Functional Loss of Chemotactic Factor Inactivator in the Adult Respiratory Distress Syndrome 1 - 4
RICHARD ROBBINS, RICHARD MAUNDER, GAIL GOSSMAN, TODD KENDALL, LEONARD HUDSON, and STEPHEN RENNARD
Introduction
The adult respiratory distress syndrome (ARDS) is an acute lung disorder characterized by hypoxemia caused by noncardiac pulmonary edema (1-4). Current concepts suggest that this syndrome may be mediated in nonimmunocompromised hosts by an accumulation of neutrophils within the ARDS lung since the presence of neutrophils correlates with the extent of pulmonary dysfunction, and these cells have the potential for initiating and maintaining pulmonary edema by the release of toxic oxygen metabolites and proteases (5-12). Therefore, those factors that lead to the accumulation of neutrophils in the ARDS lung are likely important in understanding the pulmonary dysfunction seen in this disorder (13, 14). One mechanism for the recruitment of neutrophils involvesthe complement system (15). When the complement system is activated, the fifth component of complement (C5) is cleaved to generate C5a, which can direct the migration of neutrophils (16). C5a may be converted to the less chemotactically potent C5a des Arg. However, GcGlobulin (vitamin D binding protein) can function as a cochemotaxin for C5a and C5a ,des Arg by binding to these peptides and enhancing their chemotactic potency (17). Because C5a has been identified in the lower respiratory tract of patients with ARDS, this likely represents one potential source accounting for the presence of neutrophils (6, 9). Activation of the complement system can be modulated by several inhibitors. One inhibitor of C5a-directed neutrophil chemotaxis that has been antigenically and functionally identified in human serum and bronchoalveolar lavage (BAL) fluid has been termed chemotactic factor inactivator (CFI) (17-21). A loss of the activity of this inhibitor in the ARDS lung would likely result in enhanced C5a activity and thus increased accumulation of neutrophils. To test this hypothesis,
SUMMARY The adult respiratory distress syndrome (ARDS) is often characterized by a neutrophilic al\Htolitis, which may be mediated in part by the neutrophil chemoattractant, C5a. Chemotactic factor inactivator (CFI)can decrease C5a-directed neutrophil chemotaxis. Thus, a loss of CFI activity In the ARDS lung could lead to an increased ability of C5a to attract neutrophils. Lung CFI levels were measured antlgenically and functionally In bronchoalveolar lavage (BAL) fluid obtained from 29 patients with ARDS and In 14 normal control subjects. Antigenic levels of CFI were found to be markedly elevated in ARDS BAL fluid (1,855 ± 437 ng/ml) compared with that in normal BAL (29 ± 10 ng/ml, p < 0.005), but, in contrast, CFI functional activity was markedly decreased in ARDS BAL fluid compared with that in normal BAL fluid (31 ± 7% inhibition versus 76 ± 5% inhibition, p < 0.01).Furthermore, although purified CFI readily inhibited the ability of C5a to attract neutrophils (92% inhibition), this activity was decreased when BAL fluid from patients with ARDSwas Incubated with CFI (47 ± 10%, P < 0.01). These findings suggest that patients with ARDS are functionally deficient In CFI, leading to an increased ability of C5a to attract neutrophlls. AM REV RESPIR DIS 1990; 141:1463-1468
antigenic and functional levels of CFI weremeasured in the BAL fluid obtained from patients with ARDS. In comparison to normal subjects, patients with ARDS were found to have elevated antigenic levels of CFI, but a loss of functional activity. These data suggest that one potential factor accounting for the increased presence of neutrophils in the ARDS lung may be the loss of CFI functional activity. Methods Patient Population Subjects were evaluated and studied at either the Harborview Medical Center, Omaha Veterans Administration Medical Center, or the University of Nebraska Medical Center under Institutional ReviewBoard approved protocols and after informed consent. Patients (n = 29) were considered to have ARDS by previously published criteria (6, 22). All patients had hypoxemia requiring an inspired oxygen concentration of ~ 40010 delivery by mechanical ventilation, diffuse pulmonary consolidation on chest roentgenography, and no evidence of a cardiogenic cause of pulmonary edema determined by pulmonary artery wedge pressure < 18ern H 2O. Predisposing causes of ARDS were sepsis syndrome (n = 11), trauma (n = 8), hypertransfusion (n = 8), drug overdose (n = 5), aspiration of gastric contents (n = 4), and near drowning (n = 1). Eight patients had more than one predisposing cause. Sixteen patients were smokers.
Normal nonsmoking volunteers (n = 14) were studied as the control group. None had a history of chest disease, and all normal volunteers had normal physical examinations, roentgenography, and pulmonary function testing.
Source of Biologic Materials Alveolar epithelial lining fluid was obtained by BAL as soon as clinically feasible by previously published methods (6, 23, 24). Briefly, the bronchoscope was passed into a segment of the right middle lobe, right lowerlobe, left lower lobe, or lingula, and five aliquots, each consisting of 20 to 30 ml of normal saline were injected into each of three lobes in the normal subjects and into one to three lobes in the patients with ARDS, followed by gen-
(Received in original form March 3, 1989 and in revised form November 27, 1989) 1 From the Research Service, Omaha Veterans Administration Medical Center, and the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska. 2 Supported in part by a grant from the Veterans Administration and by Grant No. HL-30542 from the National Institutes of Health. 3 Presented in part at the Annual Meeting ofthe American Thoracic Society, Kansas City, Missouri, May 11-14, 1986. 4 Correspondence and requests for reprints should be addressed to Richard A. Robbins, Pulmonary and Critical Care, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105.
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tie suctioning after each injection. The fluids from the lobes were pooled. To separate the fluid from the cells, the BAL fluid was centrifuged (500 g for 5 min), and the supernatant was frozen at - 70° C until used.
Determination of Antigenic CFI Levels An inhibition enzyme-linked immunoabsorbent assay (ELISA) was used to measure antigenic levelsof CFI (21).To accomplish this, it was first necessary to prepare pure CFI and to obtain antibodies to CFI. CFI was isolated from normal human plasma by a modification of the methods of Berenberg and Ward (18) and Kreutzer and coworkers (25). Plasma was sequentially subjected to ammonium sulfate precipitation at 45 and 65070 saturation, molecular sieve chromatography (Sephacryl S-200; Pharmacia, Piscataway, NJ), chromatofocusing (PBE 94; Pharmacia), and hydrophobic chromatography (w-hexyl agarose; Miles Laboratories, Naperville, IL). The CFI obtained by these methods gave a single precipitant line against anti-whole human serum (Meloy Laboratories, Springfield, VA)and a single band on reduced polyacrylamide gel electrophoresis when stained with Coomassie brilliant blue (21). The purified CFI did not contain carboxypeptidase activity as determined by the method of Bokisch and Muller-Eberhard (26).The CFI wasstored at - 70° C until used. Antibodies to CFI were raised in normal rabbits (Sasco, Inc., Omaha, NE) by injecting purified CFI and complete Freund adjuvant (Grand Island Biologicals, Grand Island, NY) and boosting at 2 and 4 wk with the purified CFI and Freund incomplete adjuvant (Grand Island Biologicals). The resulting antisera reacted on Ouchterlony immunodiffusion with the purified CFI and was monospecific on immunoelectrophoresis against normal human serum (21). The antisera were stored at - 70° C until used. After obtaining purified CFI and anti-CFI antibodies, an ELISA was used to determine antigenic levels of CFI (21). The amounts of CFI in the samples were calculated by comparison with known amounts of purified CFI, and the results were analyzed by the methods of Rodbard (27). Because ARDS is accompanied by the extravasation of plasma proteins into the alveolar spaces, CFI concentrations were also expressed relative to albumin (5, 23, 24). The albumin was measured in the bronchoalveolar lavage fluid using a previously described ELISA test (21, 28). Determination of C3a des Arg and C5a des Arg Because ARDS has been associated with complement activation in the lung (6, 9), C3a des Arg and C5a des Arg levels were measured in BAL fluid and in simultaneously collected EDTA-anticoagulated plasma samples from patients with ARDS using a commercially available radioimmunoassay kit (Upjohn Diagnostics, Kalamazoo, MI).
