Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock L. Axelsson, M. Bergenfeldt, P Björk, R. Olsson & K. Ohlsson To cite this article: L. Axelsson, M. Bergenfeldt, P Björk, R. Olsson & K. Ohlsson (1990) Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock, Scandinavian Journal of Clinical and Laboratory Investigation, 50:1, 35-42, DOI: 10.1080/00365519009091562 To link to this article: http://dx.doi.org/10.1080/00365519009091562
Published online: 06 Apr 2011.
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Date: 04 June 2016, At: 10:36
Scand J Clin Lab Invest 1990; 50: 35-42
Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock
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L . A X E L S S O N , M . B E R G E N F E L D T , P. B J O R K , * R . O L S S O N & K . OHLSSON Departments of Surgical Pathophysiology and *Diagnostic Radiology, University of Lund, Malrno General Hospital, Malmo, Sweden.
Axelsson L, Bergenfeldt M, Bjork P, Olsson R, Ohlsson K. Release of immunoreactive canine leukocyte elastase, normally and in endotoxin and pancreatitic shock. Scand J Clin Lab Invest 1990: 50: 35-42. A specific ELISA has been developed for the determination of al-proteinase inhibitor-bound leukocyte elastase in canine plasma and tissue fluids. Comparison of the sequence of the first 16 N-terminal amino acids of the isolated canine leukocyte elastase to other elastases indicated moderate homology with porcine pancreatic elastase and pronounced identity with human leukocyte elastase. Normal canine plasma contains about 66 pg/l leukocyte elastase measured as elastase al-proteinases inhibitor complexes. This represents about 70% of the total amount of leukocyte elastase released in plasma. The remaining 30% is bound by ala2-macroglobulin. Blood coagulation leads to a rapid release of elastase from the leukocytes. Slow intravenous infusion of a lethal dose of endotoxin into dogs is followed by a marked drop in leukocyte count and a simultaneous rapid increase in plasma leukocyte elastase concentration reaching a plateau level of 2-3 mg/l plasma. Bile-induced pancreatitis in dogs is accompanied by successive increases in leukocyte elastase levels in plasma as well as in peritoneal exudates, reaching a level of about 15 mg/l in the exudates during the late stages of disease. Professor Kjell Ohlsson, Department of Surgical Pathophysiology, Mulmo General Hospital, S-214 Mulmo, Sweden Key words: canine endotoxin shock; elastase inhibitors; leukocyte elastase (canine); az-macroglobulin, pancreatitis; a,-proteinase inhibitor
Leukocyte elastase is one of the most abundant neutral lysosomal enzymes of human [ 1-31 and dog [4] neutrophil granulocytes. Human leukocyte elastase has the capacity to degrade mucous membranes [5], connective tissue [6] and plasma proteins such as a,-antiplasmin [7, 81, antithrombin I11 [9], C3 [lo], and Factor XI11 [ll]. Normally, a,-protease inhibitor (a,PI) and a2macroglobulin (a,M) offer effective protection to tissues and plasma proteins [12] while the
secretory leukocyte protease inhibitor (SLPI) is the main inhibitor of leukocyte elastase in the mucous secretions [ 13-16]. Leukocyte elastase is released in a variety of inflammatory diseases, e.g. bronchitis [17], rheumatism [ 181, peritonitis [ 191, and septicaernia [20], and thus a local imbalance between enzyme and inhibitors may cause tissue damage and plasma protein degradation. Both radio-immunoassay [21] and ELISA 35
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[22] have been used to investigate leukocyte elastase in humans during various inflammatory diseases. Although it is clear that these conditions involve a pronounced release of leukocyte elastase, it is far from settle to what extent the released elastase is a cause of the pathophysiological changes seen. The dog has long been used to study the pathophysiology of shock. Our group has previously studied endotoxin shock [23] and acute pancreatitis in canine models [24]. We found canine leukocyte elastase also to be a major component of canine leukocytes and to have many properties in common with its human counterpart [4]. In this paper we wish to report on the development of an ELISA for immunoreactive leukocyte elastase together with determinations of plasma levels in healthy beagle dogs and during experimental pancreatitis and endotoxin shock. Leukocyte elastase levels in peritoneal exudates in experimental pancreatitis are also reported.
MATERIALS AND METHODS
Experimental animuls Fourteen beagle dogs (10-16 kg) of both sexes were used in the experimental endotoxin and pancreatitis shock studies. The experiments were performed under general anaesthesia using sodium pentothal@and artificial ventilation with air. Mean arterial blood pressure was measured by an Elema Schonander pressure transducer, through a catheter in a femoral artery. Blood samples were withdrawn in EDTA and heparin tubes every second hour via a catheter in a femoral vein. White blood cell counts were performed in a Biirker chamber. Plasma was prepared within 30 min and stored at -70 "C until analysed. The animals received 40 ml Ringer's solution per hour.
Canine endotoxin shock The endotoxin shock was induced in seven dogs. Lipopolysaccharide B E . coli 026:B6 (Difco Laboratories, Detroit, MI, USA) 2 mg/kg body weight was suspended in 10 ml 0.15 mol/l NaCl and infused intravenously over 2 h. The dogs were monitored for 14 h.
Bile-induced canine pancreatitis Pancreatitis was induced in seven dogs as described earlier by infusion of 0.5 ml/kg body weight of autologous bile into the main pancreatic duct under laparotomy [24]. The animals were followed for 24 h. Peritoneal exudate was collected from an indwelling peritoneal catheter and then analysed as the blood samples.
Chemicals The phosphatase substrates disodium p-nitrophenylphosphate, hexahydrate, succinyl-AlaAla-Ala-4-nitroanilide (Suc(Ala),-Nan), and N benzyloxy-carbonyl-L-alanine-B-napthyl-ester (Z-Ala-Nap) were obtained from Sigma Chemical Company, St Louis, MO, USA. CnBractivated Sepharose 4B, PD 10, protein ASepharose C L 4 B , and Sephadex G 200 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Canine leukocyte elastase was isolated from the granula fraction of dog granulocytes and characterized as previous described [4]. In addition, automated amino terminal sequence determination was performed with an Applied Biosystems 47DA gas-phase sequencer as described earlier [25]. Titertek Immunoassay-plate PVC was obtained from Flow Laboratories, Solna, Sweden. Canine a , P I was generously provided by Professor Carl-Bertil Laurell.
Antisera Rabbit anti-canine a , P I , rabbit anti-canine a,a,M, and sheep anti-canine leukocyte elastase were produced as previously described [26]. Alkaline phosphatase conjugated rabbit antisheep IgG was obtained from the Sigma Chemical Company.
