Biol. Chem. Hoppe-Seyler Vol. 373, pp. 119-122, March 1992

Inhibition of Human, Ovine, and Baboon Neutrophil Elastase with Eglin c and Secretory Leukocyte Proteinase Inhibitor Wolfgang Georg JUNGER*, Seth HALLSTRÖM5, Heinz REDL b , and Günther SCHLAG** a b

University of California San Diego, Department of Surgery, San Diego, USA Ludwig-Boltzmann-Institut für Experimentelle und KlinischeTraumatologie, Wien, Österreich

(Received 29 August 1991)

Summary: The association rate constants (kon) of human, ovine, and baboon neutrophil elastase with two recombinant serine proteinase inhibitors (Eglin c, secretory leukocyte proteinase inhibitor) were compared. The association rate constant of sheep leukocyte elastase (SLE) with Eglin c is about 100 times lower (kon = 2.2x 105 ivrt"1) than that of human elastase (kon = 2.4 107 NTV 1 ). Baboon elastase, however, is as effectively blocked with Eglin c (kon = 2.5 x 107 M S ) a s human elastase.

ciency; baboon elastase shows the highest association rate constant (kon = 5.6x 107 M^s"1) followed by sheep elastase (kon = 1.2 x


>7 M'',

These findings demonstrate marked differences in the inhibition kinetic properties of ovine and human elastase. Concerning a future clinical application of proteinase inhibitors, the baboon seems a more suitable model than sheep to evaluate the effects of Eglin c and SLPI, since both inhibitors block baboon and human elastase with comparable efficiency.

Secretory leukocyte proteinase inhibitor (SLPI) blocks the elastase of all three species with high effi-

Hemmung der neutrophilen Elastasen von Mensch, Schaf und Pavian mit Eglin c und Secretory Leukocyte Proteinase Inhibitor Zusammenfassung: Die Assoziationskonstanten (kon) der neutrophilen Elastasen von Mensch, Schaf und Pavian mit zwei rekombinanten Serinproteinaseinhibitoren (Eglin c, Secretory Leukocyte Proteinase Inhibitor) wurden verglichen. Die Assoziationskonstante von Schaf-Leukozytenelastase (SLE) und Eglin c ist etwa 100-fach niedriger (fcon = 2.2 x 105 M"'s'1) als die von humaner Elastase mit demselben Inhibitor (kon = 2Ax 107 M'V1). Eglin c hemmt Pavianelastase vergleichbar gut wie humane Elastase (koa = 2.5 x 107 ivTV1). Secretory Leukocyte Proteinase Inhibitor (SLPI) inhibiert die Elastasen aller drie Spezies mit großer Ef-

fizienz. Pavianelastase zeigt die höchste Assoziationskonstante (&on = 5.6 x 107 M"^"1), gefolgt von humaner (kon = 4.1 x 107 NfV1) und schließlich Schaf-Elastase (/ton = 1.2 x 107 ivrt'1). Diese Ergebnisse zeigen wichtige Unterschiede in den inhibitionskinetischen Eigenschaften der Elastasen des Schafes und Menschen auf. Im Hinblick auf eine zukünftige klinische Anwendung von Proteinaseinhibitoren scheint der Pavian ein besser geeignetes Modell zur Untersuchung der Wirkung von Eglin c und SLPI zu repräsentieren, als das Schaf, da beide Inhibitoren die Elastase des Menschen und Pavians mit vergleichbarer Effizienz hemmen.

Enzyme: Leukocyte elastase (EC Abbreviations: BLE, baboon leukocyte elastase; HLE, human leukocyte elastase; PMNs, polymorphonuclear leukocytes; SLE, sheep leukocyte elastase; SLPI, Secretory leukocyte proteinase inhibitor.

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W.O. Junger, S. Hallstr m, H. Redl and G. Schlag

Vol. 373 (1992)

Key terms: Human, sheep, and baboon neutrophil elastase inhibition, Eglin c, secretory leukocyte proteinase inhibitor.

