Role of Granulocyte Elastase in Tissue Injury in Patients with Septic Shock Complicated by
Multiple-organ Failure
HIROSHI TANAKA, M.D., HISASHI SUGIMOTO, M.D., TOSHIHARU YOSHIOKA, M.D., and TSUYOSHI SUGIMOTO, M.D.
To better understand the role of granulocyte elastase (GE) in mediating tissue injury during sepsis, GE levels were measured in plasma and bronchoalveolar lavage fluid (BALF) in patients with septic shock (n = 16) and hemorrhagic shock (n = 30). Granulocyte elastase levels were compared to levels of al-protease inhibitor (al-PI). Results show that although plasma GEal-PI complex was initially elevated in patients with hemorrhagic and septic shock, elevations in plasma GE-al-PI complex (831 + 241 sg/L) persisted in septic shock patients. al-Protease inhibitor levels in serum were increased, resulting in an inhibition of serum GE activity. Granulocyte elastase activity in BALF, however, was significantly higher in those patients with septic, as compared to hemorrhagic shock (31.4 ± 25.8 versus 3.7 ± 4.0 U/L, respectively). In addition GE levels were compared to other parameters, including respiratory index, blood neutrophil count, and plasma levels of endotoxin, fibronectin, and coagulation factor XIII. Significant correlations were observed between GE-al-PI and increased endotoxin concentration and decreased fibronectin and coagulation factor XIII levels. Significant correlation was found also between GE activity in BALF and respiratory index. These findings suggest that severe tissue damage occurred in patients with septic shock complicated by multiple-organ failure. Although GE activity appeared to be adequately inhibited by al-PI in blood, increased GE activity in local tissues, such as lung alveoli, may be responsible for significant local tissue injury during septic shock.
From the Department of Traumatology, Osaka University Medical School, Osaka, Japan
ulated by one of any number of factors, including endotoxin, opsonized immunocomplexes, or complement,4 GE is released. Therefore it plays a significant role in the degradation of foreign matter.5 Granulocyte elastase is a potent proteolytic agent, however, and is capable of lysing a wide variety of normal tissue substrates, including clotting and fibrinolytic factors, fibronectin, and tissue components.6
Granulocyte elastase also is believed to be involved in the pathogenesis of certain clinical disorders, including pulmonary emphysema due to congenital aI -protease inhibitor (a 1-PI) deficiency7 or cigarette smoking.8 Recent studies using an enzyme-linked immunosorbent assay (ELISA) found high plasma concentrations of GE-al -PI complex in patients with adult respiratory distress syndrome, rheumatoid arthritis,9 and some infectious diseases.'0 However the proteolytic activity of GE in blood has not been evaluated because the GE measured usually is complexed with a 1 -PI. Thus the role of GE in tissue injury has not been clarified fully. A new method has been developed in our laboratory to measure GE activity in tissue fluids. " We use the chromogenic synthetic substrate L-pyroglutamyl-L-prolyl-Lvaline-p-nitroanilide (L-Glu-Pro-Val-pNA), which is more sensitive and specific for GE than those previously reported. In this study we measured serial changes in the plasma levels of GE-al-PI complex in patients with septic shock complicated by MOF and patients with hemorrhagic shock, and compared these levels with several other parameters. In addition we measured GE hydrolytic activity and a l-PI activity in blood and bronchoalveolar lavage fluid (BALF) and evaluated the role of GE in tissue injury.
M s ULTIPLE-ORGAN FAILURE (MOF) sometimes occurs in patients suffering severe trauma or
burns complicated by uncontrolled infection.' However the pathophysiology of MOF is not well understood. Tissue hypoxia and such chemical mediators as oxygen radicals and proteases may be involved in the development of MOF.2'3 Granulocyte elastase (GE), a key participant in the inflammatory response, functions in the mediation of host responses to inflammation. When neutrophils are stim-
Address reprint requests to Hiroshi Tanaka, M.D., Department of Anesthesiology, 3635 Vista Avenue at Grand Boulevard, P.O. Box 15250, St. Louis, MO 63110-0250. Accepted for publication March 6, 1990.
81
82
Ann. Surg. * January 1991
TANAKA AND OTHERS
Patients and Methods Patients
Plasma levels of GE-a 1-PI complex were measured in 46 patients who were admitted to department from September 1986 to September 1988. These patients were divided into two groups. Group 1 consisted of 16 patients with septic shock complicated by MOF, and group 2 consisted of 30 patients with hemorrhagic shock secondary to severe injury. In group 1 (patients with septic shock) there were 6 trauma patients, 4 burn patients, and 6 patients with panperitonitis. Shock continued for more than 7 days and all patients in group 1 had associated multipleorgan failure. All ofthe patients in group 2 were admitted with hemorrhagic shock, and all recovered from shock within 24 hours of blood transfusion and/or surgery. The diagnosis of sepsis was based on the presence of two or more of the following criteria: (1) fever of more than 39 C for more than 3 days; (2) a leukocyte counts of more than 1 5,000/L; (3) a positive limulus test for endotoxin; (4) a positive blood culture; and (5) culture of the same organisms from an intravenous catheter tip and the focus of infection. Septic shock in septic patients was diagnosed by the following criteria: systolic blood pressure of less than 90 mmHg, decreased urine output (less than 1 mL/ kg/hr), and a cardiac index of more than 4.5 or less than 2.5 L/min/m2. A diagnosis of hemorrhagic shock was made using previously reported criteria.'2
Blood Samples In group 1 blood samples were obtained with the diagnosis of septic shock (day 0), and on days, 1, 2, 3, 5, and 7 thereafter. In group 2 samples were obtained on admission (in hemorrhagic shock) and at follow-up intervals identical to those in group 1. They were immediately separated and stored at -70 C until the time of assay.