Distribution of canine leukocyte elastase between protease inhibitors in canine serum Elastase binding to a , P I and a,a,M was studied using crossed immunoelectrophoresis [27]. Twenty-five pl of fresh canine serum fractions were incubated at room temperature with increasing amounts of leukocyte elastase (2-50 p g active enzyme) and the volumes were adjusted to 200 pl using 0.15 mol/l NaCI. Samples were incubated for 30 min at room temperature and
Releuse of canine leukocyte elustuse were then analysed with crossed immunoelectrophoresis to determine the distribution among the various inhibitors. Elastase-like activity in immunoprecipates after crossed immunoelectrophoresis using antiserum against a , a , M was localized as previously described PSI.
37
enzyme assay using Suc(Ala),Nan [29], immunoelectrophoresis [4] with antiserum against canine leukocyte elastase and a,PI, respectively, and SDS polyacrylamide gel electrophoresis [4] (results not shown).
ELlSA assay procedure
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Pul-ificarion of untihodies Rabbit anti-canine a,P1 IgG was isolated from the antiserum on a protein-A Sepharose column (1x5 cm) equilibrated with 0.1 mol/l glycine, 0.15 mol/l NaCI, 0.005 mol/l EDTA, in 0.02% NaN,, pH 6.8. After application of the antiserum the column was washed with the equilibration buffer until the absorbance at 280 nm returned to the baseline. The IgG fraction was then eluted using 3.5 mmol/l sodium thiocyanate in the same buffer. The IgC fractions were transferred to the starting buffer using a PD 10 column. Specific sheep anti-canine leukocyte elastase IgG was obtained using affinity chromatography on dog leukocyte elastase coupled to CNBr-activated Sepharose 4B. The same buffers were used as above. Finally, the IgG fraction was passed through a column of Sepharose 4B-coupled rabbit IgG, which was found to diminish unspecific binding in the assay.
Elustuse-a,PI standard The elastase-a,PI complex was produced by mixing 3.0 mg canine a,PI in 1.0 ml 0.005 molA Tris-HC1 buffer, pH 7.4, with 0.15 mol/l NaCl with 0.5 mg of active canine leukocyte elastase in 1.0 ml 0.15 mol/l NaCl. The mixture was incubated for 30 min at 37 "C. All the elastase was bound by a , P I , as controlled by
A carbonate buffer, pH 9.6 (0.015 molA Na,CO,, 0.035 mol/l NaHC03 and 0.02% NaN,) was used as coating buffer for the sheep anti-leukocyte elastase antibody. 0.05 mol/l Tris-HC1 buffer pH 7.4 containing 0.05 mol/l CaCI,, 0.02% NaN,, 0.2% BSA and 0.5 mol/l NaCf was used as sample dilution buffer. Phosphate-buffered saline (PBS) containing 0.05% Tween-20 pH 7.4 was used for washing. Between each incubation step the wells were washed 3-5 times with PBS-Tween. The wells were coated with 150 p1 sheep anticanine leukocyte elastase antibody, 5.0 mgb for 24 h at + 4 "C. Secondly, 150 p1 of sample buffer per well were incubated for at least 2 h at room temperature. Then 150 pl of diluted sample or standard were applied per well followed by overnight incubation at room temperature. The test samples were diluted 10 times routinely. The next incubation step involved 150 p1 of rabbit anti-canine a , P I antibodies, 0.02 pg/ml, in the sample buffer for 3 h at room temperature; 150 p1 of phosphatase-labelled sheep antirabbit IgC, diluted 1/1250 in sample buffer, were added and incubated for 3 h at room temperature. After a final wash 150 pl of phosphatase substrate in 10% ethanolamine buffer pH 9.8 containing MgCI, (1 mmol/l) were applied and incubated at 25 "C. The plates were then analysed in an automatic Titertek multiscan photometer at 405 nm.
10
FIG.1. Comparative N-terminal amino acid sequence of porcine pancreatic, human and canine leukocyte elastase.
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FIG.2. Precipitation pattern obtained by crossed immunoelectrophoresis with rabbit anti-caninc u,PI ( A ) and rabbit anti-canine a , a , M (B) of reaction mixtures of canine serum and increasing amounts of canine leukocyte elastase A2-A4 and B2-B4. A1 and BI show the pattern of canine serum without elastase. Direction of migration in the first run is given. The peaks with retarded electrophoretic mobility representing elastase complexes with a,P1, a , M and a,M, respectively, are indicated (+ a , t b, and + c).
RESULTS
Properties of the canine leukocyte elastase preparation The purified canine leukocyte elastase showed a single band on SDS gel electrophoresis. The sequence of the 16 N-terminal amino acids was determined and showed moderate identity with porcine pancreatic elastase and pronounced identity with human leukocyte elastase. (Fig. 1).
Binding of leukocyte elastase by a,Pl and alazMin vitro Reaction mixtures of normal canine serum and increasing amounts of canine leukocyte elastase were analysed with crossed immunoelectrophoresis. A simultaneous conversion of a , M and a,M and of a,PIinto retarded components was found, representing the complexes
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concentration (pq/l) FIG.3. Dilution curves for standard leukocyte elastase-a,PI complexes manufactured in v i m (-0) and two pools of canine plasma (U and 0-0, degree of dilution 1/2-1/32).
Release of canine leukocyte elastase with elastase (Fig. 2). The retarded a,PI component was identified as elastase-a,PI complex by also being precipitated by antibodies to elastase. The retarded aI-and a 2 M components were found to represent the complexes with elastase as judged from their strong elastase-like activity on a low molecular weight substrate. The changes in precipitate patter of a,PIfrom A3 to A4 in Figure 2 is a regular finding on saturation of a , P I with different proteases.
39
The recovery of exogenous active granulocyte elastase added to normal canine plasma was about 70%, as shown in Table I. Normal plasma range. The plasma concentration of the elastase-alPI complexes was measured in EDTA plasma from 18 randomly selected healthy beagle dogs of both sexes. The concentration of elastase in complex with alPI ranged from 26 to 146 pg/l; the mean value was 66 pg/l with a standard deviation of 37.9 pg/l.
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Canine leukocyte elastase ELISA Specificity. A standard dilution curve for the elastase-a,PI complex was compared with two dilution curves based on two plasma pools from 10 healthy dogs each. All samples were run in duplicate. The curves appeared to be parallel, indicating that the same immunoreactive substance is measured in the different dilution series (Fig. 3). Linearity was also found for individual samples. Sensitivity. The sensitivity of the assay was 1 pg/l (i.e. the absorbance was always greater than the mean absorbance value for the dilution buffer+2 SD). This was adequate to measure the elastase a,PI concentrations in samples of plasma from healthy dogs. Precision. The within-assay coefficient of variation for plasma samples (n=10) measured 12 times in one run was 5%. The betweenassay coefficient of variation measured in six consecutive assays was 8% in plasma samples (n=10). Accuracy. Aliquots of elastase - a,PI complexes produced in vitro were added to five different canine EDTA plasmas. The concentration of the complex in the native and the spiked plasma samples was assayed. The recovery in the five plasma specimens was 101%+3.0% (SEM).