Neutrophil elastase is released from polymorphonuclear leukocytes (PMNs) during shock'1·21. It is believed to contribute substantially to tissue damage leading to multi-organ failure syndrome in severely traumatized patients121. Therefore attempts are made to control liberated elastase activity using specific proteinase inhibitors. Two promising low molecular mass serine proteinase inhibitors, Eglin c and secretory leukocyte proteinase inhibitor (SLPI), have been produced in their recombinant forms and been demonstrated to effectively inhibit human leukocyte elastase in vitro13'51. In an endotoxemia model in sheep, however, Eglin c failed to prevent lung tissue injury'61. Sheep are frequently employed in research on pulmonary injury because studies of vascular permeability changes and bronchoalveolar lavage are easily performed even in chronic studies'71. Since little was known about neutrophil elastase in sheep, we have focused our interest on the biochemical properties of this enzyme in comparison to human elastase'8'91. In this brief contribution we compare the association rate constants of Eglin c and SLPI with human and ovine, as well as with baboon neutrophil elastase, since the baboon is increasingly used by our group and others for studies of elastase-induced tissue damage during shock'10"121.

pH 7.4) and incubated for 10 min at 37°C. Then 10 μΐ of a lOOmM synthetic substrate stock solution in dimethylsulfoxide (Fluka AG, CH-9470 Buchs) were added to yield a final assay concentration of ImM.The total assay volume was 1 ml. After incubation for an additional 10 min the reaction was terminated by addition of 100 μΐ of glacial acetic acid. Liberated 4-nitroaniline was measured at 405 nm. Fig. 1 shows a typical titration curve obtained for the determination of the equivalent point of an HLE solution with unknown molarity and an Eglin c solution of known molarity. Determination of association rate constants kon: Equimolar concentrations of inhibitor and elastase solutions were mixed in the stirred quartz cuvette of an Uvicon 820 double beam spectrophotometer (Kontron AG, CH-8010 Z rich).TheTris buffered saline used was the same as described above and the temperature was at 37°C. At predetermined time points the association reaction of inhibitor and elastase was terminated by addition of an excess of substrate (final assay concentration: IITIM). Final assay volume was 1 ml. Residual elastase activity in the reaction mixture was measured at 405 nm and the change of absorbane was recorded. The percentage of residual enzymatic activity was determined and expressed as the reciprocal actual enzyme concentrations 1/[E], which was calculated based on the initial enzyme concentration [E0] in the assay mixture. The 1/[E] values obtained were plotted versus the inhibition time (in seconds), the time interval elapsed between mixing of enzyme and inhibitor before substrate was added. The slopes of the resulting linear curves, depicted in Fig. 2 and 3, represent the kon values of the respective inhibitor/proteinase pairs'14·151.

Results Molar concentrations of all elastase and inhibitor solutions were determined by equivalent titration with an Eglin c solution of known molarity, or active site titrated chymotrypsin, respectively. The molar con-

Material and Methods Materials Purified human leukocyte elastase (HLE) and recombinant serine proteinase inhibitors Eglin c and secretory leukocyte proteinase inhibitor (SLPI) were kindly provided by Dr. Schnebli (Ciba-Geigy, CH-4002 Basel). Sheep leukocyte elastase (SLE) was isolated from peripheral PMNs as previously described'8'. Baboon leukocyte elastase (BLE) preparations were obtained by ultrasonication of baboon PMNs. The cells were purified by Percoll gradient centrifugation according to Hjorth et al.'13] with a slightly modified gradient from 55 to 71% instead of 55 to 74%. The baboon PMN homogenate was separated from cell debris by centrifugation with an Eppendorf centrifuge 5413 for 30 min at 4°C (Eppendorf Ger tebau, W-2000 Hamburg, Germany). This crude extract was stored at -20°C until use for kinetic determinations. The specific chromogenic substrate 7V-(methoxysuccinyl)-[L-alanyl]2-L-prolylL-Valyl-4-nitroanilide for elastase activity measurement was purchased from Protogen AG (CH-4448 L ufelfingen).

40 60 Inhibitor added M Fig. 1. Example of an equivalence titration curve.

Elastase inhibition Equivalent titration: Molar concentrations of all enzyme and inhibitor preparation were determined by direct titration according to Braun et al.' 4 '. To 10 μΐ of enzyme solutions increasing volumes (from 0 to 90 μ/) of inhibitor solution were mixed inTris-buffered saline (lOOmM NaCl, 50mMTris/HCL, 0.05% Triton X-100;

Constant volumes of a human elastase preparation of unknown molarity were mixed with different volumes of an Eglin c solution of known concentration. The residual enzymatic activity (% of initial activity) was plotted versus the amount of inhibitor (in μι) added. The equivalence point was determined by intersection of the straight line with the *-axis.

