Eur Resplr J 1991, 4, 1004-1009

Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments

C. Lafuma*, E. Frisdal**, A. Hart**, L. Robert*, W. Hornebeck*

Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments. C. Lafuma, E. Frisdal, A. Harf, L. Robert, W. Hornebeck. ABSTRACT: Heparin and its derivatives inhibit human leucocyte proteinases i.e. elastase and cathepsin G, but do not inhibit porcine pancreatic elastase and Pseudomonas aeruginosa elastase. In vitro experiments, reported here, also indicate that elastin, one of the physiological substrates of human leucocyte elastase (HLE), could decrease by 30-fold the inhibitory potential of an hexadecasaccharide heparin fragment (dp 16) isolated from CY 222. Nevertheless, the inhibitory capacity of the heparin fragment still remains elevated with IC

50 = 2.7 x 10'7 M and still inhibits HLE in Its fre.e and adsorbed

state to elastin. These overall data prompted us to evaluate the influence of CY 222 in HLE-induced emphysema.

Emphysema was Induced in mice eight weeks old, following a single Instillation of 200 J.lK of HLE. CY 222 treated animals received 2.5 mg·kg·1 subcutaneously once dally, 6 days per week during 4 weeks prior to HLE instillation, and for eight weeks following HLE instilla­tion. The heparin fragment treatment of the mice halved the mortality rate observed early following HLE instillation. After 8 weeks, surviving animals were examined for lung histological and morphometrical changes: mean linear intercept (MLI) and Internal alveolar area (ISA). The CY 222 heparin fragments exerted a protective effect against HLE·lnduced emphysema by decreasing by 70% the MLI; these heparin fragments exerted no effect on emphysema induced by pancreatic elastase In hamsters or mice.

Heparin derivatives represent a new class of physiological HLE low molecular weight inhibitors capable of preventing HLE-induced emphysema. Eur Respir J., 1991, 4, 1004-1009.

• URA CNRS 1460, Laboratoire de Biologie du Tissu Conjonctif, Universit6 Paris XII, Creteil 94010, France.

• • U 296 Inserm, Faculte de Medecine, Creteil 94010, France.

Correspondence: C. Lafuma, URA CNRS 1460, Laboratoice de Biologie du Tissu Conjonctif, Universit6 Paris XII, Creteil 94010, France.

Keywords: Elastase; emphysema; heparin.

Received: June, 1990; accepted after revision April 24, 1991.

Supported by CNRS (URA CNRS 1460), by A.N.V.A.R. (Contract No. SS 4452 00) and the conseil scientifique d l'Universit6 Paris XII.

The discovery of homozygous alpha1-proteinase

inhibitor ( ali) deficiency and the key role of elastolysis in experimental elastase-induced emphysema led to the current assessment of elastase anti-elastase imbal­ance in the lungs as the major cause of emphysema [1-3]. Human neutrophil serine elastase (EC 3. 4. 21. 37) is a potent mediator of connective tissue break­down; it is able to degrade not only elastin but also other macromolecular constituents of the extracellular matrix under physiological conditions [4-7]. The destruction of elastin fibres and the concomitant loss of elastic recoil in the emphysematous lung have been demonstrated, and attention has been focused on neu­trophil elastase as the most probable catabolic agent involved in the pathogenesis of emphysema.

inhibitors and thus restoring the elastase anti-elastase balance (8]. Nevertheless, the major drawbacks of this approach may reside in the possible undesirable side-effects of the inhibitors such as instability, toxicity or immunogenicity. These inconveniences may be minimized by using inhibitors derived from physiologi­cal molecules.