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FIG.4. a. Leukocyte count (w) in venous blood and plasma level immunoreactive leukocyte elastase (LE, U) in canine endotoxin shock (meanfSEM). b. The endotoxin-induced changes in mean arterial pressure (MAP) as a percentage of initial value (H) (mean+SEM).
TABLEI. Recovery of leukocyte elastase added to normal dog plasma as measured by ELISA (ng/ml) Exogenous elastase (ndml)
-
Elastase measured by ELISA (ndml)
Increase in elastase over endogenous material (ng/ml)
Recovery of exogenous elastase (%)
26 61 77 97 108
0 35 51 71 82
70 68 71 66
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L. Axelsson et al. 15000
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elastase increased rapidly both in plasma and peritoneal exudate, especially during the later phase of the disease. The levels increased in parallel but while the plasma level reached about 0.3 mg/l the final values in the peritoneal exudate were in the range of about 15 mg/l (Fig. 5). The animals were followed under general anaesthesia for 24 h. Five of the dogs were then dead and the two surviving dogs had a blood pressure of only 70 mmHg.
DISCUSSION For a thorough understanding of the biological
functions of leukocyte elastase, studies in aniFIG.5. Levels of immunoreactive leukocyte elastase (LE) in plasma (w)and peritoneal exudate (U) mals in vivo seem necessary. We have shown during bile-induced pancreatitis in dogs. (mean? earlier that canine leukocytes contain large SEM). amounts of an elastase highly similar to its Blood samples were collected in EDTA-containing tubes and plasma was prepared within 1 h. In plasma prepared after 3 h the corresponding value increased by about 10-20%. Serum samples allowed to coagulate for 3 h showed a 10to 20-fold increase in serum level of leukocyte elastase compared with the plasma level.
Plasma levels of leukocyte elustase in cunine endotoxin shock The endotoxin was infused over 2 h and the observation time was 14 h. Three dogs were then still alive but with blood pressures below 60% of the initial value. The plasma level of leukocyte elastase rose after the endotoxin infusion and remained elevated throughout the period of observation (Fig. 4). The final leukocyte elastase level showed a mean value of 2692 pg/l for the four dogs that died. The mean elastase level at 14 h for the three surviving dogs was 1100 pg/l. The endotoxin infusion was paralleled by marked decline in the leukocyte count. The lowest value was registered after 4 h, after which the cell count increased and exceeded the initial level at the end of the observation period (Fig. 4).
Release of leukocyte elastase in bile-induced canine puncreutitis The levels of immunoreactive leukocyte
human counterpart, especially considering enzymatic properties, such as a strong elastolytic activity [2, 41. But differences were also found. Human leukocyte elastase contains carbohydrate whereas canine leukocyte elastase does not, this probably also explains the difference in molecular weight. Canine leukocyte elastase is electrophoretically homogeneous, while the human enzyme shows several fractions. The purity of the present preparation allowed the determination of the first 16 N terminal amino acids of canine leukocyte elastase. In Figure 1 a comparison is presented of the N-terminal sequence of canine leukocyte elastase, human leukocyte elastase and porcine pancreatic elastase. In general, the sequences of the human and canine leukocyte elastase are similar with 87.5% identity, while that of the porcine enzyme is only moderately similar with 37.5% identity. The addition of increasing amounts of canine leukocyte elastase in canine plasma in vitm resulted in a simultaneous conversion of a , P I and a,a,M into leukocyte elastase-inhibitor complexes. The pattern is identical to that found on the addition of human leukocyte elastase to human plasma [12]. This indicates that a , P I is also quantitatively the major leukocyte elastase inhibitor in canine plasma. The results discussed so far indicate that human and dog leukocyte elastase are very similar, indicating that results obtained in studies on leukocyte elastase-mediated reactions in the dog should have significance for man, at least as far
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Release o f canine leukocyte elustuse as elastase in concerned. We therefore decided to develop an ELISA for the specific quantification of elastase-a,PI complexes in canine plasma and tissue fluids. The procedure follows the protocol for a solid-phase enzyme-linked sandwich immunoassay. A prerequisite was the use of immunosorbent purified antibodies against leukocyte elastase in the first step. As earlier found for human leukocyte elastase, canine elastase is rapidly released during blood coagulation, but only very slowly in EDTA-containing blood. Thus, plasma preferably separated by centrifugation within 1 h should be used in the assay. The normal plasma level of canine leukocyte elastase is similar to the reported plasma level of human leukocyte elastase 121, 221. We found that about 70% of active elastase added to normal canine plasma could be recovered by the assay. The figure agrees with the results of the partition studies showing a binding to a,P1 and a , a , M and should represent elastase bound by a,PI.a,a,M-bound elastase is not detected by the anti-elastase antibody and cannot be quantified in the assay. Furthermore, the dilution curves of canine plasma are parallel to the standard curve for the elastase- a , P I complex, indicating that plasma only contains immunoreactive leukocyte elastase in complex with a,PI. We have earlier demonstrated the release of leukocyte elastase in dogs receiving a lethal dose of endotoxin [23]. With the aid of the present ELISA we could quantitatively follow the release of leukocyte elastase in a similar dog model. We found here a drastic increase in plasma concentration simultaneously with a fall in leukocyte count in plasma. The plasma level then remained high during the experiments. The final decrease is explained by the death of the dogs with the highest concentrations. The increase in plasma level of leukocyte elastase is also accompanied by a drop in blood pressure in the animals, but to what extent the elastase release contributes to the development of shock is presently difficult to judge. It is intriguing that the plasma levels of leukocyte elastase were much higher in the dogs that died during the experiments as compared to the survivors. If released in restricted compartments, such as in the lung capillaries, a local saturation of the inhibitors may lead to elastase influence on the cascade systems, at least partly explaining the pathophysiology of the endotoxin shock.
