnormal lung microvascular permeability during resolution ofleukopenia in patients with diffuse lung injury. Am Rev Respir Dis 1985; 4:579-83 16 Thommasen H, Boyko W, Russell J, Hogg J. Thmsient leukopenia associated with adult respiratory distress syndrome. Lancet 1984; I(April-June):809-12 17 Zimmerman G, Renzetti A, Hill H. Functional and metabolic activity of granulOcytes from patients with adult respiratory distress syndrome: evidence for activated neutrophils in the pulmonary circulation. Am Rev Respir Dis 1983; 127:290-300 18 Sacks 'I: Moldow CF, Craddock PR, Bowers TIC, Jacob HS. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes: an in vitro model of immune vascular damage. J Coo Invest 1978; 61:1161-67 19 Fehr J, Moser R, Leppert D, Groscurth ~ Antiadhesive properties of biological surfaces are protective against stimulated granulocytes. J Coo Invest 1985; 76:535-42 20 Asbeck B, Hoidal J, Vercellutti G, Schwartz B, Moldow C, Jacob H. Protection against lethal hyperoxia by tracheal insufBation of erythrocytes: role of red cell glutathione. Science 1985; 227:756-59 21 Meyrick B, Hoffman L, Brigham K. Chemotaxis of granulocytes across bovine pulmonary artery intimal explants without endothelial cell injury. TIssue ~ Cell 1984; 16:1-16 22 Ward ~ lUI G, Hatherill J, Annesley'I: Kunkel R. Systemic complement activation, lung injury, and products oflipid peroxidation. J Clin Invest 1985; 76:517-27 23 Brigham K, Meyrick B. Granulocyte-dependent injury of pulmonary endothelium: a case ofmiscommunication? TIssue & Cell 1984; 16:137-55 24 Weigelt J, Norcross J, Borman K, Snyder W. Early steroid therapy for respiratory failure. Arch Surg 1985; 120:536-39

CHEST I 89 I 4 I APRIL. 1988

593