In order to prevent pulmonary tissue damage, one possible approach is to increase the antiprotease level in the lung by supplementing with suitable

Leucocyte elastase is a strong basic protein with pi near 11. It has been previously demonstrated that several anionic glycosaminoglycans could inhibit this proteinase via electrostatic interactions (9, 10). Heparin, which is a complex and heterogeneous mixture of polysaccharides varying in molecular weight and degree of sulphation [11 ], belongs to glycosaminoglycans capable of inhibiting leucocyte elastase and also cathe­psin G although to a lesser extent (12, 13]. We recently demonstrated that inhibition of leucocyte elastase by heparin fragments was correlated with the chain

EFFEcr OF HEPARIN FRAGMENTS ON ELASTASE-INDUCED EMPHYSEMA 1005

length of the oligosaccharides and was related to their degree of sulphalion [12). Previous experiments were performed using synthetic peptide elastase substrates, but we demonstrate in the present investiga­tion that elastin can greatly modulate the inhibitory potential of heparin fragments as compared with that obtained with low molecular weight synthetic substrates; however, a hexadecasacchatide heparin fragment was shown to inhibit human leucocyte elastase (HLE) both in its free form and adsorbed state to elastin.

Also, experiments indicated that heparin and its inhibi tors could influence a 1-proteinase inhibitor (ali) elastase interactions [13). When administered in vivo, heparin fragments (CY 216- CY 222) were able to increase the pancreatic elastase inhibitory capacity of hamster serum; in contrast, the rates of associations between pancreatic and leucocyte elastases and ali as determined by in vitro experiments were fou nd to decrease respectively by 2- and 40-fold [13, 14].

Therefore, several arguments seem to favour the beneficial value of heparin and its derivatives towards HLE elastolysis, whereas others pointed toward a pos­sible aggravating effect of such substances. That prompted us to evaluate the in vivo potential of these compounds as HLE elastase inhibitors in an emphysematous model.

Materials and methods

Heparin fragments

Pig mucosal standard heparin (160 USP units·mg·1;

155 anti-(factor Xa) units·mg-1) came from Sanofi/Choay (Paris, France). CY 222 polysaccharides, (mean molecular weight= 4,500; 30 USP units·mg·1; 248 anti­(factor Xa) units·mg·1) were obtained by chemical depolymerization of heparin with HN0 2 and gel filtration [12). The hexadecasaccharide (dp 16 ref. IC 84-1552) used in this study was obtained from CY 222 by gel fil tration on a Sephadex G-50 (superfine grade) column (300 cm x 2.5 cm) eluted with 0.2 M NaCl. The degree of polymerization of dp 16 was confirmed by 13C nmr analysis. The degree of sulphation (SO}·/C0

2.·) of the products were determined by

conductimetry [12, 13].

Elastase and substrates

Human leucocyte elastase (HLE) was purchased from Elastin Products Company (EPC, St. Louis Mo, USA). It was active site titrated as previously described [12]. Succinoyl trialanine paranitroanilide came from Sanofi/Choay. Insoluble elastin was prepared from calf Ligamentum nuchae; its purity was checked by amino acid analysis. It was radiolabelled with NaB3H4 (sp. radioactivity 10 ~LCi·mg·1) as detailed elsewhere [12].

Elastase assays and inhibition studies

All assays were performed using HLE (6-20 nM) in 100 mM Tris/HCl, 0.02% NaN3 pH 8.0 at 37°C. Amidolytic activity of the enzyme toward 1.25 mM Suc(Ala)

1NA was continuously recorded at 410 nM in

a Philips spectrophotometer. The elastinolytic activity of HLE was quantitated

using a 400 )Ag·mt·1 dispersion of 3H calf ligamentum insoluble elastin (10 ~-tCi·mg-1). Different chronological additions of reactants were performed: i) enzyme and heparin fragments are incubated for 20 min before the addition of elastin; ii) enzyme and substrate are incubated for 5 min before the addition of heparin fragments; iii) elastin and heparin fragments are incubated for 10 min before the addition of HLE.

Elastolysis is then allowed to proceed for up to 24 h at 37°C under constant mechanical stirring; the Eppendorf tubes are centrifuged at 10,000 rpm and aliquots of the superoatants are withdrawn for radioactivity determinations.