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Bile-induced canine pancreatitis was characterized by a pronounced release of leukocyte elastase during the later stage of disease with very high levels, especially in the peritoneal exudate. This pattern parallels our earlier observations of frequently seen heavy infiltration of polymorphonuclear leukocytes into the necrotic areas of the gland in the same model [30].The clearly demonstrable but much less pronounced increase in plasma level of leukocyte elastase is explained by the time taken for the complexes to reach the plasma and the probable elimination of large amounts of the complexes in phagocytosing cells of the RES system. Interestingly, we demonstrated recently that high levels of leukocyte elastase-a,PI complexes in the peritoneal exudate in clinical pancreatitis were an early indication of local abscess formation. This is of considerable diagnostic potential as abscess formation is a severe complication with high mortality [3I]. The present ELISA for leukocyte elastasea,PI complexes thus appears to be a powerful and convenient tool for the elucidation of the biological role of leukocyte elastase and its pathophysiological significance in septic conditions. ACKNOWLEDGMENTS The amino acid sequence analyses were performed by Ingrid Dahlqvist at the Department of Clinical Chemistry, University of Lund, Malmo General Hospital, Malmo. This study was supported by the Swedish Medical Research Council (project nos. B88-17X-03910-16A and B89 - 17K- 087 15-01A), the Medical Faculty, University of Lund, The Foundation for Medical Research administered by Malmo General Hospital, and the Swedish Society for Medical Research. REFERENCES 1 Janoff A, Scherer J. Mediators of inflammation in leukocyte lysosornes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes. J Exp Med 1968; 128: 1137-1151. 2 Ohlsson K, Olsson I. The neutral proteases of human granulocytes. Isolation and partial characterizaiton of granulocyte elastases. Eur J Biochem 1974; 42: 519-27. 3 Ohlsson K, Olsson I. The extracellular release of
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granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Scand J Haematol 1977; 19: 145-52. 4 Delshammar M, Ohlsson K. Isolation and partial characterization of elastase from dog granulocytes. Eur J Biochem 1976; 69: 125-31. 5 Tegner H, Ohlsson K, Toremalm NG, von Mecklenburg C. Effect of human leukocyte enzymes on trachael mucosa and its mucociliary activity. Rhinology 1979 XVII: 199-206. 6 Ohlsson K. Granulocyte proteases and their inhibitors in inflammation; Basic concepts and clinical implications. In: Venge P, Lindbom A eds. Inflammation - Basic Mechanisms, Tissue Injuring Principles and Clinical Models. Stockholm: Almqvist & Wiksell, 1985: 379-93. 7 Ohlsson K, Collen D. Comparison of the reactions of neutral granulocyte proteases with the major plasma protease inhibitors and with antiplasmin. Scand J Clin Lab Invest 1977; 37: 345-50. 8 Klingemann HG, Egbring R, Holst M, Gramse M, Havemann K. Digestion of alpha*-plasmin inhibitor by neutral proteases from human leukocytes. Thromb Res 1981: 24: 479-83. 9 Jochum M, Lander S, Heimburger N, Fritz H. Effect of human granulocytic elstase on isolated human antithrombin 111. Hoppe-Seyler’s Z Physiol Chem 1981; 362: 103-12. 10 Johsson U, Ohlsson K, Olsson I. Effects of granulocyte neutral proteases on complement components. Scand J Immunol 1976: 5: 421-6. 11 Henriksson P, Nilsson IM, Ohlsson K, Stenberg P. Granulocyte elastase activation and degradation of factor XIII. Thromb Res 1980; 18: 343-51. 12 Ohlsson K, Olsson I. Neutral proteases of human granulocytes. 111. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand J Clin Lab Invest 1974; 34: 349-55. 13 Tegner H. Inhibitors of granulocyte proteases in bronchial secretion. Thesis, University of Lund, 1977. 14 Schiessler H, Arnhold M, Ohlsson K, Fritz H. Inhibitors of acrosin and granulocyte proteinases from human genital tract secretions. HoppeSeyler’s Z Physiol Chem 1976; 357: 1251-60. 15 Ohlsson K, Tegner H, Fritz H, Schiessler H. Immunologic similarity between low molecular weight trypsin-chymotrypsin inhibitors from human bronchial secretion and seminal plasma. Hoppe-Seyler’s Z Physiol Chem 1976; 357: 124 1-44. 16 Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci, USA 1986; 83: 6692-6. 17 Fryksmark U, Prellner T, Tegner H, Ohlsson K. Studies on the role of antileukoprotease in respira-
tory tracts diseases. Eur J Respir Dis 1984: 6.5: 201-9. 18 Ekerot L, Ohlsson, K. lmmunoreactive granulocyte elastase in rheumatoid synovial fluid and membrane. Scand J Plast Reconstr Sur 1982: 16: 117-22. 19 Ohlsson K. Collagenase and elastase released during peritonitis are complexed by plasma protease inhibitors. Surgery 1976; 79: 652-57. 20 Egbring R, Schmidt W, Fuchs G, Havemann K. Demonstration of granulocyte proteases in plasma of patients with acute leukemia and septicemia with coagulation defects. Blood 1977: 49: 219-3 I . 21 Ohlsson K, Olsson A-S. Immunoreactive granulocyte elastase in human serum. Hoppe-Seyler’s Z Physiol Chem 1978; 3.59: 1521-9. 22 Neumann S, Hennrich N , Gunzer G, Lang H. Enzyme-linked immunoassay for human granulocyte elastase in complex wtih alphal-proteinase inhibitor. In: Proteases, Potential Role in Health and Disease. New York: Plenum Press, 1984: 379-90. 23 Aasen AO, Ohlsson K. Release of granulocyte elastase in lethal canine endotoxin shock. HoppeSeyler’s Z Physiol Chem 1978: 359: 683-90. 24 Ohlsson K, Eddeland A. Release of proteolytic enzymes in bile-induced pancreatitis in dogs. Gastroenterology 1975; 69: 669-75. 25 Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequencer. J Biol Chem 1981; 256: 7990-7. 26 Ohlsson K. Isolation and partial characterization of two related trypsin binding alpha-macroglobulins of dog plasma. Biochim Biophys Acta 1971; 236: 84-91. 27 Ganrot PO. Crossed immunoelectrophoresis. Scand J Clin Lab Invest 1972; 29 (Suppl. 124): 39-47. 28 Hfiansson H-0, Ohlsson K. Interactions in vitro and in vivo between human and porcine cationic pancreatic elstase and plasma protease inhibitors. Biol Chem Hoppe-Seyler 1988; 369: 309-15. 29 Bieth J, Spiess B, Wermuth CG. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem Med 1974; 11: 4: 350-7. 30 Ohlsson K, Olsson R, Bjork P et a/. Local administration of human pancreatic secretory trypsin inhibitor prevents the development of experimental acute pancreatisis in rats and dogs. Scand J Gastroenterol 1989; 24; (in press) 31 Balldin G, Ohlsson K. Early diagnosis of a pancreatic abscess in acute pancreatitis. Scand J Gastroenterol 1986: 21(125): 32-34. Received 12 April 1989 Accepted 23 June 1989
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock L. Axelsson, M. Bergenfeldt, P Björk, R. Olsson & K. Ohlsson To cite this article: L. Axelsson, M. Bergenfeldt, P Björk, R. Olsson & K. Ohlsson (1990) Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock, Scandinavian Journal of Clinical and Laboratory Investigation, 50:1, 35-42, DOI: 10.1080/00365519009091562 To link to this article: http://dx.doi.org/10.1080/00365519009091562
Published online: 06 Apr 2011.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [Australian National University]
Date: 04 June 2016, At: 10:36
Scand J Clin Lab Invest 1990; 50: 35-42
Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock
Downloaded by [Australian National University] at 10:36 04 June 2016
L . A X E L S S O N , M . B E R G E N F E L D T , P. B J O R K , * R . O L S S O N & K . OHLSSON Departments of Surgical Pathophysiology and *Diagnostic Radiology, University of Lund, Malrno General Hospital, Malmo, Sweden.