added to 200 ,uL of sample and the reaction was initiated by the addition of200 uL ofsubstrate solution containing L-Glu-Pro-Val-pNA at 37 C. After incubation for 3 hours, diazotization was performed to enhance sensitivity after the addition of 500 ,L of each of the following: 0.04% (w/v) sodium nitrite in 0.48 mol/L (molar) HCI, 0.3% (w/v) ammonium sulfamate, and 0.07% (w/v) N-l-naphthylethylenediamine dihydrochloride. Absorbance was measured at 545 nm after diazotization. Bronchoalveolar lavage fluid was obtained on day 7 after diagnosis of shock from 11 patients in group 1 and 12 in group 2, according to the method of Lee et al.'3 Twenty-five milliliters of sterile 0.9% NaCl was instilled through an endotracheal tube and aspirated by suction. The cells, debris, and mucus were removed by centrifugation at 3000 RPM for 10 minutes.
alI-PI Activity and Concentration a 1-Protease inhibitor activity in serum and BALF was measured as follows. Fifty microliters of sample was diluted with 1000 ,tL of buffer (0.5 mol/L TRIS, pH 8.0) containing 0.15 mol/L methylamine hydrochloride. The latter was used to hamper the effect of a2-macroglobulin. An excess of trypsin (20 ,mol/L [micromolar], 2 mmol/ L HC1, 10 mmol/L CaCl2) was added to 50 ,uL of diluted sample. During the incubation that followed, trypsin was inhibited by 1-PI in the sample. The remaining trypsin was measured with the use of the chromogenic substrate L-benzoylarginine p-nitroanilide. After incubation for 20 minutes, absorbance was measured at 410 nm. Serum al-PI concentration was measured by single radial immunodiffusion. a
Other Parameters Several other parameters were measured at the same time intervals after the diagnosis of shock. The respiratory index was calculated by AaDO2/PaO2, where AaDO2 is the alveolar-arterial oxygen difference, and PaO2 is the
Plasma GE-a l-PI Complex Plasma levels of GE-al-PI complex were determined with an ELISA kit (E. Merck, Darmstadt, FRG). The values are reported as the concentration of complexed elastase inhibited by l-PI and not active.
pg/l 1,000-
GE Activity in Serum and BALF
L-Glu-Pro-Val-pNA (Kabi Diagnostica, Stockholm, Sweden) was dissolved in dimethyl sulfoxide and diluted four times with distilled water. The final concentration of the substrate was 2 mmol/L (millimolar). Granulocyte elastase activity was assayed by a new method developed in our laboratory. Two hundred microliters of buffer (0.1 mol/L [molar] TRIS, 0.96 mol/L NaCl, pH 8.3) was "
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FIG. 1. Plasma levels of GE-a I-PI (Ag/L) in groups 1 (0) and 2 (0) measured at the diagnosis of shock (day 0), and days 1, 2, 3, 5, and 7 thereafter. Normal GE-a 1-PI values are represented by the shaded area.
mg/di
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83
GRANULOCYTE ELASTASE IN TISSUE INJURY
Vol. 213 * No. I
FL
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5
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6 ~ 7d ay FIG. 2. Serial changes in a I-PI concentration (a) and activitiy (0) after diagnosis of shock in group 1. Normal values are represented by the shaded area. 3
5
arterial partial pressure of oxygen.'4 Neutrophils were counted by an automated blood cell counter (CC-120, Sysmex, Towa Medical, Hyogo, Japan) and WrightGiemsa staining. Endotoxin was measured in plasma by conventional chromogenic limulus testing'5 (Seikagaku Kogyo Co., Ltd., Tokyo, Japan). Plasma fibronectin was measured by single radial immunodiffusion and coagulation factor XIII was measured by the method of Bohn. 16
Statistical Analysis All values are expressed as mean plus the standard deviation (SD). The data were analyzed by means of linear regression analysis and Student's t test. Differences were considered significant at the p < 0.05 level.
Results GE-a l-PI Complex in Blood Normal plasma GE-al-PI values ranged from 21 to 165 ,ug/L. Figure 1 shows the serial changes in GE-a 1-PI in both patient group. Levels in group 1 were elevated (831 ± 241 ,Ag/L) at diagnosis of septic shock and remained high during the ensuing week. In group 2 GEa l-PI levels were high during hemorrhagic shock (574 + 131 ug/L) but quickly returned to normal at recovery.