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
Determination of Functional Activity of CFI Functional activity of CFI was determined by the ability of partially purified CFI from the BAL fluid to inhibit C5a-induced neutrophil chemotaxis (21, 29). To accomplish this, C5a was generated from normal human serum by incubating the serum for 45 min at 37° C in the presence of 1.0 M epsilonaminocaproic acid (Sigma Chemical, St. Louis, MO) and zymosan (Sigma) according to the methods of Kreutzer and coworkers (30). The complement-activated serum was then fractionated by gel filtration over Sephadex G-75 (Pharmacia), and the C5a-containing fractions were identified by radioimmunoassay according to the methods of Chenoweth and Hugli (31).Partially purified C5a prepared in this manner contains the cochemotaxin, GcGlobulin, at a concentration sufficient to enhance C5a- or C5a des Arg-directed neutrophil chemotaxis (32). To determine functional activity of CFI, the C5a was combined with partially purified CFI obtained from randomly selected BAL fluids (20, 28). The CFI was partially purified by ammonium sulfate precipitation at 45 and 65070 saturation. The resultant pellet was redissolved in distilled water at onefifth the original volume of BAL fluid and subjected to dialysis overnight against phosphate-buffered saline (PBS). Equal volumes of the partially purified CFI from the BAL fluid and C5a (75 to 100ng/ml) were incubated at 37° C for 30 min. The samples were subsequently assayed for chemotactic activity using purified human neutrophils and a blindwell chamber technique (6, 21, 29, 33). The results were expressed as the number of cells migrating through the filter per high power field. Percent inhibition of C5a-induced neutrophil chemotaxis was determined by comparing the chemotactic activity to C5a in PBS by the methods of Berenberg and Ward (18), and the results werecompared using Student's two-tailed t test. Depletion of CFI from BAL Fluid Todetermine if CFI was an important inhibitor of C5a-induced neutrophil chemotaxis, BAL fluid was first sequentially fractionated by ammonium sulfate fractionation at 45 and 65070 saturation. The three fractions « 45070, 45-65070, and> 65070 saturation) were dissolved in distilled water at one-fifth the original volume and dialyzed overnight against PBS to remove any undissolved ammonium sulfate. Subsequently, the 45-65070 ammonium sulfate fraction was depleted of CFI using anti-CFI IgG bound to Sepharose beads. This was done by preparing rabbit antihuman CFI IgG by precipitating whole antiserum using ammonium sulfate. The antihuman CFI IgG was subsequently coupled to Sepharose-CNBr 4B beads (Pharmacia) according to the manufacturer's directions. The 45-65070 ammonium sulfate saturation fraction was then incubated with an equal volume of the anti-CFI beads for 30 min at 37° C under constant agi-
tation. The beads and the BAL fluid werethen separated by centrifugation, and the CFIdepleted, 45-65070 fraction supernatant was removed. The CFI depletion removed> 90% of the CFI from the 45-65% fraction when evaluated by ELISA. The < 45070, 45-65%, and> 65% ammonium sulfate fractions as wellas the CFI -depleted 45-65% fraction were then evaluated for inhibition of C5a-induced neutrophil chemotaxis as described above.
Determination of the A bility of ARDS BAL Fluid to Functionally Inactivate CFI Because CFI seemed to be partially inactivated in ARDS BAL fluid, the ability of ARDS lavage fluid to inactivate CFI was evaluated. To do this, 30 III of normal or ARDS BAL fluid was incubated with 150 III of partially purified CFI for 30 min at 37° C. Subsequently, 150IIIofC5a (75 to 100ng/ml) were added to the lavage fluid and CFI and again incubated for 30 min at 37° C. The samples were subsequently evaluated for chemotactic activity as described above. The results were expressed as the percent inhibition of C5a-induced neutrophil chemotaxis (17).
Results
Patient Characteristics Bronchoalveolar lavage was performed 31 times in 29 patients an average of 1.8 days after the onset of ARDS (range, 3 h to 6 days). There were 22 men and seven women 19 to 83 yr of age (average, 47 yr); 16 patients were known current cigarette smokers. The average Pao 2/ FIo 2ratio was 169 ± 78, with patients receiving an average F102 of 0.60 ± 0.18 and positive end-expiratory pressure of 11.6 ± 7 em H 20 . Nineteen patients eventually died (66070 mortality). Nonsurvivors had received ventilation an average of 11.6 days and survivors 24 days. BAL Characteristics The average volume of lavage fluid recovered from patients with ARDS did not differ from that of the normal subjects (54 ± 14070 versus 60 ± 10070, p > 0.2). The total inflammatory cell count averaged 72 ± 84 million cells, of which 65 ± 29070 were neutrophils and 30 ± 29070 were macrophages. The average C3a des Arg concentration in BAL fluid was 104 ± 112ng/ml, and in plasma, 371 ± 218 ng/ml. C5a des Arg levels were below the limit of detection in unconcentrated lavage fluid and plasma from most patients with ARDS and undetectable in the normal control subjects « 10 ng/ml). Quantification oj Antigenic CFI Levels The amount of CFI present in BAL fluid was quantified using an ELISA. The
1465
FUNCTION LOSS OF CHEMOTACTIC FACTOR INACTIVATOR IN ARDS
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on's rank sum). Because ARDS is a disorder characterized by an increase in protein concentrations in BAL fluid, CFI levels were also expressed relative to albumin. When CFI levels were expressed in this manner, the patients with ARDS also had significantly greater amounts of CFI than did the normal subjects (figure 1, panel B) (8.1 ± 2.6 versus 0.46 ± 0.1 ug/mg albumin, mean ± SEM; p < 0.01 by Wilcoxon's rank sum). The antigenic elevation of CFI observed in the patients with ARDS could not be explained by degradation of the CFI bound to the polystyrene plates. Incubation of CFI bound to the plates with ARDS BAL fluid for 30 min after removingthe BAL fluid did not change the resultant ELISA curve using purified CFI (data not shown). This suggests that if degradation occurs, it does not alter the major antigenic determinants of CFI recognized by the antiCFI antisera.
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Functional Amounts of CFI Partially purified CFI obtained from the lavage fluids of patients with ARDS and from normal volunteers was found to inhibit the ability of C5a to attract human neutrophils (figure 2). However, in contrast to the antigenic amounts of CFI that were elevated in the ARDS BAL fluid, the functional amounts of CFI were decreased (p < 0.01 compared with those in the normal subjects). Furthermore, there was no correlation between antigenic and functional amounts of CFI (r = 0.15).
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Fig. 3. Ability of bronchoalveolar lavage fluid (SAL) to inhibit C5a-induced chemotaxis before and after depletion of chemotactic factor inactivator (CFI). Percent inhibition of C5a-induced chemotaxis is on the ordinate, and SAL alone and SAL with CFI depleted are on the abscissa (see METHODS for details). Closed circles represent normal SAL and closed triangles represent ARDS SAL. Open circles represent normal SAL after depletion of CFI and open triangles represent ARDS BAL after depletion of CFI.