IC50

with both substrates are defined as the concen­tration of substance that gives 50 per cent of inhibition under our experimental conditions.

HLE-induced emphysema in mice

Four groups of 2 month old male white Swiss mice (30±3 g body wt) from IFA CREDO Laboratories (Lyon, France) were used. The control group (I) (n=12) received an intratracheal injection of 200 ~-tl saline. The second group (11) (n=ll) was injected subcutane­ously with 250 ~-t l of heparin fragments CY 222 (2.5 mg·kg·l), once daily, five days per week, during 12 consecutive weeks; this group received an intra­tracheal saline injection (250 J.lJ) following 4 weeks of treatment with heparin fragments. A group (Ill) (n=12) was instillated intratracheally with HLE in saline solution (200 ~lg in 200 J.ll). A group (IV) (n=12) was injected subcutaneously with heparin fragments CY 222 as in the second group, but also received a single intratracheal injection of 200 f..lg HLE just following the 4 weeks of treatment with CY 222. The last injection of CY 222 was administered 2 h before HLE instillation.

At the end of the post-instillation treatment period with CY 222, mice were killed by bleeding from their abdominal aorta and their lungs were examined for histological and morphometrical changes.

Lung histology and morphometry

Lungs were deflated and fixed at a pressure of 20 cmHp by instillation of 2.5% glutaraldehyde through a polyethylene catheter inserted into the trachea. The volume (VL) of fixed lungs was measured by saline displacement.

1006 C. LAFUMA ET AL.

100

~ 80 .~

~ 60

40

20

0.0001 0.001 0.01 0.1 10 100

Concentraction I!Q·mr1

Fig. 1. - Inhibition of human leucocyte elastase (HLE) (6 nM) by a hexadecasaccharide heparin fragment (dp 16). +--+: substrate Suc(Ala)1NA (1.25 mM); eJ---el: substrate )H elastin (400 j.lg·mJ-1 sp. act: 10 j.lCi·mg-1; abscissa: concentration of dp 16 in f.!g·ml (logarithmic scale); ordinate: percent of residual activity.

Lungs were then dehydrated, degassed, and embedded in paraffin. Sagittal sections (6 !!m) cut from whole lungs were stained with haematoxylin-eosin and processed for histological studies.

200 ~m

Light microscopy stereological studies of lung parenchyma were carried out on both lungs. Points and intersections were recorded in 20 randomized microscopic fields per lung, using a Zeiss ocular test system with five lines at a magnification of 10. The linear shrinkage factor was estimated equal to 1.3. The mean linear intercept (MLI) and internal alveolar area (ISA) were calculated as previously de­scribed [14].

Statistical analysis

For each parameter, one way variance analysis was performed and intergroup comparisons were made. Moreover, data were statistically investigated with a variance analysis for repeated measurements (BMPD statistical software).

Results

CY 222, polydispersed heparin fragments, had IC

5 equal to 40 ng·ml'1 when Suc(Ala)

3NA is used as

an HLE substrate [13]; giving the average molecular weight of the substance, this inhibitory value is close

Fig. 2. - Light microscopy of inflation-fixed lungs. Lungs from control mice (A), CY 222 treated mice (B) and CY 222 and human leucocyte elastase (HLE) treated mice (C) show normal alveolar morphological f~atures. Discrete emphysema was evidence in mice treated with HLE (D).