> 20-
'-p cm
_
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1 group; group 2 group 1 BALF Serum FIG. 4. Mean hydrolytic GE activity (U/L) in BALF and serum in the two groups on day 7 after diagnosis of shock. *p < 0.05, group 1 versus group 2.
al-PI Concentration and Activity in Blood Figure 2 illustrates the serial changes in a 1-PI concentration and activity in group 1. Both measures increased to twice the normal range at the time of the diagnosis of shock. In group 2 both measures decreased during shock but increased after recovery and thereafter persisted at twice the normal levels (Fig. 3). GE and al-PI Activity in BALF and Blood Figure 4 shows the hydrolytic GE activity in BALF and serum in the two groups on day 7. Mean GE activity in BALF was significantly higher in group 1 (31.4 ± 25.8 U/ L) than in group 2 (3.7 ± 4.0 U/L). Granulocyte elastase activity in blood was barely detected in either group. 0
7-
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-a
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area.
2o
40 GE activity
60
80 u/I
FIG. 5. Correlation between GE activity and the respiratory index. *, 1; 0, group 2; r = 0.86, p < 0.05.
group
Ann. Surg. * January 1991 TANAKA AND OTHERS 84 TABLE 1. Relationships Between Plasma GE-a l-PI Concentration and Granulocyte elastase activity in BALF and the respiratory Neutrophil Count, Endotoxin, Fibronectin, and index, as shown in Figure 5, were significantly and posiCoagulation Factor XIII Levels tively correlated (r = 0.86, p < 0.05). Figure 6 shows the GE-a 1-PI Group 2 Group 1 al-PI activity in BALF and serum on the seventh day following the diagnosis of shock. In both groups, al-PI Neutrophil activity was lower in BALF than in serum. NS (Xl03/mm3) 9413 ± 5437 8057 ± 3553
Neutrophils, Endotoxin, Fibronectin, and Coagulation Factor XIII in Blood Table 1 summarizes the averaged data from days 3 and 7 for blood neutrophil counts, and plasma endotoxin, fibronectin, and coagulation factor XIII and their relationship to plasma GE-al-PI levels. There was no significant difference in the number of neutrophils between the two groups and no relationship between the levels of GE-al PI and the neutrophil counts. However the ratio of GEal-PI to neutrophils was significantly higher in group 1, as was the mean endotoxin concentration in blood (33 + 12 pg/mL). In group 2 the latter was within the normal range at 6.4 ± 6.3 pg/mL. In addition there was a significant correlation between the endotoxin and GE-al-PI levels (r = 0.68, p < 0.05). In group 1 the fibronectin level decreased to 16.7 ± 6.1 mg/dL, while fibronectin remained within the normal range (27.6 ± 6.6 mg/dL) in group 2. There was a significant negative correlation between fibronectin and GEal-PI (r = -0.71, p < 0.05). In group 1 coagulation factor XIII decreased during shock to 34% ± 10.1%. In group 2 this factor decreased during shock but soon recovered to 46.4% ± 10.7%. There was a significant negative correlation between coagulation factor XIII and GE-a l-PI (r = -0.81, p < 0.05). Discussion The results of this study demonstrate that plasma GEal-PI complex is elevated in patients with shock. In hemu/ml
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group 2 group 1 group 2 group 1 Serum BALF FIG. 6. Mean l-PI activity (U/mL) in BALF and serum in the two groups on day 7 after diagnosis of shock. a
Endotoxin (pg/mL) Fibronectin (mg/dL) Factor XIII (%)
33.2 ± 12.2*
6.4 ± 6.3
16.7 ± 6.1* 34.0 ± 10.1*
27.6 ± 6.6 46.4 ± 10.7
r = 0.68, p < 0.05 r = -0.71, p < 0.05 r = -0.81, p < 0.05
p < 0.05, group 1 versus group 2. Values are expressed as the mean ± SD of blood samples obtained from all patients on days 3 and 7 after diagnosis of shock. *
orrhagic shock due to severe injury (group 2), plasma GEa l-PI increased during shock but returned to normal at recovery. In contrast a marked increase in plasma GEal -PI was observed in patients in septic shock complicated by MOF but GE activity was not measurable in the blood in either group. This indicates that GE activity was neutralized by a 1-PI, an acute-phase reactant and potent inhibitor of GE.'7 In healthy individuals GE is regulated by their ample inhibitory capacity outside the cell. However neutrophils, when stimulated, release oxygen radicals that create a zone of oxidation around them, forming a microenvironment within which alI-PI is inactivated.'8 In our earlier study we demonstrated that GE hydrolytic activity increases in the fluid of inflamed tissue, such as synovial fluid of patients with rheumatoid arthritis and blister fluid in burn patients." In this study we found an increase in GE activity in BALF in patients with septic shock complicated by MOF. These patients had a high respiratory index, and the correlation between GE activity and the respiratory index was significant. In addition there was little a l-PI activity in BALF. These findings suggest that although GE activity is suppressed by increased a 1PI in blood, it may be significantly active in tissues in which it is not neutralized by a specific inhibitor. Respiratory failure occurs frequently in septic shock. It is thought that neutrophils migrate to the lung in response to a chemotactic factor, where they then release mediators, such as GE or superoxide, that cause destruction of lung tissues. There was no relationship between the GE-al -PI level and the neutrophil count. Therefore the release of GE into the circulation is unrelated to an increase in circulating neutrophils. We found a significant correlation between the GE-a l-PI concentration and the amount of endotoxin in blood. In patients suffering from sepsis, endotoxin may stimulate neutrophils directly to release GE. Smedly et al.'9 demonstrated that neutrophils stimulated by a low concentration of lipopolysaccharide with C5a or chemotactic factor produced marked endothelial injury. They concluded that these factors caused the release of GE from neutrophils.