Relationship of CFI to Clinical and Lavage Characteristics There was no relationship between functional or antigenic CFI levelsand underlying risk condition (sepsis, trauma, etc.), severityof ARDS, or outcome. There was a correlation between the albumin levels and the CFI levels in the ARDS BAL lavage fluid (r = 0.382, p < 0.05). Quantification of the Ability of CFI-depleted BAL Fluid to Inhibit C5a-induced Chemotaxis To determine if CFI was an important inhibitor of C5a-induced chemotaxis in BAL, normal and ARDS BAL fluids werefractionated at < 45070, 45-65010, and > 65% ammonium sulfate saturation. None ofthe < 45% and> 65010 fractions obtained from normal and from ARDS lavage fluids caused significant inhibition of C5a-induced neutrophil chemotaxis (data not shown, p > 0.2). In contrast, the 45-65070 fraction containing CFI caused a significant inhibition of C5ainduced neutrophil chemotaxis (P < 0.001 for each BAL) (figure 3). Depleting the 45-65010 fraction of CFI resulted in a significant decrease in the inhibitory activity compared with the 45-65070 fraction that was not depleted of CFI (p < 0.05, all comparisons). This suggeststhat CFI is
1466
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
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0.2). In contrast, ARDS BAL fluid resulted in a marked loss in the ability of CFI to functionally inhibit C5a-induced neutrophil chemotactic activity (47 ± 100/0 inhibition, p < 0.01). Discussion
Activation of the complement systemcan lead to the cleavage of C5. C5a, a fragment derived from cleavage of C5, at-
tracts neutrophils toward sites of inflammation (16). However, accompanying the influx of neutrophils into inflammatory sites is the extravasation of plasma, which would presumably contain complement proteins including C5. Enzymes released from the neutrophil can cleave C5 to C5a (34), which would result in a spiraling sequence of complement activation and neutrophil accumulation. If this action is unopposed, a massive inflammatory reaction would occur. Because ARDS is a disorder where a massive acute inflammatory response often occurs in the lung (5-7), it seems likely that the mechanism(s) that would normally oppose the acute inflammatory response and subsequent inflammatory response may be inactive. In this context, this study describes the functional inactivation of one inhibitor of C5a, CFI, in the ARDS lung. C5a, a potent neutrophil chemotactic factor, has been proposed to be an important mechanism by which neutrophils are recruited to the ARDS lung (6, 13). The potential for C5a to induce a marked inflammatory response has been documented in the lungs of animals (35-37), and, recently, C5a has been detected in the BAL fluid of patients with ARDS (6, 9). Furthermore, ARDS BAL fluid can generate C5a from C5 in vitro (6, 8). These findings suggest that C5a may be responsible, at least in part, for the massive inflammatory response seen in the lungs of patients with ARDS. If this is true, modulation of C5a activity would be important in the pathogenesis of ARDS. Several inhibitors of C5a have been described. One inhibitor described in normal human plasma and BAL fluid has been determined to be CFI (17-21). Alterations of CFI and other C5a inhibitors have been described in a number of disorders (26, 29, 38-45). It has been proposed that a loss of a C5a inhibitor can contribute to a neutrophil accumulation at sites of inflammation. In support of this concept, Matzner and Brezinski (46) have shown that a deficiency of a C5a inhibitor in patients with familial Mediterranean fever can result in a large inflammatory reaction in peritoneal and joint fluids. CFI may playa similar role in regulating the inflammatory response in the ARDS lung. Inhibitors of C5a other than CFI have been described. Bokischand Muller-Eberhard (26) have described an inhibitor of C5a, anaphylatoxin inactivator (AI), which is a carboxypeptidase and rapidly abolishes the anaphylatoxin activity of
C5a. It would appear that CFI is distinct from AI (18-21,47). Furthermore, the action of AI converts C5a to C5a des Arg, and C5a des Arg retains chemotactic activity for neutrophils; the chemotactic activity of C5a des Arg may be restored to approximate C5a by binding to the cochemotaxin, GcGlobulin (17,47). Thus, it seems likely that inactivation of C5a may require the action of more than a single inactivator. Because CFI was the only C5a inhibitor examined in this study, conclusions as to role of other inhibitors are not possible at this time. The mechanism of CFI inhibition of C5a-directed neutrophil chemotaxis is not known. CFI has been reported to act as an aminopeptidase (48),but recent evidence suggests that CFI may function in binding to GcGlobulin and preventing GcGlobulin from functioning as a cochemotaxin (32). GcGlobulin has been identified in bronchoalveolar lavage fluid, and lavage fluid GcGlobulin can function as a cochemotaxin for C5a (49). Lavage fluid CFI can also bind to GcGlobulin (Robbins RA, unpublished observations), and it seemslikelythat CFI binding to GcGlobulin may explain the capacity of CFI to modulate C5a-directed neutrophil chemotaxis. The source of CFI in the lungs is unknown. CFI is contained in serum or plasma (18-21). Current concepts of ARDS suggest that leakage of plasma into the interstitium and alveolar structure occurs (1-4). This suggests that CFI may be present in the ARDS lung secondary to increased pulmonary vascular permeability. However,local sources of CFI production may also be important. The mechanism of inactivation of CFI in the ARDS lung is also unknown. CFI can be activated by a variety of mechanisms including alterations in pH, neutrophil elastase, and oxidation (31, 50). Although the contribution of each of these factors to CFI inactivation in the lower respiratory tract of patients with ARDS is unknown, the 0 bservation that CFI is inactivated by incubating with ARDS BAL suggests the mechanism is at least in part due to a mechanism other than oxidation. In support of this concept, recent evidence has suggested that CFI may be cleaved in the lower respiratory tract of patients with ARDS, suggesting an enzymatic mechanism of inactivation (50). It is unknown if the inactivation of CFI is specific for ARDS or can occur in other pathophysiologic processes. Recent evidence suggests CFI may be inactivated
FUNCTION LOSS OF CHEMOTACTIC FACTOR INACTIVATOR IN ARDS
by cigarette smoke, but, surprisingly, may be functionally active in the bronchial inflammatory disorder cysticfibrosis (Robbins RA, unpublished observations). In the context of ARDS, the activity of CFI in the lower respiratory tract of intubated patients, in patients with cardiogenic pulmonary edema, and in lower respiratory tract inflammation caused by disorders other than ARDS is unknown and would require further study. Although the present study demonstrated that CFI obtained from BAL fluid of patients with ARDS exhibits a decreased capacity to inhibit C5a-induced neutrophil chemotaxis in vitro, it did not demonstrate that administration of CFI would attenuate ARDS. However,the observation that CFI can suppress the leukocyte infiltration, permeability changes, and hemorrhage associated with acute lung injury in rats provides support for the role of CFI as an important regulator of lung inflammation (51). Furthermore, the present study examined only patients with ARDS. Because C5a likely directs inflammatory reactions in lung disorders other than ARDS, ~ loss of CFI functional activity may accompany other lung disorders. Because ARDS is a clinical disorder often characterized by a large and continued inflammatory response in the lung, loss of CFI activity may be important in understanding the pathogenesis of this disorder. Acknowledgment The writers wish to acknowledge the secretarial assistance of Phyllis Siracusano in the preparation of the manuscript.