Axelsson L, Bergenfeldt M, Bjork P, Olsson R, Ohlsson K. Release of immunoreactive canine leukocyte elastase, normally and in endotoxin and pancreatitic shock. Scand J Clin Lab Invest 1990: 50: 35-42. A specific ELISA has been developed for the determination of al-proteinase inhibitor-bound leukocyte elastase in canine plasma and tissue fluids. Comparison of the sequence of the first 16 N-terminal amino acids of the isolated canine leukocyte elastase to other elastases indicated moderate homology with porcine pancreatic elastase and pronounced identity with human leukocyte elastase. Normal canine plasma contains about 66 pg/l leukocyte elastase measured as elastase al-proteinases inhibitor complexes. This represents about 70% of the total amount of leukocyte elastase released in plasma. The remaining 30% is bound by ala2-macroglobulin. Blood coagulation leads to a rapid release of elastase from the leukocytes. Slow intravenous infusion of a lethal dose of endotoxin into dogs is followed by a marked drop in leukocyte count and a simultaneous rapid increase in plasma leukocyte elastase concentration reaching a plateau level of 2-3 mg/l plasma. Bile-induced pancreatitis in dogs is accompanied by successive increases in leukocyte elastase levels in plasma as well as in peritoneal exudates, reaching a level of about 15 mg/l in the exudates during the late stages of disease. Professor Kjell Ohlsson, Department of Surgical Pathophysiology, Mulmo General Hospital, S-214 Mulmo, Sweden Key words: canine endotoxin shock; elastase inhibitors; leukocyte elastase (canine); az-macroglobulin, pancreatitis; a,-proteinase inhibitor
Leukocyte elastase is one of the most abundant neutral lysosomal enzymes of human [ 1-31 and dog [4] neutrophil granulocytes. Human leukocyte elastase has the capacity to degrade mucous membranes [5], connective tissue [6] and plasma proteins such as a,-antiplasmin [7, 81, antithrombin I11 [9], C3 [lo], and Factor XI11 [ll]. Normally, a,-protease inhibitor (a,PI) and a2macroglobulin (a,M) offer effective protection to tissues and plasma proteins [12] while the
secretory leukocyte protease inhibitor (SLPI) is the main inhibitor of leukocyte elastase in the mucous secretions [ 13-16]. Leukocyte elastase is released in a variety of inflammatory diseases, e.g. bronchitis [17], rheumatism [ 181, peritonitis [ 191, and septicaernia [20], and thus a local imbalance between enzyme and inhibitors may cause tissue damage and plasma protein degradation. Both radio-immunoassay [21] and ELISA 35
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[22] have been used to investigate leukocyte elastase in humans during various inflammatory diseases. Although it is clear that these conditions involve a pronounced release of leukocyte elastase, it is far from settle to what extent the released elastase is a cause of the pathophysiological changes seen. The dog has long been used to study the pathophysiology of shock. Our group has previously studied endotoxin shock [23] and acute pancreatitis in canine models [24]. We found canine leukocyte elastase also to be a major component of canine leukocytes and to have many properties in common with its human counterpart [4]. In this paper we wish to report on the development of an ELISA for immunoreactive leukocyte elastase together with determinations of plasma levels in healthy beagle dogs and during experimental pancreatitis and endotoxin shock. Leukocyte elastase levels in peritoneal exudates in experimental pancreatitis are also reported.
MATERIALS AND METHODS
Experimental animuls Fourteen beagle dogs (10-16 kg) of both sexes were used in the experimental endotoxin and pancreatitis shock studies. The experiments were performed under general anaesthesia using sodium pentothal@and artificial ventilation with air. Mean arterial blood pressure was measured by an Elema Schonander pressure transducer, through a catheter in a femoral artery. Blood samples were withdrawn in EDTA and heparin tubes every second hour via a catheter in a femoral vein. White blood cell counts were performed in a Biirker chamber. Plasma was prepared within 30 min and stored at -70 "C until analysed. The animals received 40 ml Ringer's solution per hour.
Canine endotoxin shock The endotoxin shock was induced in seven dogs. Lipopolysaccharide B E . coli 026:B6 (Difco Laboratories, Detroit, MI, USA) 2 mg/kg body weight was suspended in 10 ml 0.15 mol/l NaCl and infused intravenously over 2 h. The dogs were monitored for 14 h.
Bile-induced canine pancreatitis Pancreatitis was induced in seven dogs as described earlier by infusion of 0.5 ml/kg body weight of autologous bile into the main pancreatic duct under laparotomy [24]. The animals were followed for 24 h. Peritoneal exudate was collected from an indwelling peritoneal catheter and then analysed as the blood samples.
Chemicals The phosphatase substrates disodium p-nitrophenylphosphate, hexahydrate, succinyl-AlaAla-Ala-4-nitroanilide (Suc(Ala),-Nan), and N benzyloxy-carbonyl-L-alanine-B-napthyl-ester (Z-Ala-Nap) were obtained from Sigma Chemical Company, St Louis, MO, USA. CnBractivated Sepharose 4B, PD 10, protein ASepharose C L 4 B , and Sephadex G 200 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Canine leukocyte elastase was isolated from the granula fraction of dog granulocytes and characterized as previous described [4]. In addition, automated amino terminal sequence determination was performed with an Applied Biosystems 47DA gas-phase sequencer as described earlier [25]. Titertek Immunoassay-plate PVC was obtained from Flow Laboratories, Solna, Sweden. Canine a , P I was generously provided by Professor Carl-Bertil Laurell.
Antisera Rabbit anti-canine a , P I , rabbit anti-canine a,a,M, and sheep anti-canine leukocyte elastase were produced as previously described [26]. Alkaline phosphatase conjugated rabbit antisheep IgG was obtained from the Sigma Chemical Company.