Vol. 213-No.l
GRANULOCYTE ELASTASE IN TISSUE INJURY
In this study the increase in GE-a 1-PI was significantly correlated with decreases in the levels of fibronectin and coagulation factor XIII. In vitro, GE has been shown to degrade various connective tissue components, including elastin, collagen, and proteoglycan,20,21 as well as fibrinolytic and coagulation factors, complement, fibronectin, and some transport proteins.22'23 Fibronectin, a glycoprotein, plays a major role in cell conjugation and cell-mediated tissue repair.24 Fibronectin levels were within the normal range in our hemorrhagic shock patients but were significantly low in those with septic shock. In addition there was a significant negative correlation between the GE-al-PI level and that of fibronectin. Coagulation factor XIII is an important factor in tissue repair. 25 Coagulation factor XIII was low in group 1 and was also significantly negatively related to levels of GEa 1-PT. These findings suggest that GE may degrade these substrates in vivo and thereby cause tissue damage. Severe tissue injury occurred in patients with septic shock complicated by MOF. In these patients fibronectin and coagulation factor XIII were decreased, in which case, tissue repair and regrowth could not be expected. Although al-PI increased and GE activity was inhibited in blood, GE activity was enhanced in BALF. These results suggest that GE plays a significant role in the local destruction ofinflamed tissue and causes severe tissue damage in patients with septic shock and MOF.
References 1. Fry DE, Pearlstein L, Fulton RL, et al. Multiple system organ failure: the role of uncontrolled infection. Arch Surg 1980; 115:136-140. 2. Eiseman B, Sloan R, Hansbrough J, et al. Multiple organ failure: clinical and experimental. Am Surg 1980; 46:14-19. 3. Carrico CJ, Meakins JL, Marshall JC, et al. Multiple-organ-failure syndrome. Arch Surg 1986; 121:196-208. 4. Weissmann G, Zurier RB, Hoffstein S. Leukocytic proteases and the immunologic release of lysosomal enzymes. Am J Pathol 1972; 68:539-559. 5. Fritz H, Jochum M, Duswald KH, et al. Granulocyte proteinases as mediators of unspecific proteolysis in inflammation. A review. In Proteinases in Inflammation and Tumor Invasion. New York: Walter De Gruyter, 1986, pp 1-24. 6. Janoff A. Elastase in tissue injury. Annu Rev Med 1985; 36:207216. 7. Kaplan PD, Kuhn C, Pierce JA. The induction of emphysema with elastase. 1. The evolution of the lesion and the influence of serum. J Lab Clin Med 1973; 82:349-357.
85
8. Smith SF, Guz A, Cooke NT, et al. Extracellular elastolytic activity in human lung lavage: a comparative study between smokers and non-smokers. Clin Sci 1985; 69:17-27. 9. Adeyemi EO, Hull RG, Chadwick VS, et al. Circulating human leukocyte elastase in rheumatoid arthritis. Rheumatol Int 1986; 6:57-60. 10. Duswald KH, Jochum M, Fritz H, et al. Released granulocytic elastase: an indicator of pathobiochemical alterations in septicemia after abdominal surgery. Surgery 1985; 98:892-899. 11. Tanaka H, Yoshioka T, Sugimoto T, et al. A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Chin Chim Acta 1990; 187:173-180. 12. Sugimoto T, Ogawa M, Shimazaki S, et al. Klinische Untersuchung uber die Schockorgane in Beziehung zur Schockdauer. Anaesthesist 1976; 25:51-55. 13. Lee C, Fein AM, Lippmann M, et al. Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome. N Engl J Med 1981; 304:192-196. 14. Goldfarb MA, Ciurej TF, McAslan TC, et al. Tracking respiratory therapy in the trauma patient. Am J Surg 1975; 129:255-258. 15. Ikegami K, Ikemura K, Sugimoto T, et al. Early diagnosis of invasive candidiasis and rapid evaluation of antifungal therapy by combined use of conventional chromogenic limulus test and a newly developed endotoxin specific assay. J Trauma 1988; 28:11181126. 16. Bohn H. Isolierung und Charakterisierung des fibrinstabilisierenden Faktors aus menschlichen Thrombozyten. Thrombos Diathes Haemorrh 1970; 23:454-468. 17. Ohlsson K, Olsson I. The neutral proteases of human granulocytes: isolation and partial characterization of granulocyte elastases. Eur J Biochem 1974; 42:519-527. 18. Carrell RW. al-Antitrypsin: molecular pathology, leukocytes, and tissue damage. J Clin Invest 1986; 78:1427-1431. 19. Smedly LA, Tonnesen MG, Worthen GS, et al. Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 1986; 77:12331243. 20. Keiser H, Greenwald RA, Janoff A. Degradation of cartilage proteoglycan by human leukocyte granule neutral protease. A model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan. J Clin Invest 1976; 57:625-632. 21. Mainardi CL, Hasty DL, Kang AH. Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase. J Biol Chem 1980; 255:12006-12010. 22. Jochum M, Witte J, Fritz H, et al. Clotting and other plasma factors in experimental endotoxemia: inhibition of degradation by exogenous proteinase inhibitors. Eur Surg Res 1981; 13:152-168. 23. Gramse M, Havemann K, Egbring R. Alpha-2-plasmin-inhibitor inactivation by human granulocyte elastase. Adv Exp Med Biol 1984; 167:253-261. 24. Plosher DF, Schad PE, Kleinman HK. Cross-linking of fibronectin to collagen by blood coagulation factor XIII. J Clin Invest 1979; 64:781-787. 25. Mosher DF. Action of fibrin-stabilizing factor on cold-insoluble globulin and a2-macroglobulin in clotting plasma. J Biol Chem 1976; 251:1639-1645.