References 1. Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distressin adults. Lancet 1967; 2:319-23. 2. Petty TL, Newman JH. Adult respiratory distress syndrome (medicalprogress).WestJ Med 1978; 128:399-407. 3. Fowler AA, Hamman RF, Good JT, et al. Adult respiratory distress syndrome: risk with common predispositions. Ann Intern Med 1983; 98:593-7. 4. Hyers TM. Pathogenesis of the adult respiratory distress syndrome: current concepts. Semin Respir Med 1982; 2:104-8. 5. Weiland JE, Davis WB, Holter JF, Mahammad lR, Dorinsky PM, Gadek IE. Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance. Am Rev Respir Dis 1986; 133:218-25. 6. Robbins RA, Russ WD, Rasmussen JK, Clayton MM. Complement activation in the adult respiratory distress syndrome. Am Rev Respir Dis 1987; 135:651-8. 7. Lee CT, Fein AM, Lepprun M, Holtzman H, . Kimbel P, Weinbaum G. Elastolytic activity in pulmonary lavage fluid from patients with the adult respiratory distress syndrome. N Engl J Med 1981;
304:192-6. 8. McGuire WW, Spragg RG, Cohen AM, Cochrane CG. Studies on the pathogenesis of the adult respiratory distress syndrome. J Clin Invest 1982; 69:543-53. 9. Idell S, Kuich U, Fein A, et al. Neutrophil elastase-releasing factors in bronchoalveolar lavage from patients with respiratory distress syndrome. Am Rev Respir Dis 1985; 132:1098-105. 10. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes: an in vitro model of immune vascular damage. 1 Clin Invest 1978; 61:1161-7. 11. Martin WJ, Gadek lE, Hunninghake GW, Crystal RG. Oxidant injury of lung parenchyma cells. J Clin Invest 1981; 68:1277-88. 12. Cochrane CG, Spragg R, Revak SD. Pathogenesis of the adult respiratory distress syndrome: evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 1983; 71:754-61. 13. Tate RM, Repine lE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 128:552-9. 14. Rinaldo lE, Rogers RM. Adult respiratory distress syndrome: changing concepts of lung injury and repair. N Engl 1 Med 1982; 306:900-9. 15. Ward PA, Cochrane CB, Muller-Eberhard IH. The role of serum complement in chemotaxis of leukocytes. 1 Exp Med 1965; 122:327-46. ' 16. Ward PA, Newman LJ. A neutrophil chemotactic factor from human C5. 1 Immunoll969; 102: 93-9. 17. Kew RR, Webster RD. GcGlobulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J Clin Invest 1988; 82:364-9. 18. Berenberg n, Ward PA. Chemotactic factor inactivator in normal human serum. 1 Clin Invest 1973; 52:1200-6. 19. Till G, Ward PA. lWo distinct chemotactic factor inactivators in human serum. 1 Immunol 1975; 114:843-7. 20. Fantone J, Senior RM, Kreutzer DL, Jones ML, Ward PA. Biochemical quantification of the chemotactic factor inactivator in human serum. J Lab Clin Med 1979; 93:17-24. 21. Robbins RA, Rasmussen lK, Clayton ME, Gossman GL, Kendall Tl, Rennard SI. Antigenic detection of chemotactic factor inactivator in normal human serum and bronchoalveolar lavagefluid. 1 Lab Clin Med 1987; 110:292-9. 22. Pepe PE, P~tkin RT, Reus DH, Hudson LD, Carrico CJ. Clinical predictors of the adult respiratory distress syndrome. Am J Surg 1982; 144: 124-30. 23. Reynolds HY, Newball HH. Analysis of proteins and respiratory cells obtained by bronchial lavage. 1 Lab Clin Med 1974; 84:559-73. 24. Hunninghake GW, Gadek lE, Kawanami 0, Ferrans Vl, Crystal RG. Inflammatory and immune processes in the human lung in health and disease. Am 1 Pathol 1979; 97:149-206. 25. Kreutzer DL, Claypool WD, Jones ML, Ward PA. Isolation by hydrophobic chromatography of the chemotactic factor inactivator from human serum. Clin Immunol Immunopathol 1979; 12: 162-76. 26. Bokisch VA,Miiller-Bberhard Hl. Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase. 1 Clin Invest 1970; 49:2427-36. 27. Rodbard D. Statistical aspects of radioimmunoassays. In: Odell WD, Daughaday WH, eds. Principles of competitive protein-binding assays. Philadelphia: JB Lippincott, 1971; 204-59. 28. Rennard SI, Crystal RG. Fibronectin in hu-
1467 man bronchoalveolar lavage fluid: elevation in patients with interstitial lung disease. 1 Clin Invest 1982; 69:113-22. 29. Robbins RA, Zetterman RK, KendallTJ, Gossman GL, Monsour HP, Rennard SI. Elevation of chemotactic factor inactivator in alcoholic liver disease. Hepatology 1987; 7:872-7. 30. Kreutzer DL, O'Flaherty JT, Orr FW, Showell HJ, Becker EL, Ward PA. Quantitative comparison of the various biological responses of neutrophils to different active and inactive chemotactic factors. Immunopharmacology 1979; 1:39-47. 31. Chenoweth DE, Hugli TE. Techniquesand significance of C3a and C5a measurement. In: Nukumura RM, ed. Future perspectives in clinical laboratory immunoassays. New York: Alan R. Liss, 1980; 443-60. 32. Robbins RA, Gossman GL, NelsonKJ. Chemotactic factor inactivator interaction with GcGlobulin. A mechanism of modulating the chemotactic activity of C5a (abstract). Clin Res 1989;37:418A. 33. Boyum A. Isolation of mononuclear cellsand granulocytes from human blood. Scand J Clin Invest 1968; 21:77-98. 34. Orr FW, Varani 1, Kreutzer DL, Senior RM, Ward PA. Digestion of-the fifth component of complement by leukocyte enzymes. Sequential generation of chemotactic activities for leukocytes and tumor cells. Am 1 Pathol 1979; 94:75-83. 35. Desai 1, Kreutzer DL, Showell CV, Arroyave CU, Ward PA. Acute inflammatory reactions induced by chemotactic factors. Am J Pathol 1979; 96:71-83. 36. Henson PM, McCarthy K, Larsen GL, et al. Complement fragments, alveolar macrophages, and alveolitis. Am 1 Pathol 1979; 97:93-105. 37. Shaw 10, Henson PM, Henson J, WebsterRO. Lung inflammation induced by complementderived chemotactic factors in the alveolus. Lab Invest 1980; 42:547-58. 38. Maderazo EG, Ward PA, Wornick CL, Kubick J, DeGraff AC. Leukotactic dysfunction in sarcoidosis. Ann Intern Med 1976; 84:414-9. 39. Ward PA, Berenberg lL. Defective regulation of inflammatory mediators in Hodgkin's disease. Supernormal levels of chemotactic factor inactivator. N Engl J Med 1974; 190:76-80. 40. Ward PA, Goralnick S, Bullock WE. Defective leukotaxis in patients with lepromatous leprosy. J Lab Clin Med 1976; 87:1025-32. 41. Maderazo EG, Ward PA, Quintiliani R. Defective regulation of chemotaxis in cirrhosis. J Lab Clin Med 1975; 621-30. 42. Ward PA, Thlamo RC. Deficiency of the chemotactic factor inactivator in human sera with ai-antitrypsin deficiency. J Clin Invest 1973; 52: 516-9. 43. Perez HD, Liptor M, Goldstein 1M. A specific inhibitor of complement (C5)-derived chemotactic activity in serum from patients with systemic lupus erythematosus. J Clin Invest 1978;62:29-38. 44. Van Epps DE, Strickland RG, Williams RC. Inhibitors ofleukocyte chemotaxis in alcoholic liver disease. Am 1 Med 1975; 59:200-7. 45. Kreutzer DL, McCormick JR, Thrall RS, Hupp lR, Moore VL, Fink IN. Elevation of serum chemotactic factor inactivator activity during acute inflammatory reactions in patients with hypersensitivity pneumonitis. Am Rev Respir Dis 1982; 125:612-4. 46. Matzner Y, Brezinski A. C5a-inhibitor deficiencyin peritoneal fluids from patients with familial Mediterranean fever. N Eng! 1 Med 1984; 311: 287-90. 47. Webster RO, Hong SR, Johnston RB, Henson PM. Biological effects of the human complement fragments C5a and C5a des Arg on neutrophil
1468 function. Immunopharmacology 1980; 2:201-19. 48. Ward PA, Ozols J. Characterization of the protease activity in the chemotactic factor inactivator. J Clin Invest 1976; 58:123-9. 49. Metcalf JP, Thompson AB, Rennard SI, Robbins RA. GcGlobulin functions as a cochemotax-
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
in in the lower respiratory tract (abstract). Chest 1989; 96:131S. 50. Robbins RA, Haley K, Gossman GL, et at. Functional inhibition of chemotactic factor inactivator by neutrophil elastase (abstract). Clin Res 1988; 36:446A.
51. Johnson KJ, Anderson TP, Ward PA. Suppression of immune complex-induced inflammation by the chemotactic factor inactivator. J Clin Invest 1977; 59:951-8.