Distribution of canine leukocyte elastase between protease inhibitors in canine serum Elastase binding to a , P I and a,a,M was studied using crossed immunoelectrophoresis [27]. Twenty-five pl of fresh canine serum fractions were incubated at room temperature with increasing amounts of leukocyte elastase (2-50 p g active enzyme) and the volumes were adjusted to 200 pl using 0.15 mol/l NaCI. Samples were incubated for 30 min at room temperature and
Releuse of canine leukocyte elustuse were then analysed with crossed immunoelectrophoresis to determine the distribution among the various inhibitors. Elastase-like activity in immunoprecipates after crossed immunoelectrophoresis using antiserum against a , a , M was localized as previously described PSI.
37
enzyme assay using Suc(Ala),Nan [29], immunoelectrophoresis [4] with antiserum against canine leukocyte elastase and a,PI, respectively, and SDS polyacrylamide gel electrophoresis [4] (results not shown).
ELlSA assay procedure
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Pul-ificarion of untihodies Rabbit anti-canine a,P1 IgG was isolated from the antiserum on a protein-A Sepharose column (1x5 cm) equilibrated with 0.1 mol/l glycine, 0.15 mol/l NaCI, 0.005 mol/l EDTA, in 0.02% NaN,, pH 6.8. After application of the antiserum the column was washed with the equilibration buffer until the absorbance at 280 nm returned to the baseline. The IgG fraction was then eluted using 3.5 mmol/l sodium thiocyanate in the same buffer. The IgC fractions were transferred to the starting buffer using a PD 10 column. Specific sheep anti-canine leukocyte elastase IgG was obtained using affinity chromatography on dog leukocyte elastase coupled to CNBr-activated Sepharose 4B. The same buffers were used as above. Finally, the IgG fraction was passed through a column of Sepharose 4B-coupled rabbit IgG, which was found to diminish unspecific binding in the assay.
Elustuse-a,PI standard The elastase-a,PI complex was produced by mixing 3.0 mg canine a,PI in 1.0 ml 0.005 molA Tris-HC1 buffer, pH 7.4, with 0.15 mol/l NaCl with 0.5 mg of active canine leukocyte elastase in 1.0 ml 0.15 mol/l NaCl. The mixture was incubated for 30 min at 37 "C. All the elastase was bound by a , P I , as controlled by
A carbonate buffer, pH 9.6 (0.015 molA Na,CO,, 0.035 mol/l NaHC03 and 0.02% NaN,) was used as coating buffer for the sheep anti-leukocyte elastase antibody. 0.05 mol/l Tris-HC1 buffer pH 7.4 containing 0.05 mol/l CaCI,, 0.02% NaN,, 0.2% BSA and 0.5 mol/l NaCf was used as sample dilution buffer. Phosphate-buffered saline (PBS) containing 0.05% Tween-20 pH 7.4 was used for washing. Between each incubation step the wells were washed 3-5 times with PBS-Tween. The wells were coated with 150 p1 sheep anticanine leukocyte elastase antibody, 5.0 mgb for 24 h at + 4 "C. Secondly, 150 p1 of sample buffer per well were incubated for at least 2 h at room temperature. Then 150 pl of diluted sample or standard were applied per well followed by overnight incubation at room temperature. The test samples were diluted 10 times routinely. The next incubation step involved 150 p1 of rabbit anti-canine a , P I antibodies, 0.02 pg/ml, in the sample buffer for 3 h at room temperature; 150 p1 of phosphatase-labelled sheep antirabbit IgC, diluted 1/1250 in sample buffer, were added and incubated for 3 h at room temperature. After a final wash 150 pl of phosphatase substrate in 10% ethanolamine buffer pH 9.8 containing MgCI, (1 mmol/l) were applied and incubated at 25 "C. The plates were then analysed in an automatic Titertek multiscan photometer at 405 nm.
10
FIG.1. Comparative N-terminal amino acid sequence of porcine pancreatic, human and canine leukocyte elastase.
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FIG.2. Precipitation pattern obtained by crossed immunoelectrophoresis with rabbit anti-caninc u,PI ( A ) and rabbit anti-canine a , a , M (B) of reaction mixtures of canine serum and increasing amounts of canine leukocyte elastase A2-A4 and B2-B4. A1 and BI show the pattern of canine serum without elastase. Direction of migration in the first run is given. The peaks with retarded electrophoretic mobility representing elastase complexes with a,P1, a , M and a,M, respectively, are indicated (+ a , t b, and + c).
RESULTS
Properties of the canine leukocyte elastase preparation The purified canine leukocyte elastase showed a single band on SDS gel electrophoresis. The sequence of the 16 N-terminal amino acids was determined and showed moderate identity with porcine pancreatic elastase and pronounced identity with human leukocyte elastase. (Fig. 1).
Binding of leukocyte elastase by a,Pl and alazMin vitro Reaction mixtures of normal canine serum and increasing amounts of canine leukocyte elastase were analysed with crossed immunoelectrophoresis. A simultaneous conversion of a , M and a,M and of a,PIinto retarded components was found, representing the complexes
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concentration (pq/l) FIG.3. Dilution curves for standard leukocyte elastase-a,PI complexes manufactured in v i m (-0) and two pools of canine plasma (U and 0-0, degree of dilution 1/2-1/32).
Release of canine leukocyte elastase with elastase (Fig. 2). The retarded a,PI component was identified as elastase-a,PI complex by also being precipitated by antibodies to elastase. The retarded aI-and a 2 M components were found to represent the complexes with elastase as judged from their strong elastase-like activity on a low molecular weight substrate. The changes in precipitate patter of a,PIfrom A3 to A4 in Figure 2 is a regular finding on saturation of a , P I with different proteases.
39
The recovery of exogenous active granulocyte elastase added to normal canine plasma was about 70%, as shown in Table I. Normal plasma range. The plasma concentration of the elastase-alPI complexes was measured in EDTA plasma from 18 randomly selected healthy beagle dogs of both sexes. The concentration of elastase in complex with alPI ranged from 26 to 146 pg/l; the mean value was 66 pg/l with a standard deviation of 37.9 pg/l.
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Canine leukocyte elastase ELISA Specificity. A standard dilution curve for the elastase-a,PI complex was compared with two dilution curves based on two plasma pools from 10 healthy dogs each. All samples were run in duplicate. The curves appeared to be parallel, indicating that the same immunoreactive substance is measured in the different dilution series (Fig. 3). Linearity was also found for individual samples. Sensitivity. The sensitivity of the assay was 1 pg/l (i.e. the absorbance was always greater than the mean absorbance value for the dilution buffer+2 SD). This was adequate to measure the elastase a,PI concentrations in samples of plasma from healthy dogs. Precision. The within-assay coefficient of variation for plasma samples (n=10) measured 12 times in one run was 5%. The betweenassay coefficient of variation measured in six consecutive assays was 8% in plasma samples (n=10). Accuracy. Aliquots of elastase - a,PI complexes produced in vitro were added to five different canine EDTA plasmas. The concentration of the complex in the native and the spiked plasma samples was assayed. The recovery in the five plasma specimens was 101%+3.0% (SEM).