EFFECf OF HEPARIN FRAGMENTS ON ELASTASE-INDUCED EMPHYSEMA 1007

to that obtained with a hexadecasaccharide (dp 16) isolated from CY 222: dp 16 IC

50 is equal to 0.8 nM

(fig. 1). We therefore investigated the influence of the substrate using dp 16 as a defined heparin fragment. Whatever the substrate used i.e. Suc(Ala)3NA or 3H insoluble elastin, dp 16 and HLE (0.6 nM) were preincubated for 20 min at 3rc before the addition of the substrate. Figure 1 shows that the efficiency of dp 16 as an HLE inhibitor is markedly decreased when elastin is used as a substrate as compared with that obtained using Suc(Ala)

3NA. The IC

50 of the

compound is equal to 2.7x10·7 M when elastin is used as substrate, that representing a 30-fold decrease in its inhibitory capacity as compared with experiments performed with the synthetic elastase substrate.

However, when elastin is used as a substrate, a nearly complete inhibition of HLE activity is observed in contrast with that found using Suc(Ala)~NA where, as previously observed, only partial inh1bition was attained [12). When elastase was preincubated with elastin in conditions where all added enzyme bound to its substrate, the IC

50 of dp 16 is found equal to

2.3xt0·7 M indicating that dp 16 inhibited HLE whether the enzyme was free or adsorbed to its substrate with little difference.

MU 90

80

70

60

50

40

**

c CV CY+HLE HLE

Fig. 3. - Mean linear intercept (MLI) values (mean±SEM) for the 4 groups of mice, as described in the text (animals). The MLI mean value of the human leucocyte elastase (HLE) treated groups is sig­nificantly different from the control (C) group and is indicated by two asterisks. (••: p<O.OOl). The MLI mean value of the CY 222 HLE groups Is sianificantly different from the (HLE) group and is indicated by one asterisk (•: p<0.02).

Although reduced in the presence of elastin, the inhibitory capacity of dp 16 (0.1 ~g·ml·1 : 2.7xl0·7 M) and CY 222 fragments (0.25 J.tg·ml·') could be consid­ered in the range of "good" elastase inhibitors [4, 8] substantiating further in vivo studies.

Human leucocyte elastase-induced emphysema in mice, and effects of heparin fragments

Intratracheal injection of HLE in mice produced a high mortality rate (60%) due to haemorrhage within minutes following the instillation. Treatment with CY 222 heparin fragments reduced the mortality rate by half. T he lungs of mice treated w ith hepari n fragments were comparable to those of control mice (fig. 2A, 2B). Lungs of surviving mice injected with HLE exhibited several regions of mild emphysema characterized by discrete air space enlargement (fig. 20). Emphysematous lesions produced by HLE appeared reduced by treatment with CY 222 (fig. 2C).

MLI was significantly increased in HLE treated mice when compared to the control group (fig. 3). CY 222 treatment by itself did not modify MLI or ISA, but MLI and ISA returned near control values in mice injected with HLE and treated with CY 222.

Discussion

The potential role of HLE in the pathogenesis of emphysema is now well documented. This enzyme appears at a hig h local concentration in alveolar spaces or interstitial sputu m during infl am matory pulmonary injury [ 4-6]. The active site of this leuco­cyte enzyme is known and its tertiary structure in complex with inhibitors has been elucidated [15-17]; therefore, potent inhibitors of HLE can be designed. HLE is a very basic protein and positively charged arginine residues appear in a cluster array on the envelope of the molecule [17}. This particular arrange­ment of arginine probably accounts for the interactions of the enzyme with glycosaminoglycans in a non­competitive hyperbolic way (12]. A minimal number of 12 saccharide units was necessary to obtain values of Ki<J.tM and the inhibitory potential of heparin fragments could be further increased by oversulphation of these heparin derivatives [12, 13].

Inhibition of HLE by heparin and its fragments could be abolished by increasing the ionic strength of buffer assays; we also demonstrate here the substrate dependency of this inhibition. R educed inhibitory efficiency of hexadecasaccharide was obtained when a synthetic peptidic elastase substrate i.e. Suc(Ala)

3NA

was replaced by elastin. Since enzyme and heparin fragments were preincubated in each case before adding the subslrate we can reasonably assume that elastin was able to partly dissociate heparin fragments bound to HLE or alternatively to modify the hyperbolic nature of the inhibition i.e. influencing the ~ component of the inhibition [12]. Nearly complete inhibition of HLE was attained when using 3H insoluble elastin as substrate. In contrast, it was reported that elastin was unable to displace HLE complexed with its main physiological inhib itors a li or mucus proteinase inhibitor (MPI) [18]. In contrast to a li and similarly to MPI, a low MW heparin fragment was able to inhibit HLE indifferently in its free and adsorbed state on elastin [19).