RICHARD ROBBINS, RICHARD MAUNDER, GAIL GOSSMAN, TODD KENDALL, LEONARD HUDSON, and STEPHEN RENNARD
Introduction
The adult respiratory distress syndrome (ARDS) is an acute lung disorder characterized by hypoxemia caused by noncardiac pulmonary edema (1-4). Current concepts suggest that this syndrome may be mediated in nonimmunocompromised hosts by an accumulation of neutrophils within the ARDS lung since the presence of neutrophils correlates with the extent of pulmonary dysfunction, and these cells have the potential for initiating and maintaining pulmonary edema by the release of toxic oxygen metabolites and proteases (5-12). Therefore, those factors that lead to the accumulation of neutrophils in the ARDS lung are likely important in understanding the pulmonary dysfunction seen in this disorder (13, 14). One mechanism for the recruitment of neutrophils involvesthe complement system (15). When the complement system is activated, the fifth component of complement (C5) is cleaved to generate C5a, which can direct the migration of neutrophils (16). C5a may be converted to the less chemotactically potent C5a des Arg. However, GcGlobulin (vitamin D binding protein) can function as a cochemotaxin for C5a and C5a ,des Arg by binding to these peptides and enhancing their chemotactic potency (17). Because C5a has been identified in the lower respiratory tract of patients with ARDS, this likely represents one potential source accounting for the presence of neutrophils (6, 9). Activation of the complement system can be modulated by several inhibitors. One inhibitor of C5a-directed neutrophil chemotaxis that has been antigenically and functionally identified in human serum and bronchoalveolar lavage (BAL) fluid has been termed chemotactic factor inactivator (CFI) (17-21). A loss of the activity of this inhibitor in the ARDS lung would likely result in enhanced C5a activity and thus increased accumulation of neutrophils. To test this hypothesis,
SUMMARY The adult respiratory distress syndrome (ARDS) is often characterized by a neutrophilic al\Htolitis, which may be mediated in part by the neutrophil chemoattractant, C5a. Chemotactic factor inactivator (CFI)can decrease C5a-directed neutrophil chemotaxis. Thus, a loss of CFI activity In the ARDS lung could lead to an increased ability of C5a to attract neutrophils. Lung CFI levels were measured antlgenically and functionally In bronchoalveolar lavage (BAL) fluid obtained from 29 patients with ARDS and In 14 normal control subjects. Antigenic levels of CFI were found to be markedly elevated in ARDS BAL fluid (1,855 ± 437 ng/ml) compared with that in normal BAL (29 ± 10 ng/ml, p < 0.005), but, in contrast, CFI functional activity was markedly decreased in ARDS BAL fluid compared with that in normal BAL fluid (31 ± 7% inhibition versus 76 ± 5% inhibition, p < 0.01).Furthermore, although purified CFI readily inhibited the ability of C5a to attract neutrophils (92% inhibition), this activity was decreased when BAL fluid from patients with ARDSwas Incubated with CFI (47 ± 10%, P < 0.01). These findings suggest that patients with ARDS are functionally deficient In CFI, leading to an increased ability of C5a to attract neutrophlls. AM REV RESPIR DIS 1990; 141:1463-1468
antigenic and functional levels of CFI weremeasured in the BAL fluid obtained from patients with ARDS. In comparison to normal subjects, patients with ARDS were found to have elevated antigenic levels of CFI, but a loss of functional activity. These data suggest that one potential factor accounting for the increased presence of neutrophils in the ARDS lung may be the loss of CFI functional activity. Methods Patient Population Subjects were evaluated and studied at either the Harborview Medical Center, Omaha Veterans Administration Medical Center, or the University of Nebraska Medical Center under Institutional ReviewBoard approved protocols and after informed consent. Patients (n = 29) were considered to have ARDS by previously published criteria (6, 22). All patients had hypoxemia requiring an inspired oxygen concentration of ~ 40010 delivery by mechanical ventilation, diffuse pulmonary consolidation on chest roentgenography, and no evidence of a cardiogenic cause of pulmonary edema determined by pulmonary artery wedge pressure < 18ern H 2O. Predisposing causes of ARDS were sepsis syndrome (n = 11), trauma (n = 8), hypertransfusion (n = 8), drug overdose (n = 5), aspiration of gastric contents (n = 4), and near drowning (n = 1). Eight patients had more than one predisposing cause. Sixteen patients were smokers.
Normal nonsmoking volunteers (n = 14) were studied as the control group. None had a history of chest disease, and all normal volunteers had normal physical examinations, roentgenography, and pulmonary function testing.
Source of Biologic Materials Alveolar epithelial lining fluid was obtained by BAL as soon as clinically feasible by previously published methods (6, 23, 24). Briefly, the bronchoscope was passed into a segment of the right middle lobe, right lowerlobe, left lower lobe, or lingula, and five aliquots, each consisting of 20 to 30 ml of normal saline were injected into each of three lobes in the normal subjects and into one to three lobes in the patients with ARDS, followed by gen-
(Received in original form March 3, 1989 and in revised form November 27, 1989) 1 From the Research Service, Omaha Veterans Administration Medical Center, and the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska. 2 Supported in part by a grant from the Veterans Administration and by Grant No. HL-30542 from the National Institutes of Health. 3 Presented in part at the Annual Meeting ofthe American Thoracic Society, Kansas City, Missouri, May 11-14, 1986. 4 Correspondence and requests for reprints should be addressed to Richard A. Robbins, Pulmonary and Critical Care, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105.
1463
1464
tie suctioning after each injection. The fluids from the lobes were pooled. To separate the fluid from the cells, the BAL fluid was centrifuged (500 g for 5 min), and the supernatant was frozen at - 70° C until used.
Determination of Antigenic CFI Levels An inhibition enzyme-linked immunoabsorbent assay (ELISA) was used to measure antigenic levelsof CFI (21).To accomplish this, it was first necessary to prepare pure CFI and to obtain antibodies to CFI. CFI was isolated from normal human plasma by a modification of the methods of Berenberg and Ward (18) and Kreutzer and coworkers (25). Plasma was sequentially subjected to ammonium sulfate precipitation at 45 and 65070 saturation, molecular sieve chromatography (Sephacryl S-200; Pharmacia, Piscataway, NJ), chromatofocusing (PBE 94; Pharmacia), and hydrophobic chromatography (w-hexyl agarose; Miles Laboratories, Naperville, IL). The CFI obtained by these methods gave a single precipitant line against anti-whole human serum (Meloy Laboratories, Springfield, VA)and a single band on reduced polyacrylamide gel electrophoresis when stained with Coomassie brilliant blue (21). The purified CFI did not contain carboxypeptidase activity as determined by the method of Bokisch and Muller-Eberhard (26).The CFI wasstored at - 70° C until used. Antibodies to CFI were raised in normal rabbits (Sasco, Inc., Omaha, NE) by injecting purified CFI and complete Freund adjuvant (Grand Island Biologicals, Grand Island, NY) and boosting at 2 and 4 wk with the purified CFI and Freund incomplete adjuvant (Grand Island Biologicals). The resulting antisera reacted on Ouchterlony immunodiffusion with the purified CFI and was monospecific on immunoelectrophoresis against normal human serum (21). The antisera were stored at - 70° C until used. After obtaining purified CFI and anti-CFI antibodies, an ELISA was used to determine antigenic levels of CFI (21). The amounts of CFI in the samples were calculated by comparison with known amounts of purified CFI, and the results were analyzed by the methods of Rodbard (27). Because ARDS is accompanied by the extravasation of plasma proteins into the alveolar spaces, CFI concentrations were also expressed relative to albumin (5, 23, 24). The albumin was measured in the bronchoalveolar lavage fluid using a previously described ELISA test (21, 28). Determination of C3a des Arg and C5a des Arg Because ARDS has been associated with complement activation in the lung (6, 9), C3a des Arg and C5a des Arg levels were measured in BAL fluid and in simultaneously collected EDTA-anticoagulated plasma samples from patients with ARDS using a commercially available radioimmunoassay kit (Upjohn Diagnostics, Kalamazoo, MI).