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Vol. 373 (1992)

Inhibition of Human, Ovine, and Baboon Neutrophil Elastase

centration of (active) Eglin had initially been determined with an active-site-titrated chymotrypsin solution. A linear regression of the linear part of titration curves as shown in Fig. 1 was applied to determine the



• HLE/Eglin c O BLE/Eglin c D SLE/Egli



'ο ^ 50

10 15 20 Inhibition time [s]



Fig.2. Plot used for calculation of the association rate constants of elastases with Eglin c. Association rate constants A:on of the inhibition reactions of SLE, BLE, and HLE with Eglin c.The values of kon were determined from the slopes shown in the diagram according to following equation: kon = ([E]~l - [Eo]~l)/t.


10 15 20 Inhibition time [s]



Fig. 3. Determination of the association rate constants of the elastases with SLPI. The association rate constants of the inhibition of SLE, BLE, and HLE with SLPI were determined as described in the text and in legend of Fig. 2.

Table. Association rate constants (kon) of sheep, human, and baboon neutrophil elastases with Eglin c and SLPI. Association rate constant kon [M ls J ] Inhibitors

Eglin c SLPI

Neutrophil Elastases Human



2.4 xlO 7 4.1X10 7

2.5 χ 107 5.6X10 7

2.2 x 105 1.2X107


volume of inhibitor solution needed to inhibit the elastase solution. The coefficient of correlation was constantly higher than 0.98. Association rate constants were calculated from graphs as depicted in Figs. 2 and 3, according to the equation 1/[E] = (1/[E0]) + kon x t. The kon values determined for all elastase/inhibitor systems are shown in the table. Sheep elastase was poorly inhibited by the recombinant serine proteinase inhibitor Eglin c (&on = 2.2 x 105 Μ *s l). In contrast the kon of this inhibitor is more than 100 times higher with human elastase and 115 times higher with baboon elastase (table). Secretory leukocyte proteinase inhibitor effectively inhibited human, baboon, and sheep elastase with similar association rate constants. Ovine elastase again was found to have the lowest association rate constant of all three elastases tested. SLPI inhibits HLE with an association rate constant 3 times higher than that of SLE and 1.4 times lower than that of baboon elastase (see table). Discussion The immediate inhibition of elastase released during shock could be one possible way of reducing tissue damage and subsequent development of multiorgan failure syndrome. Eglin c and SLPI, two promising proteinase inhibitors with high potential to block human elastase in vitro are available in their recombinant form. A final answer about the usefulness of in vivo inhibition of elastase activity may be provided by in vivo experiments in animal models. The ability of the inhibitors to block the appropriate elastase with adequate effectiveness in vitro, however, is a prerequisite for such studies. In this contribution we determined the association rate constants of Eglin c and SLPI with human elastase and compared these data with values obtained with ovine and baboon elastases, respectively, as these two animal species serve as convenient models in shock research. Inhibition of sheep elastase by the recombinant serine proteinase inhibitor Eglin c is much less effective than inhibition of human and baboon elastases by Eglin c. This may explain the in vivo finding, that Eglin c does not ameliorate endotoxin shock in an ovine model of endotoxemia[6], because high concentrations of inhibitor would be needed to block elastase activity. Secretory leukocyte proteinase inhibitor seems to be more suitable for studying inhibition of elastase in sheep, since the association rate constant of SLE with SLPI is more than 50 times higher than that of the SLE/Eglin c pair, but only 4 times lower than that of HLE/SLPI. To our knowledge SLPI has Brought to you by | Purdue University Libraries Authenticated Download Date | 5/26/15 6:06 PM


W.G. Junger, S. Hallström, H. Redl and G. Schlag

not been tested in an ovine model although our in vitro findings indicate that this inhibitor may better reflect the conditions to be expected in patients. Baboon models are increasingly used in shock research by several groups of investigators[10~12]. Since the inhibition kinetic properties of baboon elastase with the two proteinase inhibitors are very similar to those of HLE, this model appears to be most suitable for studying these inhibitors. Before proceeding with in vivo experiments to test the feasibility of proteinase inhibitor in the baboon, in vitro testing of such inhibitors should be performed with purified elastase of baboon neutrophils to corroborate the data we obtained with crude PMN extracts. Despite many similarities between the biochemical properties of sheep and human elastase molecules, and between the elastase contents of neutrophils from both species^, a significant difference was found in the inhibition kinetics of these enzymes with the recombinant serine proteinase inhibitors Eglin c and SLPI. This difference between sheep and human elastase should be considered when using sheep as animal models to study ways to prevent proteolytic tissue damage with elastase inhibitors. The baboon, however, promises to be a more relevant model overall for such experiments.