Multiple-organ Failure
HIROSHI TANAKA, M.D., HISASHI SUGIMOTO, M.D., TOSHIHARU YOSHIOKA, M.D., and TSUYOSHI SUGIMOTO, M.D.
To better understand the role of granulocyte elastase (GE) in mediating tissue injury during sepsis, GE levels were measured in plasma and bronchoalveolar lavage fluid (BALF) in patients with septic shock (n = 16) and hemorrhagic shock (n = 30). Granulocyte elastase levels were compared to levels of al-protease inhibitor (al-PI). Results show that although plasma GEal-PI complex was initially elevated in patients with hemorrhagic and septic shock, elevations in plasma GE-al-PI complex (831 + 241 sg/L) persisted in septic shock patients. al-Protease inhibitor levels in serum were increased, resulting in an inhibition of serum GE activity. Granulocyte elastase activity in BALF, however, was significantly higher in those patients with septic, as compared to hemorrhagic shock (31.4 ± 25.8 versus 3.7 ± 4.0 U/L, respectively). In addition GE levels were compared to other parameters, including respiratory index, blood neutrophil count, and plasma levels of endotoxin, fibronectin, and coagulation factor XIII. Significant correlations were observed between GE-al-PI and increased endotoxin concentration and decreased fibronectin and coagulation factor XIII levels. Significant correlation was found also between GE activity in BALF and respiratory index. These findings suggest that severe tissue damage occurred in patients with septic shock complicated by multiple-organ failure. Although GE activity appeared to be adequately inhibited by al-PI in blood, increased GE activity in local tissues, such as lung alveoli, may be responsible for significant local tissue injury during septic shock.
From the Department of Traumatology, Osaka University Medical School, Osaka, Japan
ulated by one of any number of factors, including endotoxin, opsonized immunocomplexes, or complement,4 GE is released. Therefore it plays a significant role in the degradation of foreign matter.5 Granulocyte elastase is a potent proteolytic agent, however, and is capable of lysing a wide variety of normal tissue substrates, including clotting and fibrinolytic factors, fibronectin, and tissue components.6
Granulocyte elastase also is believed to be involved in the pathogenesis of certain clinical disorders, including pulmonary emphysema due to congenital aI -protease inhibitor (a 1-PI) deficiency7 or cigarette smoking.8 Recent studies using an enzyme-linked immunosorbent assay (ELISA) found high plasma concentrations of GE-al -PI complex in patients with adult respiratory distress syndrome, rheumatoid arthritis,9 and some infectious diseases.'0 However the proteolytic activity of GE in blood has not been evaluated because the GE measured usually is complexed with a 1 -PI. Thus the role of GE in tissue injury has not been clarified fully. A new method has been developed in our laboratory to measure GE activity in tissue fluids. " We use the chromogenic synthetic substrate L-pyroglutamyl-L-prolyl-Lvaline-p-nitroanilide (L-Glu-Pro-Val-pNA), which is more sensitive and specific for GE than those previously reported. In this study we measured serial changes in the plasma levels of GE-al-PI complex in patients with septic shock complicated by MOF and patients with hemorrhagic shock, and compared these levels with several other parameters. In addition we measured GE hydrolytic activity and a l-PI activity in blood and bronchoalveolar lavage fluid (BALF) and evaluated the role of GE in tissue injury.
M s ULTIPLE-ORGAN FAILURE (MOF) sometimes occurs in patients suffering severe trauma or
burns complicated by uncontrolled infection.' However the pathophysiology of MOF is not well understood. Tissue hypoxia and such chemical mediators as oxygen radicals and proteases may be involved in the development of MOF.2'3 Granulocyte elastase (GE), a key participant in the inflammatory response, functions in the mediation of host responses to inflammation. When neutrophils are stim-
Address reprint requests to Hiroshi Tanaka, M.D., Department of Anesthesiology, 3635 Vista Avenue at Grand Boulevard, P.O. Box 15250, St. Louis, MO 63110-0250. Accepted for publication March 6, 1990.