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
Determination of Functional Activity of CFI Functional activity of CFI was determined by the ability of partially purified CFI from the BAL fluid to inhibit C5a-induced neutrophil chemotaxis (21, 29). To accomplish this, C5a was generated from normal human serum by incubating the serum for 45 min at 37° C in the presence of 1.0 M epsilonaminocaproic acid (Sigma Chemical, St. Louis, MO) and zymosan (Sigma) according to the methods of Kreutzer and coworkers (30). The complement-activated serum was then fractionated by gel filtration over Sephadex G-75 (Pharmacia), and the C5a-containing fractions were identified by radioimmunoassay according to the methods of Chenoweth and Hugli (31).Partially purified C5a prepared in this manner contains the cochemotaxin, GcGlobulin, at a concentration sufficient to enhance C5a- or C5a des Arg-directed neutrophil chemotaxis (32). To determine functional activity of CFI, the C5a was combined with partially purified CFI obtained from randomly selected BAL fluids (20, 28). The CFI was partially purified by ammonium sulfate precipitation at 45 and 65070 saturation. The resultant pellet was redissolved in distilled water at onefifth the original volume of BAL fluid and subjected to dialysis overnight against phosphate-buffered saline (PBS). Equal volumes of the partially purified CFI from the BAL fluid and C5a (75 to 100ng/ml) were incubated at 37° C for 30 min. The samples were subsequently assayed for chemotactic activity using purified human neutrophils and a blindwell chamber technique (6, 21, 29, 33). The results were expressed as the number of cells migrating through the filter per high power field. Percent inhibition of C5a-induced neutrophil chemotaxis was determined by comparing the chemotactic activity to C5a in PBS by the methods of Berenberg and Ward (18), and the results werecompared using Student's two-tailed t test. Depletion of CFI from BAL Fluid Todetermine if CFI was an important inhibitor of C5a-induced neutrophil chemotaxis, BAL fluid was first sequentially fractionated by ammonium sulfate fractionation at 45 and 65070 saturation. The three fractions « 45070, 45-65070, and> 65070 saturation) were dissolved in distilled water at one-fifth the original volume and dialyzed overnight against PBS to remove any undissolved ammonium sulfate. Subsequently, the 45-65070 ammonium sulfate fraction was depleted of CFI using anti-CFI IgG bound to Sepharose beads. This was done by preparing rabbit antihuman CFI IgG by precipitating whole antiserum using ammonium sulfate. The antihuman CFI IgG was subsequently coupled to Sepharose-CNBr 4B beads (Pharmacia) according to the manufacturer's directions. The 45-65070 ammonium sulfate saturation fraction was then incubated with an equal volume of the anti-CFI beads for 30 min at 37° C under constant agi-
tation. The beads and the BAL fluid werethen separated by centrifugation, and the CFIdepleted, 45-65070 fraction supernatant was removed. The CFI depletion removed> 90% of the CFI from the 45-65% fraction when evaluated by ELISA. The < 45070, 45-65%, and> 65% ammonium sulfate fractions as wellas the CFI -depleted 45-65% fraction were then evaluated for inhibition of C5a-induced neutrophil chemotaxis as described above.
Determination of the A bility of ARDS BAL Fluid to Functionally Inactivate CFI Because CFI seemed to be partially inactivated in ARDS BAL fluid, the ability of ARDS lavage fluid to inactivate CFI was evaluated. To do this, 30 III of normal or ARDS BAL fluid was incubated with 150 III of partially purified CFI for 30 min at 37° C. Subsequently, 150IIIofC5a (75 to 100ng/ml) were added to the lavage fluid and CFI and again incubated for 30 min at 37° C. The samples were subsequently evaluated for chemotactic activity as described above. The results were expressed as the percent inhibition of C5a-induced neutrophil chemotaxis (17).
Results
Patient Characteristics Bronchoalveolar lavage was performed 31 times in 29 patients an average of 1.8 days after the onset of ARDS (range, 3 h to 6 days). There were 22 men and seven women 19 to 83 yr of age (average, 47 yr); 16 patients were known current cigarette smokers. The average Pao 2/ FIo 2ratio was 169 ± 78, with patients receiving an average F102 of 0.60 ± 0.18 and positive end-expiratory pressure of 11.6 ± 7 em H 20 . Nineteen patients eventually died (66070 mortality). Nonsurvivors had received ventilation an average of 11.6 days and survivors 24 days. BAL Characteristics The average volume of lavage fluid recovered from patients with ARDS did not differ from that of the normal subjects (54 ± 14070 versus 60 ± 10070, p > 0.2). The total inflammatory cell count averaged 72 ± 84 million cells, of which 65 ± 29070 were neutrophils and 30 ± 29070 were macrophages. The average C3a des Arg concentration in BAL fluid was 104 ± 112ng/ml, and in plasma, 371 ± 218 ng/ml. C5a des Arg levels were below the limit of detection in unconcentrated lavage fluid and plasma from most patients with ARDS and undetectable in the normal control subjects « 10 ng/ml). Quantification oj Antigenic CFI Levels The amount of CFI present in BAL fluid was quantified using an ELISA. The
1465
FUNCTION LOSS OF CHEMOTACTIC FACTOR INACTIVATOR IN ARDS
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curves generated by the purified CFI, normal BAL fluid, and ARDS BAL fluid gave the typical S-shaped curve seen with an inhibition ELISA test (data not shown). The similarity of the shape of the curves suggests that purified CFI and the CFI present in the BAL fluids are antigenically similar. CFI concentrations were significantly higher in the BAL fluid of patients with ARDS than in that of normal subjects (figure 1, panel A) (p < 0.005 by Wilcox100 CI)
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on's rank sum). Because ARDS is a disorder characterized by an increase in protein concentrations in BAL fluid, CFI levels were also expressed relative to albumin. When CFI levels were expressed in this manner, the patients with ARDS also had significantly greater amounts of CFI than did the normal subjects (figure 1, panel B) (8.1 ± 2.6 versus 0.46 ± 0.1 ug/mg albumin, mean ± SEM; p < 0.01 by Wilcoxon's rank sum). The antigenic elevation of CFI observed in the patients with ARDS could not be explained by degradation of the CFI bound to the polystyrene plates. Incubation of CFI bound to the plates with ARDS BAL fluid for 30 min after removingthe BAL fluid did not change the resultant ELISA curve using purified CFI (data not shown). This suggests that if degradation occurs, it does not alter the major antigenic determinants of CFI recognized by the antiCFI antisera.
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Functional Amounts of CFI Partially purified CFI obtained from the lavage fluids of patients with ARDS and from normal volunteers was found to inhibit the ability of C5a to attract human neutrophils (figure 2). However, in contrast to the antigenic amounts of CFI that were elevated in the ARDS BAL fluid, the functional amounts of CFI were decreased (p < 0.01 compared with those in the normal subjects). Furthermore, there was no correlation between antigenic and functional amounts of CFI (r = 0.15).
BAl -CFI
Fig. 3. Ability of bronchoalveolar lavage fluid (SAL) to inhibit C5a-induced chemotaxis before and after depletion of chemotactic factor inactivator (CFI). Percent inhibition of C5a-induced chemotaxis is on the ordinate, and SAL alone and SAL with CFI depleted are on the abscissa (see METHODS for details). Closed circles represent normal SAL and closed triangles represent ARDS SAL. Open circles represent normal SAL after depletion of CFI and open triangles represent ARDS BAL after depletion of CFI.