References 1 2 3 4 5 6 7 8 9 10 11

12 13

We thank Dr. H.P. Nick, Ciba-Geigy AG, Pharma Division, Basel, Switzerland, for his valuable help, and Lucy Schwab for carefully correcting the manuscript. This study was supported in part by a grant of the Nanonalbunkfcmds.

Vol. 373(1992)

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Duswald, K.-H., Jochum, M., Schramm, W. & Fritz, H. (1985) Surgery 98, 892-899. Nuytinck, J.K.S., Goris, R.J.A., Redl, H., Schlag, G. & van Munster P.J.J. (1986) Arch. Surg. 121, 886-890. Braun, N.J. & Schnebli, H.P. (1986) Eur. J. Respir. Dis. 146,541-547. Braun, N.J., Bodmer, J.L., Virca, G.D., Metz-Virca, G., Maschler, R., Bieth, J.G. & Schnebli, H.P. (1987) Biol. Chem. Hoppe-Seyler368, 299-308. Vogelmeier, C., Buhl, R., Hoyt, R.F., Wilson, E., Fells, G.A., Hubbard, R.C., Schnebli, H.P,Thompson, R.C. & Crystal, R.G. (1990)/. Appl. Physiol. 69,1843-1848. Redl, H., Schlag, G., Vogl, C., Schiesser, A., Paul, E., Thurnher, M., Junger, W., Traber, L.D. & Traber, D.L. (1988) Biol. Chem. Hoppe-Seyler369, Suppl. 153-156. Staub, N.C., Bland, R.D., Brigham, K.L., Demling, R., Erdman, A.J. &Woolverton, C.W. (1975)/. Surg. Res. 19, 315-320. Junger, W, Hallström, S., Redl, H. & Schlag, G. (1988) Biol. Chem. Hoppe-Seyler369, Suppl. 63-68. Junger, W, Hallström, S., Redl, H. & Schlag, G. (1988) Am. Rev. Respir. Dis. 138,1358. Pretorius, J.P., Schlag, G., Redl, H., Botha, W.S., Goosen, D.J., Bosman, H. & van Eeden, A.F. (1987) /. Trauma 27, 1344-1353. Herman, C.M., McKee, A.E., Schilling, P.W, Dickson, L.G., Horowitz, D.L., Coran, A.G., Cryer, P.E. & Kopriva, C.J. (1972) in Shock in Low- and High-Flow States (Forscher, B.K., Lillehei, R.C. & Stubbs, S.S., eds.) pp. 42-48, Excerpta Medica, Amsterdam. Taylor, F.B.Jr, Emerson,T.E.Jr., Jordan, R., Chang, A.K. & Blick, K.E. (1988) Circ. Shock26,227-235. Hjorth, R., Jonsson, A.-K. & Vretblad, R. (1981) /. Immunol Methods 43, 95-101. Meyer, J.F., Bieth, J.G. & Metais, P. (1975) Clin. Chim. Acta62,43-61. Vincent, J.-P & Lazdunski, M. (1972) Biochemistry 11, 2967 2977.

W.G. Junger*, University of California San Diego, Department of Surgery 8236, Division of Trauma, 225 Dickinson Street, San Diego, CA92103, USA; S. Hallström, H. Redl, G. Schlag, Ludwig-Boltzmann-Institut für Experimentelle und KlinischeTraumatologie, Donaueschingenstr. 13, A-1200Wien, Österreich. * To whom correspondence should be addressed.

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Inhibition of human, ovine, and baboon neutrophil elastase with Eglin c and secretory leukocyte proteinase inhibitor.

The association rate constants (kon) of human, ovine, and baboon neutrophil elastase with two recombinant serine proteinase inhibitors (Eglin c, secre...
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