81
82
Ann. Surg. * January 1991
TANAKA AND OTHERS
Patients and Methods Patients
Plasma levels of GE-a 1-PI complex were measured in 46 patients who were admitted to department from September 1986 to September 1988. These patients were divided into two groups. Group 1 consisted of 16 patients with septic shock complicated by MOF, and group 2 consisted of 30 patients with hemorrhagic shock secondary to severe injury. In group 1 (patients with septic shock) there were 6 trauma patients, 4 burn patients, and 6 patients with panperitonitis. Shock continued for more than 7 days and all patients in group 1 had associated multipleorgan failure. All ofthe patients in group 2 were admitted with hemorrhagic shock, and all recovered from shock within 24 hours of blood transfusion and/or surgery. The diagnosis of sepsis was based on the presence of two or more of the following criteria: (1) fever of more than 39 C for more than 3 days; (2) a leukocyte counts of more than 1 5,000/L; (3) a positive limulus test for endotoxin; (4) a positive blood culture; and (5) culture of the same organisms from an intravenous catheter tip and the focus of infection. Septic shock in septic patients was diagnosed by the following criteria: systolic blood pressure of less than 90 mmHg, decreased urine output (less than 1 mL/ kg/hr), and a cardiac index of more than 4.5 or less than 2.5 L/min/m2. A diagnosis of hemorrhagic shock was made using previously reported criteria.'2
Blood Samples In group 1 blood samples were obtained with the diagnosis of septic shock (day 0), and on days, 1, 2, 3, 5, and 7 thereafter. In group 2 samples were obtained on admission (in hemorrhagic shock) and at follow-up intervals identical to those in group 1. They were immediately separated and stored at -70 C until the time of assay.
added to 200 ,uL of sample and the reaction was initiated by the addition of200 uL ofsubstrate solution containing L-Glu-Pro-Val-pNA at 37 C. After incubation for 3 hours, diazotization was performed to enhance sensitivity after the addition of 500 ,L of each of the following: 0.04% (w/v) sodium nitrite in 0.48 mol/L (molar) HCI, 0.3% (w/v) ammonium sulfamate, and 0.07% (w/v) N-l-naphthylethylenediamine dihydrochloride. Absorbance was measured at 545 nm after diazotization. Bronchoalveolar lavage fluid was obtained on day 7 after diagnosis of shock from 11 patients in group 1 and 12 in group 2, according to the method of Lee et al.'3 Twenty-five milliliters of sterile 0.9% NaCl was instilled through an endotracheal tube and aspirated by suction. The cells, debris, and mucus were removed by centrifugation at 3000 RPM for 10 minutes.
alI-PI Activity and Concentration a 1-Protease inhibitor activity in serum and BALF was measured as follows. Fifty microliters of sample was diluted with 1000 ,tL of buffer (0.5 mol/L TRIS, pH 8.0) containing 0.15 mol/L methylamine hydrochloride. The latter was used to hamper the effect of a2-macroglobulin. An excess of trypsin (20 ,mol/L [micromolar], 2 mmol/ L HC1, 10 mmol/L CaCl2) was added to 50 ,uL of diluted sample. During the incubation that followed, trypsin was inhibited by 1-PI in the sample. The remaining trypsin was measured with the use of the chromogenic substrate L-benzoylarginine p-nitroanilide. After incubation for 20 minutes, absorbance was measured at 410 nm. Serum al-PI concentration was measured by single radial immunodiffusion. a
Other Parameters Several other parameters were measured at the same time intervals after the diagnosis of shock. The respiratory index was calculated by AaDO2/PaO2, where AaDO2 is the alveolar-arterial oxygen difference, and PaO2 is the
Plasma GE-a l-PI Complex Plasma levels of GE-al-PI complex were determined with an ELISA kit (E. Merck, Darmstadt, FRG). The values are reported as the concentration of complexed elastase inhibited by l-PI and not active.
pg/l 1,000-
GE Activity in Serum and BALF
L-Glu-Pro-Val-pNA (Kabi Diagnostica, Stockholm, Sweden) was dissolved in dimethyl sulfoxide and diluted four times with distilled water. The final concentration of the substrate was 2 mmol/L (millimolar). Granulocyte elastase activity was assayed by a new method developed in our laboratory. Two hundred microliters of buffer (0.1 mol/L [molar] TRIS, 0.96 mol/L NaCl, pH 8.3) was "
4T
i4
a
ts
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II
a. 0
500r
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1
2
3
4
5
6
7 day
FIG. 1. Plasma levels of GE-a I-PI (Ag/L) in groups 1 (0) and 2 (0) measured at the diagnosis of shock (day 0), and days 1, 2, 3, 5, and 7 thereafter. Normal GE-a 1-PI values are represented by the shaded area.
mg/di
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u/ml 20
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:::::::::::::::-::::::::::::::::::::: ::-:
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83
GRANULOCYTE ELASTASE IN TISSUE INJURY
Vol. 213 * No. I
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arterial partial pressure of oxygen.'4 Neutrophils were counted by an automated blood cell counter (CC-120, Sysmex, Towa Medical, Hyogo, Japan) and WrightGiemsa staining. Endotoxin was measured in plasma by conventional chromogenic limulus testing'5 (Seikagaku Kogyo Co., Ltd., Tokyo, Japan). Plasma fibronectin was measured by single radial immunodiffusion and coagulation factor XIII was measured by the method of Bohn. 16
Statistical Analysis All values are expressed as mean plus the standard deviation (SD). The data were analyzed by means of linear regression analysis and Student's t test. Differences were considered significant at the p < 0.05 level.