Relationship of CFI to Clinical and Lavage Characteristics There was no relationship between functional or antigenic CFI levelsand underlying risk condition (sepsis, trauma, etc.), severityof ARDS, or outcome. There was a correlation between the albumin levels and the CFI levels in the ARDS BAL lavage fluid (r = 0.382, p < 0.05). Quantification of the Ability of CFI-depleted BAL Fluid to Inhibit C5a-induced Chemotaxis To determine if CFI was an important inhibitor of C5a-induced chemotaxis in BAL, normal and ARDS BAL fluids werefractionated at < 45070, 45-65010, and > 65% ammonium sulfate saturation. None ofthe < 45% and> 65010 fractions obtained from normal and from ARDS lavage fluids caused significant inhibition of C5a-induced neutrophil chemotaxis (data not shown, p > 0.2). In contrast, the 45-65070 fraction containing CFI caused a significant inhibition of C5ainduced neutrophil chemotaxis (P < 0.001 for each BAL) (figure 3). Depleting the 45-65010 fraction of CFI resulted in a significant decrease in the inhibitory activity compared with the 45-65070 fraction that was not depleted of CFI (p < 0.05, all comparisons). This suggeststhat CFI is
1466
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
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0.2). In contrast, ARDS BAL fluid resulted in a marked loss in the ability of CFI to functionally inhibit C5a-induced neutrophil chemotactic activity (47 ± 100/0 inhibition, p < 0.01). Discussion
Activation of the complement systemcan lead to the cleavage of C5. C5a, a fragment derived from cleavage of C5, at-
tracts neutrophils toward sites of inflammation (16). However, accompanying the influx of neutrophils into inflammatory sites is the extravasation of plasma, which would presumably contain complement proteins including C5. Enzymes released from the neutrophil can cleave C5 to C5a (34), which would result in a spiraling sequence of complement activation and neutrophil accumulation. If this action is unopposed, a massive inflammatory reaction would occur. Because ARDS is a disorder where a massive acute inflammatory response often occurs in the lung (5-7), it seems likely that the mechanism(s) that would normally oppose the acute inflammatory response and subsequent inflammatory response may be inactive. In this context, this study describes the functional inactivation of one inhibitor of C5a, CFI, in the ARDS lung. C5a, a potent neutrophil chemotactic factor, has been proposed to be an important mechanism by which neutrophils are recruited to the ARDS lung (6, 13). The potential for C5a to induce a marked inflammatory response has been documented in the lungs of animals (35-37), and, recently, C5a has been detected in the BAL fluid of patients with ARDS (6, 9). Furthermore, ARDS BAL fluid can generate C5a from C5 in vitro (6, 8). These findings suggest that C5a may be responsible, at least in part, for the massive inflammatory response seen in the lungs of patients with ARDS. If this is true, modulation of C5a activity would be important in the pathogenesis of ARDS. Several inhibitors of C5a have been described. One inhibitor described in normal human plasma and BAL fluid has been determined to be CFI (17-21). Alterations of CFI and other C5a inhibitors have been described in a number of disorders (26, 29, 38-45). It has been proposed that a loss of a C5a inhibitor can contribute to a neutrophil accumulation at sites of inflammation. In support of this concept, Matzner and Brezinski (46) have shown that a deficiency of a C5a inhibitor in patients with familial Mediterranean fever can result in a large inflammatory reaction in peritoneal and joint fluids. CFI may playa similar role in regulating the inflammatory response in the ARDS lung. Inhibitors of C5a other than CFI have been described. Bokischand Muller-Eberhard (26) have described an inhibitor of C5a, anaphylatoxin inactivator (AI), which is a carboxypeptidase and rapidly abolishes the anaphylatoxin activity of
C5a. It would appear that CFI is distinct from AI (18-21,47). Furthermore, the action of AI converts C5a to C5a des Arg, and C5a des Arg retains chemotactic activity for neutrophils; the chemotactic activity of C5a des Arg may be restored to approximate C5a by binding to the cochemotaxin, GcGlobulin (17,47). Thus, it seems likely that inactivation of C5a may require the action of more than a single inactivator. Because CFI was the only C5a inhibitor examined in this study, conclusions as to role of other inhibitors are not possible at this time. The mechanism of CFI inhibition of C5a-directed neutrophil chemotaxis is not known. CFI has been reported to act as an aminopeptidase (48),but recent evidence suggests that CFI may function in binding to GcGlobulin and preventing GcGlobulin from functioning as a cochemotaxin (32). GcGlobulin has been identified in bronchoalveolar lavage fluid, and lavage fluid GcGlobulin can function as a cochemotaxin for C5a (49). Lavage fluid CFI can also bind to GcGlobulin (Robbins RA, unpublished observations), and it seemslikelythat CFI binding to GcGlobulin may explain the capacity of CFI to modulate C5a-directed neutrophil chemotaxis. The source of CFI in the lungs is unknown. CFI is contained in serum or plasma (18-21). Current concepts of ARDS suggest that leakage of plasma into the interstitium and alveolar structure occurs (1-4). This suggests that CFI may be present in the ARDS lung secondary to increased pulmonary vascular permeability. However,local sources of CFI production may also be important. The mechanism of inactivation of CFI in the ARDS lung is also unknown. CFI can be activated by a variety of mechanisms including alterations in pH, neutrophil elastase, and oxidation (31, 50). Although the contribution of each of these factors to CFI inactivation in the lower respiratory tract of patients with ARDS is unknown, the 0 bservation that CFI is inactivated by incubating with ARDS BAL suggests the mechanism is at least in part due to a mechanism other than oxidation. In support of this concept, recent evidence has suggested that CFI may be cleaved in the lower respiratory tract of patients with ARDS, suggesting an enzymatic mechanism of inactivation (50). It is unknown if the inactivation of CFI is specific for ARDS or can occur in other pathophysiologic processes. Recent evidence suggests CFI may be inactivated
FUNCTION LOSS OF CHEMOTACTIC FACTOR INACTIVATOR IN ARDS
by cigarette smoke, but, surprisingly, may be functionally active in the bronchial inflammatory disorder cysticfibrosis (Robbins RA, unpublished observations). In the context of ARDS, the activity of CFI in the lower respiratory tract of intubated patients, in patients with cardiogenic pulmonary edema, and in lower respiratory tract inflammation caused by disorders other than ARDS is unknown and would require further study. Although the present study demonstrated that CFI obtained from BAL fluid of patients with ARDS exhibits a decreased capacity to inhibit C5a-induced neutrophil chemotaxis in vitro, it did not demonstrate that administration of CFI would attenuate ARDS. However,the observation that CFI can suppress the leukocyte infiltration, permeability changes, and hemorrhage associated with acute lung injury in rats provides support for the role of CFI as an important regulator of lung inflammation (51). Furthermore, the present study examined only patients with ARDS. Because C5a likely directs inflammatory reactions in lung disorders other than ARDS, ~ loss of CFI functional activity may accompany other lung disorders. Because ARDS is a clinical disorder often characterized by a large and continued inflammatory response in the lung, loss of CFI activity may be important in understanding the pathogenesis of this disorder. Acknowledgment The writers wish to acknowledge the secretarial assistance of Phyllis Siracusano in the preparation of the manuscript.