1008 C. LAFUMA BT AL.

The in vivo effect of heparin fragments on elastase­induced emphysema was then investigated. Chronic subcutaneous pretreatment and post-treatment with CY 222 was carried out since this route of administra­tion allowed a greater half life of heparin derivatives [20]. Since previous data have shown that subcutaneous injections of CY 222 in hamsters were able to increase the pancreatic elastase inhibitory potential of the sera of the animals, probably stimulating the synthesis of all [13], we first investigated the effect of these heparin fragment treatments on pancreatic elastase­induced emphysema in hamsters (not shown). Hamsters were pretreated subcutaneously with CY 222 (2.5 mg·kg·1 body weight) similarly as mice and received a single dose of 50 J,tg of porcine pancreatic elastase (PPE). Morphometric studies as well as inves­tigations of lung mechanics clearly showed that treatment with CY 222 was totally inefficient in preventing pancreatic elastase-induced emphysema. Preliminary investigations also indicated that similar CY 222 treatment applied to mice did not affect the development of emphysematous lesions induced by intratracheal instillation of 20 J,tg of pancreatic enzyme. This result is in keeping with the lack of any in vitro inhibitory effect of heparin fragments against PPE (12]; it also indicated that a 30-40% enhancement of elastase inhibitory activity of sera following CY 222 treatment did not influence to any extent the deleterious effect of the proteinase toward parenchyma! structures at least in our experimental conditions and with the dose of enzyme instilled.

We initially tried to induce emphysema in hamsters by administering human leucocyte elastase intratracheally. Even administration of amounts of enzyme as high as 300 J,tg proved to be totally ineffi­cient in producing any characteristic emphysematous lesions in hamsters. A similar discrepancy between the capacity of PPE and HLE to induce emphysema had also been reported by other investigators [3, 21-23). It could be interpreted partly by the high rate of association of HLE with ali [24), and also partly by the presence of specific HLE inhibitors as bronchial inhibitors in alveolar spaces [25]; it can also be attributed to the broader specificity of HLE as compared to PPE targeting the leucocyte enzyme to macromolecules other than elastin. In addition, porcine pancreatic elastase, but not HLE was shown to be carried across the alveolar wall in pinocytotic vesicles therefore possibly explaining its rapid access to elastin fibres and the functional inefficiency of proteinase inhibitors [25]. We therefore performed experiments in smaller animals such as mice, and were able to induce emphysematous lesions 8 weeks following a single intratracheal injection of HLE (200 J.l8 per mouse). Chronic subcutaneous injections of CY 222 were able to significantly reduce the HLE-induced lung emphysema in mice by decreasing the MLI by 70%. This treatment was also able to reduce the high mortality rate induced by HLE instilla­tion by 50%. This high mortality rate is probably due to bleeding, cell disruption and denudation of

basement membranes caused by HLE. The 50% reduc­tion in the mortality rate observed in the CY 222 treated mice may also be partly related to an inhibitory effect of these compounds toward the degradation of antithrombin Ill by HLE [26).

Although the in vivo protecting effect of heparin fragments towards lung elastolysis induced by HLE seemed to be correlated to the in vitro inhibitory capacity of CY 222 towards elastase, other biological effects of these substances could perhaps be superim­posed. Glycosaminoglycans, for instance, have been shown to bind to elastin and may provide a physical barrier to elastolysis catalysed by HLE [27). Recent data indicate that heparin can accelerate the interaction between HLE and mucus bronchial inhibitor (MBI) (14) a substance which can regulate proteolysis at the neutrophil substrate interface [28).