Results GE-a l-PI Complex in Blood Normal plasma GE-al-PI values ranged from 21 to 165 ,ug/L. Figure 1 shows the serial changes in GE-a 1-PI in both patient group. Levels in group 1 were elevated (831 ± 241 ,Ag/L) at diagnosis of septic shock and remained high during the ensuing week. In group 2 GEa l-PI levels were high during hemorrhagic shock (574 + 131 ug/L) but quickly returned to normal at recovery.
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1 group; group 2 group 1 BALF Serum FIG. 4. Mean hydrolytic GE activity (U/L) in BALF and serum in the two groups on day 7 after diagnosis of shock. *p < 0.05, group 1 versus group 2.
al-PI Concentration and Activity in Blood Figure 2 illustrates the serial changes in a 1-PI concentration and activity in group 1. Both measures increased to twice the normal range at the time of the diagnosis of shock. In group 2 both measures decreased during shock but increased after recovery and thereafter persisted at twice the normal levels (Fig. 3). GE and al-PI Activity in BALF and Blood Figure 4 shows the hydrolytic GE activity in BALF and serum in the two groups on day 7. Mean GE activity in BALF was significantly higher in group 1 (31.4 ± 25.8 U/ L) than in group 2 (3.7 ± 4.0 U/L). Granulocyte elastase activity in blood was barely detected in either group. 0
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FIG. 5. Correlation between GE activity and the respiratory index. *, 1; 0, group 2; r = 0.86, p < 0.05.
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Ann. Surg. * January 1991 TANAKA AND OTHERS 84 TABLE 1. Relationships Between Plasma GE-a l-PI Concentration and Granulocyte elastase activity in BALF and the respiratory Neutrophil Count, Endotoxin, Fibronectin, and index, as shown in Figure 5, were significantly and posiCoagulation Factor XIII Levels tively correlated (r = 0.86, p < 0.05). Figure 6 shows the GE-a 1-PI Group 2 Group 1 al-PI activity in BALF and serum on the seventh day following the diagnosis of shock. In both groups, al-PI Neutrophil activity was lower in BALF than in serum. NS (Xl03/mm3) 9413 ± 5437 8057 ± 3553
Neutrophils, Endotoxin, Fibronectin, and Coagulation Factor XIII in Blood Table 1 summarizes the averaged data from days 3 and 7 for blood neutrophil counts, and plasma endotoxin, fibronectin, and coagulation factor XIII and their relationship to plasma GE-al-PI levels. There was no significant difference in the number of neutrophils between the two groups and no relationship between the levels of GE-al PI and the neutrophil counts. However the ratio of GEal-PI to neutrophils was significantly higher in group 1, as was the mean endotoxin concentration in blood (33 + 12 pg/mL). In group 2 the latter was within the normal range at 6.4 ± 6.3 pg/mL. In addition there was a significant correlation between the endotoxin and GE-al-PI levels (r = 0.68, p < 0.05). In group 1 the fibronectin level decreased to 16.7 ± 6.1 mg/dL, while fibronectin remained within the normal range (27.6 ± 6.6 mg/dL) in group 2. There was a significant negative correlation between fibronectin and GEal-PI (r = -0.71, p < 0.05). In group 1 coagulation factor XIII decreased during shock to 34% ± 10.1%. In group 2 this factor decreased during shock but soon recovered to 46.4% ± 10.7%. There was a significant negative correlation between coagulation factor XIII and GE-a l-PI (r = -0.81, p < 0.05). Discussion The results of this study demonstrate that plasma GEal-PI complex is elevated in patients with shock. In hemu/ml
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group 2 group 1 group 2 group 1 Serum BALF FIG. 6. Mean l-PI activity (U/mL) in BALF and serum in the two groups on day 7 after diagnosis of shock. a
Endotoxin (pg/mL) Fibronectin (mg/dL) Factor XIII (%)
33.2 ± 12.2*
6.4 ± 6.3
16.7 ± 6.1* 34.0 ± 10.1*
27.6 ± 6.6 46.4 ± 10.7
r = 0.68, p < 0.05 r = -0.71, p < 0.05 r = -0.81, p < 0.05
p < 0.05, group 1 versus group 2. Values are expressed as the mean ± SD of blood samples obtained from all patients on days 3 and 7 after diagnosis of shock. *
orrhagic shock due to severe injury (group 2), plasma GEa l-PI increased during shock but returned to normal at recovery. In contrast a marked increase in plasma GEal -PI was observed in patients in septic shock complicated by MOF but GE activity was not measurable in the blood in either group. This indicates that GE activity was neutralized by a 1-PI, an acute-phase reactant and potent inhibitor of GE.'7 In healthy individuals GE is regulated by their ample inhibitory capacity outside the cell. However neutrophils, when stimulated, release oxygen radicals that create a zone of oxidation around them, forming a microenvironment within which alI-PI is inactivated.'8 In our earlier study we demonstrated that GE hydrolytic activity increases in the fluid of inflamed tissue, such as synovial fluid of patients with rheumatoid arthritis and blister fluid in burn patients." In this study we found an increase in GE activity in BALF in patients with septic shock complicated by MOF. These patients had a high respiratory index, and the correlation between GE activity and the respiratory index was significant. In addition there was little a l-PI activity in BALF. These findings suggest that although GE activity is suppressed by increased a 1PI in blood, it may be significantly active in tissues in which it is not neutralized by a specific inhibitor. Respiratory failure occurs frequently in septic shock. It is thought that neutrophils migrate to the lung in response to a chemotactic factor, where they then release mediators, such as GE or superoxide, that cause destruction of lung tissues. There was no relationship between the GE-al -PI level and the neutrophil count. Therefore the release of GE into the circulation is unrelated to an increase in circulating neutrophils. We found a significant correlation between the GE-a l-PI concentration and the amount of endotoxin in blood. In patients suffering from sepsis, endotoxin may stimulate neutrophils directly to release GE. Smedly et al.'9 demonstrated that neutrophils stimulated by a low concentration of lipopolysaccharide with C5a or chemotactic factor produced marked endothelial injury. They concluded that these factors caused the release of GE from neutrophils.