References 1. Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distressin adults. Lancet 1967; 2:319-23. 2. Petty TL, Newman JH. Adult respiratory distress syndrome (medicalprogress).WestJ Med 1978; 128:399-407. 3. Fowler AA, Hamman RF, Good JT, et al. Adult respiratory distress syndrome: risk with common predispositions. Ann Intern Med 1983; 98:593-7. 4. Hyers TM. Pathogenesis of the adult respiratory distress syndrome: current concepts. Semin Respir Med 1982; 2:104-8. 5. Weiland JE, Davis WB, Holter JF, Mahammad lR, Dorinsky PM, Gadek IE. Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance. Am Rev Respir Dis 1986; 133:218-25. 6. Robbins RA, Russ WD, Rasmussen JK, Clayton MM. Complement activation in the adult respiratory distress syndrome. Am Rev Respir Dis 1987; 135:651-8. 7. Lee CT, Fein AM, Lepprun M, Holtzman H, . Kimbel P, Weinbaum G. Elastolytic activity in pulmonary lavage fluid from patients with the adult respiratory distress syndrome. N Engl J Med 1981;
304:192-6. 8. McGuire WW, Spragg RG, Cohen AM, Cochrane CG. Studies on the pathogenesis of the adult respiratory distress syndrome. J Clin Invest 1982; 69:543-53. 9. Idell S, Kuich U, Fein A, et al. Neutrophil elastase-releasing factors in bronchoalveolar lavage from patients with respiratory distress syndrome. Am Rev Respir Dis 1985; 132:1098-105. 10. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes: an in vitro model of immune vascular damage. 1 Clin Invest 1978; 61:1161-7. 11. Martin WJ, Gadek lE, Hunninghake GW, Crystal RG. Oxidant injury of lung parenchyma cells. J Clin Invest 1981; 68:1277-88. 12. Cochrane CG, Spragg R, Revak SD. Pathogenesis of the adult respiratory distress syndrome: evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 1983; 71:754-61. 13. Tate RM, Repine lE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 128:552-9. 14. Rinaldo lE, Rogers RM. Adult respiratory distress syndrome: changing concepts of lung injury and repair. N Engl 1 Med 1982; 306:900-9. 15. Ward PA, Cochrane CB, Muller-Eberhard IH. The role of serum complement in chemotaxis of leukocytes. 1 Exp Med 1965; 122:327-46. ' 16. Ward PA, Newman LJ. A neutrophil chemotactic factor from human C5. 1 Immunoll969; 102: 93-9. 17. Kew RR, Webster RD. GcGlobulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J Clin Invest 1988; 82:364-9. 18. Berenberg n, Ward PA. Chemotactic factor inactivator in normal human serum. 1 Clin Invest 1973; 52:1200-6. 19. Till G, Ward PA. lWo distinct chemotactic factor inactivators in human serum. 1 Immunol 1975; 114:843-7. 20. Fantone J, Senior RM, Kreutzer DL, Jones ML, Ward PA. Biochemical quantification of the chemotactic factor inactivator in human serum. J Lab Clin Med 1979; 93:17-24. 21. Robbins RA, Rasmussen lK, Clayton ME, Gossman GL, Kendall Tl, Rennard SI. Antigenic detection of chemotactic factor inactivator in normal human serum and bronchoalveolar lavagefluid. 1 Lab Clin Med 1987; 110:292-9. 22. Pepe PE, P~tkin RT, Reus DH, Hudson LD, Carrico CJ. Clinical predictors of the adult respiratory distress syndrome. Am J Surg 1982; 144: 124-30. 23. Reynolds HY, Newball HH. Analysis of proteins and respiratory cells obtained by bronchial lavage. 1 Lab Clin Med 1974; 84:559-73. 24. Hunninghake GW, Gadek lE, Kawanami 0, Ferrans Vl, Crystal RG. Inflammatory and immune processes in the human lung in health and disease. Am 1 Pathol 1979; 97:149-206. 25. Kreutzer DL, Claypool WD, Jones ML, Ward PA. Isolation by hydrophobic chromatography of the chemotactic factor inactivator from human serum. Clin Immunol Immunopathol 1979; 12: 162-76. 26. Bokisch VA,Miiller-Bberhard Hl. Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase. 1 Clin Invest 1970; 49:2427-36. 27. Rodbard D. Statistical aspects of radioimmunoassays. In: Odell WD, Daughaday WH, eds. Principles of competitive protein-binding assays. Philadelphia: JB Lippincott, 1971; 204-59. 28. Rennard SI, Crystal RG. Fibronectin in hu-
1467 man bronchoalveolar lavage fluid: elevation in patients with interstitial lung disease. 1 Clin Invest 1982; 69:113-22. 29. Robbins RA, Zetterman RK, KendallTJ, Gossman GL, Monsour HP, Rennard SI. Elevation of chemotactic factor inactivator in alcoholic liver disease. Hepatology 1987; 7:872-7. 30. Kreutzer DL, O'Flaherty JT, Orr FW, Showell HJ, Becker EL, Ward PA. Quantitative comparison of the various biological responses of neutrophils to different active and inactive chemotactic factors. Immunopharmacology 1979; 1:39-47. 31. Chenoweth DE, Hugli TE. Techniquesand significance of C3a and C5a measurement. In: Nukumura RM, ed. Future perspectives in clinical laboratory immunoassays. New York: Alan R. Liss, 1980; 443-60. 32. Robbins RA, Gossman GL, NelsonKJ. Chemotactic factor inactivator interaction with GcGlobulin. A mechanism of modulating the chemotactic activity of C5a (abstract). Clin Res 1989;37:418A. 33. Boyum A. Isolation of mononuclear cellsand granulocytes from human blood. Scand J Clin Invest 1968; 21:77-98. 34. Orr FW, Varani 1, Kreutzer DL, Senior RM, Ward PA. Digestion of-the fifth component of complement by leukocyte enzymes. Sequential generation of chemotactic activities for leukocytes and tumor cells. Am 1 Pathol 1979; 94:75-83. 35. Desai 1, Kreutzer DL, Showell CV, Arroyave CU, Ward PA. Acute inflammatory reactions induced by chemotactic factors. Am J Pathol 1979; 96:71-83. 36. Henson PM, McCarthy K, Larsen GL, et al. Complement fragments, alveolar macrophages, and alveolitis. Am 1 Pathol 1979; 97:93-105. 37. Shaw 10, Henson PM, Henson J, WebsterRO. Lung inflammation induced by complementderived chemotactic factors in the alveolus. Lab Invest 1980; 42:547-58. 38. Maderazo EG, Ward PA, Wornick CL, Kubick J, DeGraff AC. Leukotactic dysfunction in sarcoidosis. Ann Intern Med 1976; 84:414-9. 39. Ward PA, Berenberg lL. Defective regulation of inflammatory mediators in Hodgkin's disease. Supernormal levels of chemotactic factor inactivator. N Engl J Med 1974; 190:76-80. 40. Ward PA, Goralnick S, Bullock WE. Defective leukotaxis in patients with lepromatous leprosy. J Lab Clin Med 1976; 87:1025-32. 41. Maderazo EG, Ward PA, Quintiliani R. Defective regulation of chemotaxis in cirrhosis. J Lab Clin Med 1975; 621-30. 42. Ward PA, Thlamo RC. Deficiency of the chemotactic factor inactivator in human sera with ai-antitrypsin deficiency. J Clin Invest 1973; 52: 516-9. 43. Perez HD, Liptor M, Goldstein 1M. A specific inhibitor of complement (C5)-derived chemotactic activity in serum from patients with systemic lupus erythematosus. J Clin Invest 1978;62:29-38. 44. Van Epps DE, Strickland RG, Williams RC. Inhibitors ofleukocyte chemotaxis in alcoholic liver disease. Am 1 Med 1975; 59:200-7. 45. Kreutzer DL, McCormick JR, Thrall RS, Hupp lR, Moore VL, Fink IN. Elevation of serum chemotactic factor inactivator activity during acute inflammatory reactions in patients with hypersensitivity pneumonitis. Am Rev Respir Dis 1982; 125:612-4. 46. Matzner Y, Brezinski A. C5a-inhibitor deficiencyin peritoneal fluids from patients with familial Mediterranean fever. N Eng! 1 Med 1984; 311: 287-90. 47. Webster RO, Hong SR, Johnston RB, Henson PM. Biological effects of the human complement fragments C5a and C5a des Arg on neutrophil
1468 function. Immunopharmacology 1980; 2:201-19. 48. Ward PA, Ozols J. Characterization of the protease activity in the chemotactic factor inactivator. J Clin Invest 1976; 58:123-9. 49. Metcalf JP, Thompson AB, Rennard SI, Robbins RA. GcGlobulin functions as a cochemotax-
ROBBINS, MAUNDER, GOSSMAN, KENDALL, HUDSON, AND RENNARD
in in the lower respiratory tract (abstract). Chest 1989; 96:131S. 50. Robbins RA, Haley K, Gossman GL, et at. Functional inhibition of chemotactic factor inactivator by neutrophil elastase (abstract). Clin Res 1988; 36:446A.
51. Johnson KJ, Anderson TP, Ward PA. Suppression of immune complex-induced inflammation by the chemotactic factor inactivator. J Clin Invest 1977; 59:951-8.