Whatever the in vivo mechanism involved, our data show that heparin derivatives are potential physiologi­cal drugs in the treatment of emphysema.

Acknowledgements: We are grateful to Dr M. Levame for helpful scientific assistance, Mr Discours for his excellent technical assistance and to Drs J. Choay and M. Petitou for providing us with heparin fractions and Dr J. Bieth for helpful discussion.

References

1. Laurell CB, Erickson S. - The electrophoretic alpha 1 globulin pattern of serum in alpha 1 antitrypsin deficiency. Scand J Clin Invest, 1983, 15, 132-140. 2. Janoff A. - Elastases and emphysema. Current assess­ment of the protease-anti proteases hypothesis. Am Rev Respir Dis, 1985, 132, 417-433. 3. Senior RM, Tegner H, Kuhn C, Ohlsson K, Starcher BC, Pierce JA. - The induction of pulmonary emphysema with leucocyte elastase. Am Rev Respir Dis, 1977, 116, 469-475. 4. Bieth J. - Elastases: structure, function and pathologi­cal role. In: Front Matrix Bioi, 1978, 6, 1- 82. 5. Gadek JE, Fells GA, Wright DG, Crystal R. - Human neutrophil elastase function as a type Ill collagen "Collagenase". Biochem Biophys Res Comm, 1980, 95, 4, 1815-1822. 6. Mainardi C, Dixit S, Kang A. - Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocytes granules. J Bioi Chem, 1980, 255, 11, 5435- 5441. 7. McDonald J, Kelley D. - Degradation of fibronectin by human leukocyte elastase. J Bioi Chem, 1980, 255, 18, 8848-8858. 8. Powers JC, Bengali. - Elastase inhibitors for treatment of emphysema. Approaches to synthesis and biological evalu­ation. Am Rev Respir Dis, 1986, 134, 1097-1100. 9. Baici A, Salgam P, Ferh K, Boni A. - Inhibition of human elastase polymorphonuclear leucocytes by a glycosaminoglycan polysulphate (Arteparon). B iochem Pharmacol, 1980, 29, 1723-1728. 10. Baici A, Bradamante P. - Interactions between human leucocyte elastase and chondroitin sulfate. Chem Bioi Inter­act, 1984, 51, 1-11. 11. Jaques LB. - Heparin: an old drug with new paradigm. Science, 1979, 206, 528-533.