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FIG.4. a. Leukocyte count (w) in venous blood and plasma level immunoreactive leukocyte elastase (LE, U) in canine endotoxin shock (meanfSEM). b. The endotoxin-induced changes in mean arterial pressure (MAP) as a percentage of initial value (H) (mean+SEM).
TABLEI. Recovery of leukocyte elastase added to normal dog plasma as measured by ELISA (ng/ml) Exogenous elastase (ndml)
-
Elastase measured by ELISA (ndml)
Increase in elastase over endogenous material (ng/ml)
Recovery of exogenous elastase (%)
26 61 77 97 108
0 35 51 71 82
70 68 71 66
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L. Axelsson et al. 15000
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elastase increased rapidly both in plasma and peritoneal exudate, especially during the later phase of the disease. The levels increased in parallel but while the plasma level reached about 0.3 mg/l the final values in the peritoneal exudate were in the range of about 15 mg/l (Fig. 5). The animals were followed under general anaesthesia for 24 h. Five of the dogs were then dead and the two surviving dogs had a blood pressure of only 70 mmHg.
DISCUSSION For a thorough understanding of the biological
functions of leukocyte elastase, studies in aniFIG.5. Levels of immunoreactive leukocyte elastase (LE) in plasma (w)and peritoneal exudate (U) mals in vivo seem necessary. We have shown during bile-induced pancreatitis in dogs. (mean? earlier that canine leukocytes contain large SEM). amounts of an elastase highly similar to its Blood samples were collected in EDTA-containing tubes and plasma was prepared within 1 h. In plasma prepared after 3 h the corresponding value increased by about 10-20%. Serum samples allowed to coagulate for 3 h showed a 10to 20-fold increase in serum level of leukocyte elastase compared with the plasma level.
Plasma levels of leukocyte elustase in cunine endotoxin shock The endotoxin was infused over 2 h and the observation time was 14 h. Three dogs were then still alive but with blood pressures below 60% of the initial value. The plasma level of leukocyte elastase rose after the endotoxin infusion and remained elevated throughout the period of observation (Fig. 4). The final leukocyte elastase level showed a mean value of 2692 pg/l for the four dogs that died. The mean elastase level at 14 h for the three surviving dogs was 1100 pg/l. The endotoxin infusion was paralleled by marked decline in the leukocyte count. The lowest value was registered after 4 h, after which the cell count increased and exceeded the initial level at the end of the observation period (Fig. 4).
Release of leukocyte elastase in bile-induced canine puncreutitis The levels of immunoreactive leukocyte
human counterpart, especially considering enzymatic properties, such as a strong elastolytic activity [2, 41. But differences were also found. Human leukocyte elastase contains carbohydrate whereas canine leukocyte elastase does not, this probably also explains the difference in molecular weight. Canine leukocyte elastase is electrophoretically homogeneous, while the human enzyme shows several fractions. The purity of the present preparation allowed the determination of the first 16 N terminal amino acids of canine leukocyte elastase. In Figure 1 a comparison is presented of the N-terminal sequence of canine leukocyte elastase, human leukocyte elastase and porcine pancreatic elastase. In general, the sequences of the human and canine leukocyte elastase are similar with 87.5% identity, while that of the porcine enzyme is only moderately similar with 37.5% identity. The addition of increasing amounts of canine leukocyte elastase in canine plasma in vitm resulted in a simultaneous conversion of a , P I and a,a,M into leukocyte elastase-inhibitor complexes. The pattern is identical to that found on the addition of human leukocyte elastase to human plasma [12]. This indicates that a , P I is also quantitatively the major leukocyte elastase inhibitor in canine plasma. The results discussed so far indicate that human and dog leukocyte elastase are very similar, indicating that results obtained in studies on leukocyte elastase-mediated reactions in the dog should have significance for man, at least as far
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Release o f canine leukocyte elustuse as elastase in concerned. We therefore decided to develop an ELISA for the specific quantification of elastase-a,PI complexes in canine plasma and tissue fluids. The procedure follows the protocol for a solid-phase enzyme-linked sandwich immunoassay. A prerequisite was the use of immunosorbent purified antibodies against leukocyte elastase in the first step. As earlier found for human leukocyte elastase, canine elastase is rapidly released during blood coagulation, but only very slowly in EDTA-containing blood. Thus, plasma preferably separated by centrifugation within 1 h should be used in the assay. The normal plasma level of canine leukocyte elastase is similar to the reported plasma level of human leukocyte elastase 121, 221. We found that about 70% of active elastase added to normal canine plasma could be recovered by the assay. The figure agrees with the results of the partition studies showing a binding to a,P1 and a , a , M and should represent elastase bound by a,PI.a,a,M-bound elastase is not detected by the anti-elastase antibody and cannot be quantified in the assay. Furthermore, the dilution curves of canine plasma are parallel to the standard curve for the elastase- a , P I complex, indicating that plasma only contains immunoreactive leukocyte elastase in complex with a,PI. We have earlier demonstrated the release of leukocyte elastase in dogs receiving a lethal dose of endotoxin [23]. With the aid of the present ELISA we could quantitatively follow the release of leukocyte elastase in a similar dog model. We found here a drastic increase in plasma concentration simultaneously with a fall in leukocyte count in plasma. The plasma level then remained high during the experiments. The final decrease is explained by the death of the dogs with the highest concentrations. The increase in plasma level of leukocyte elastase is also accompanied by a drop in blood pressure in the animals, but to what extent the elastase release contributes to the development of shock is presently difficult to judge. It is intriguing that the plasma levels of leukocyte elastase were much higher in the dogs that died during the experiments as compared to the survivors. If released in restricted compartments, such as in the lung capillaries, a local saturation of the inhibitors may lead to elastase influence on the cascade systems, at least partly explaining the pathophysiology of the endotoxin shock.