Vol. 213-No.l
GRANULOCYTE ELASTASE IN TISSUE INJURY
In this study the increase in GE-a 1-PI was significantly correlated with decreases in the levels of fibronectin and coagulation factor XIII. In vitro, GE has been shown to degrade various connective tissue components, including elastin, collagen, and proteoglycan,20,21 as well as fibrinolytic and coagulation factors, complement, fibronectin, and some transport proteins.22'23 Fibronectin, a glycoprotein, plays a major role in cell conjugation and cell-mediated tissue repair.24 Fibronectin levels were within the normal range in our hemorrhagic shock patients but were significantly low in those with septic shock. In addition there was a significant negative correlation between the GE-al-PI level and that of fibronectin. Coagulation factor XIII is an important factor in tissue repair. 25 Coagulation factor XIII was low in group 1 and was also significantly negatively related to levels of GEa 1-PT. These findings suggest that GE may degrade these substrates in vivo and thereby cause tissue damage. Severe tissue injury occurred in patients with septic shock complicated by MOF. In these patients fibronectin and coagulation factor XIII were decreased, in which case, tissue repair and regrowth could not be expected. Although al-PI increased and GE activity was inhibited in blood, GE activity was enhanced in BALF. These results suggest that GE plays a significant role in the local destruction ofinflamed tissue and causes severe tissue damage in patients with septic shock and MOF.
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8. Smith SF, Guz A, Cooke NT, et al. Extracellular elastolytic activity in human lung lavage: a comparative study between smokers and non-smokers. Clin Sci 1985; 69:17-27. 9. Adeyemi EO, Hull RG, Chadwick VS, et al. Circulating human leukocyte elastase in rheumatoid arthritis. Rheumatol Int 1986; 6:57-60. 10. Duswald KH, Jochum M, Fritz H, et al. Released granulocytic elastase: an indicator of pathobiochemical alterations in septicemia after abdominal surgery. Surgery 1985; 98:892-899. 11. Tanaka H, Yoshioka T, Sugimoto T, et al. A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Chin Chim Acta 1990; 187:173-180. 12. Sugimoto T, Ogawa M, Shimazaki S, et al. Klinische Untersuchung uber die Schockorgane in Beziehung zur Schockdauer. Anaesthesist 1976; 25:51-55. 13. Lee C, Fein AM, Lippmann M, et al. Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome. N Engl J Med 1981; 304:192-196. 14. Goldfarb MA, Ciurej TF, McAslan TC, et al. Tracking respiratory therapy in the trauma patient. Am J Surg 1975; 129:255-258. 15. Ikegami K, Ikemura K, Sugimoto T, et al. Early diagnosis of invasive candidiasis and rapid evaluation of antifungal therapy by combined use of conventional chromogenic limulus test and a newly developed endotoxin specific assay. J Trauma 1988; 28:11181126. 16. Bohn H. Isolierung und Charakterisierung des fibrinstabilisierenden Faktors aus menschlichen Thrombozyten. Thrombos Diathes Haemorrh 1970; 23:454-468. 17. Ohlsson K, Olsson I. The neutral proteases of human granulocytes: isolation and partial characterization of granulocyte elastases. Eur J Biochem 1974; 42:519-527. 18. Carrell RW. al-Antitrypsin: molecular pathology, leukocytes, and tissue damage. J Clin Invest 1986; 78:1427-1431. 19. Smedly LA, Tonnesen MG, Worthen GS, et al. Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 1986; 77:12331243. 20. Keiser H, Greenwald RA, Janoff A. Degradation of cartilage proteoglycan by human leukocyte granule neutral protease. A model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan. J Clin Invest 1976; 57:625-632. 21. Mainardi CL, Hasty DL, Kang AH. Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase. J Biol Chem 1980; 255:12006-12010. 22. Jochum M, Witte J, Fritz H, et al. Clotting and other plasma factors in experimental endotoxemia: inhibition of degradation by exogenous proteinase inhibitors. Eur Surg Res 1981; 13:152-168. 23. Gramse M, Havemann K, Egbring R. Alpha-2-plasmin-inhibitor inactivation by human granulocyte elastase. Adv Exp Med Biol 1984; 167:253-261. 24. Plosher DF, Schad PE, Kleinman HK. Cross-linking of fibronectin to collagen by blood coagulation factor XIII. J Clin Invest 1979; 64:781-787. 25. Mosher DF. Action of fibrin-stabilizing factor on cold-insoluble globulin and a2-macroglobulin in clotting plasma. J Biol Chem 1976; 251:1639-1645.