41
Bile-induced canine pancreatitis was characterized by a pronounced release of leukocyte elastase during the later stage of disease with very high levels, especially in the peritoneal exudate. This pattern parallels our earlier observations of frequently seen heavy infiltration of polymorphonuclear leukocytes into the necrotic areas of the gland in the same model [30].The clearly demonstrable but much less pronounced increase in plasma level of leukocyte elastase is explained by the time taken for the complexes to reach the plasma and the probable elimination of large amounts of the complexes in phagocytosing cells of the RES system. Interestingly, we demonstrated recently that high levels of leukocyte elastase-a,PI complexes in the peritoneal exudate in clinical pancreatitis were an early indication of local abscess formation. This is of considerable diagnostic potential as abscess formation is a severe complication with high mortality [3I]. The present ELISA for leukocyte elastasea,PI complexes thus appears to be a powerful and convenient tool for the elucidation of the biological role of leukocyte elastase and its pathophysiological significance in septic conditions. ACKNOWLEDGMENTS The amino acid sequence analyses were performed by Ingrid Dahlqvist at the Department of Clinical Chemistry, University of Lund, Malmo General Hospital, Malmo. This study was supported by the Swedish Medical Research Council (project nos. B88-17X-03910-16A and B89 - 17K- 087 15-01A), the Medical Faculty, University of Lund, The Foundation for Medical Research administered by Malmo General Hospital, and the Swedish Society for Medical Research. REFERENCES 1 Janoff A, Scherer J. Mediators of inflammation in leukocyte lysosornes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes. J Exp Med 1968; 128: 1137-1151. 2 Ohlsson K, Olsson I. The neutral proteases of human granulocytes. Isolation and partial characterizaiton of granulocyte elastases. Eur J Biochem 1974; 42: 519-27. 3 Ohlsson K, Olsson I. The extracellular release of
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granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Scand J Haematol 1977; 19: 145-52. 4 Delshammar M, Ohlsson K. Isolation and partial characterization of elastase from dog granulocytes. Eur J Biochem 1976; 69: 125-31. 5 Tegner H, Ohlsson K, Toremalm NG, von Mecklenburg C. Effect of human leukocyte enzymes on trachael mucosa and its mucociliary activity. Rhinology 1979 XVII: 199-206. 6 Ohlsson K. Granulocyte proteases and their inhibitors in inflammation; Basic concepts and clinical implications. In: Venge P, Lindbom A eds. Inflammation - Basic Mechanisms, Tissue Injuring Principles and Clinical Models. Stockholm: Almqvist & Wiksell, 1985: 379-93. 7 Ohlsson K, Collen D. Comparison of the reactions of neutral granulocyte proteases with the major plasma protease inhibitors and with antiplasmin. Scand J Clin Lab Invest 1977; 37: 345-50. 8 Klingemann HG, Egbring R, Holst M, Gramse M, Havemann K. Digestion of alpha*-plasmin inhibitor by neutral proteases from human leukocytes. Thromb Res 1981: 24: 479-83. 9 Jochum M, Lander S, Heimburger N, Fritz H. Effect of human granulocytic elstase on isolated human antithrombin 111. Hoppe-Seyler’s Z Physiol Chem 1981; 362: 103-12. 10 Johsson U, Ohlsson K, Olsson I. Effects of granulocyte neutral proteases on complement components. Scand J Immunol 1976: 5: 421-6. 11 Henriksson P, Nilsson IM, Ohlsson K, Stenberg P. Granulocyte elastase activation and degradation of factor XIII. Thromb Res 1980; 18: 343-51. 12 Ohlsson K, Olsson I. Neutral proteases of human granulocytes. 111. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand J Clin Lab Invest 1974; 34: 349-55. 13 Tegner H. Inhibitors of granulocyte proteases in bronchial secretion. Thesis, University of Lund, 1977. 14 Schiessler H, Arnhold M, Ohlsson K, Fritz H. Inhibitors of acrosin and granulocyte proteinases from human genital tract secretions. HoppeSeyler’s Z Physiol Chem 1976; 357: 1251-60. 15 Ohlsson K, Tegner H, Fritz H, Schiessler H. Immunologic similarity between low molecular weight trypsin-chymotrypsin inhibitors from human bronchial secretion and seminal plasma. Hoppe-Seyler’s Z Physiol Chem 1976; 357: 124 1-44. 16 Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci, USA 1986; 83: 6692-6. 17 Fryksmark U, Prellner T, Tegner H, Ohlsson K. Studies on the role of antileukoprotease in respira-
tory tracts diseases. Eur J Respir Dis 1984: 6.5: 201-9. 18 Ekerot L, Ohlsson, K. lmmunoreactive granulocyte elastase in rheumatoid synovial fluid and membrane. Scand J Plast Reconstr Sur 1982: 16: 117-22. 19 Ohlsson K. Collagenase and elastase released during peritonitis are complexed by plasma protease inhibitors. Surgery 1976; 79: 652-57. 20 Egbring R, Schmidt W, Fuchs G, Havemann K. Demonstration of granulocyte proteases in plasma of patients with acute leukemia and septicemia with coagulation defects. Blood 1977: 49: 219-3 I . 21 Ohlsson K, Olsson A-S. Immunoreactive granulocyte elastase in human serum. Hoppe-Seyler’s Z Physiol Chem 1978; 3.59: 1521-9. 22 Neumann S, Hennrich N , Gunzer G, Lang H. Enzyme-linked immunoassay for human granulocyte elastase in complex wtih alphal-proteinase inhibitor. In: Proteases, Potential Role in Health and Disease. New York: Plenum Press, 1984: 379-90. 23 Aasen AO, Ohlsson K. Release of granulocyte elastase in lethal canine endotoxin shock. HoppeSeyler’s Z Physiol Chem 1978: 359: 683-90. 24 Ohlsson K, Eddeland A. Release of proteolytic enzymes in bile-induced pancreatitis in dogs. Gastroenterology 1975; 69: 669-75. 25 Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequencer. J Biol Chem 1981; 256: 7990-7. 26 Ohlsson K. Isolation and partial characterization of two related trypsin binding alpha-macroglobulins of dog plasma. Biochim Biophys Acta 1971; 236: 84-91. 27 Ganrot PO. Crossed immunoelectrophoresis. Scand J Clin Lab Invest 1972; 29 (Suppl. 124): 39-47. 28 Hfiansson H-0, Ohlsson K. Interactions in vitro and in vivo between human and porcine cationic pancreatic elstase and plasma protease inhibitors. Biol Chem Hoppe-Seyler 1988; 369: 309-15. 29 Bieth J, Spiess B, Wermuth CG. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem Med 1974; 11: 4: 350-7. 30 Ohlsson K, Olsson R, Bjork P et a/. Local administration of human pancreatic secretory trypsin inhibitor prevents the development of experimental acute pancreatisis in rats and dogs. Scand J Gastroenterol 1989; 24; (in press) 31 Balldin G, Ohlsson K. Early diagnosis of a pancreatic abscess in acute pancreatitis. Scand J Gastroenterol 1986: 21(125): 32-34. Received 12 April 1989 Accepted 23 June 1989