EFFECI' OF HEPARIN FRAGMENTS ON ELASTASE-INDUCED EMPHYSEMA 1009

12. Redini F, Tixier JM, Petitou M, Choay J, Robert L, Hornebeck W. - Inhibition of leucocyte elastase by heparin and its derivatives. Biochem J, 1988, 252, 515-519. 13. Redini F, Lafuma C, Hornebeck W, Choay J, Robert L. - Influence of heparin fragments on the biological activities of serum elastase and alpha 1 proteinase inhibitor. Biochem Pharmacal, 1988, 37, 22, 4257-4261. 14. Bieth J. - Effect of heparin on the rate of inhibition of neutrophil elastase by a proteinase and mucus proteinase inhibitor. In: Internationa) Symposium on peptide inhibitors of proteolytic enzymes, Montpellier, France, 1990. 15. Li de la Sierr I, Papamichael E, Sakarlllos C, Dimicoli JL, Praogi T. - Interaction of the peptide CF3-Leu-AJaNH­C6M4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A. J Mol Recognition, 1990, 3(1) 36-44. 16. Bode W, Wei AZ, Hubert R, Meyer E, Travis J, Neuman. - X-ray crystal structure of the complex of human leukocyte elastase (PMN-elastase) and the third domain of the turkey ovomucoid inhibitor. Embo J, 1986, 5, 2453- 2458. 17. Navia MA, Springer JP, Lin TY, Williams HR, Fire­stone RA, Pisano JM, Doherty JB, Finke PE, Hoogsteen K. - Crystallographic study of a ~-lactam inhibitor complex with elastase at 1.8 A resolution. Nature, 1987, 327, 79-82. 18. Bruch M, Bieth JG. - Influence of elastin on the inhi­bition of leukocyte elastase by a1 proteinase inhibitor and bronchial inhibitor. Potent inhibition of elastin-bound elastase by bronchial inhibitor. Biochem J, 1986, 238, 269-273. 19. Hornebeck W, Schnebli MP. - Effect of different elastase inhibitors on leukocyte elastase preadsorbed to elas­tin. Z Physiol Chem, 1982, 363, 455-458. 20. Fareed J, Kumar A, Walenga JM, Emanuelei RM, Williamson K, Hoppensteadt D. - Antithrombotic actions and pharmacokinetics of heparin fractions and fragments. Nouv Rev Fr Hematol, 1984, 26, 267-275. 21. Kleinerman J, Ranga V, Rynbrandt D, Sorensen J, Powers J. - Effect of the specific elastase inhibitor, alanyl alanyl prolyl alanine chloromethylketone, on elastase induced emphysema. Am Rev Respir Dis, 1980, 121, 381-387. 22. Stone P, Lucey E, Colore J, Snider G, Franzblau C, Castillo M, Powers J. - The moderation of elastase induced emphysema in the hamster by intratracheal pretreatment or post-treatment with succinyl-alanyl-alanyl prolyl­valine chlorometyl ketone. Am Rev Respir Dis, 1981, 124, 56-59. 23. Gadapaty S, Liener I, Hoidal J, Padmanablan R, Niewoehner D, Abel J. - The prevention of elastase induced emphysema in hamsters by the intratracheal administration of a synthetic elastase inhibitor bound to . albumin microspheres. Am Rev Respir Dis, 1985, 163, 132-159. 24. Bieth JG. - In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem Med, 1984, 32, 387-392. 25. Lucey EC, Stone PJ, Ciccolella DE, Brener R, Christenseur TG, Thomson RC, Snider GL. ·- Recombinant human secretory leukocyte-protease inhibitor: In vitro properties and amelioration of human neutrophil elastase

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Prevention par les fragments d'heparine de l'emphyseme induit par /'elastase /eucocytaire chez /es sour is. C. Lafuma, E. Frisdal, A. Harf. L. Robert, W. Hornebeck. RESUME: L'Mparine et ses d~riv~s inhibent les prot~inases leucocytaires humaines, c'est-a-dire l'tlastase et la cathepsine G, mais non !'elastase pancrtatique porcine ou !'elastase de pseudomonas aeruginosa.

Les exptrimentations in vitro, mentionnees ici, indiquent egalcment, toutefois, que l'elastine, un des substiats physiologiques de !'elastase leucocytaire bumaine (HLE), pourrait diminuer de 30 fois le potentiel inhibiteur d'un fragmen t bexadecasaccbaridique de l'Mparine (dp 16) isol6 de CY 222. Neanmolns, la capacite inhibitrice du fragment d'Mparine reste encore ~levee, avec IC

5 = 2.7xlO·' M et

inhibe toujours HLE, qu'elle soit libre ou a~sorb6e a l'elastine. L'ensemble de ces donnees nous a pousse a evaluer !'influence de CY 222 dans l'empbys~me induit par HLE. L'emphys~me a ete provoque chez des souris ligees de huit

semaines par une seule instillation de 200 J.tg de HLE. Les animaux traites par CY 222 ont re~u par voie sous-cutanee 2.5 mg!kg de poids une fois par jour, 6 jours par semaine pendant 4 semaines, prealablement a !'instillation de HLE, et penda.nt huit semaines apras cette instillation. Le traitement avec des fragments d'Mparine des souris a rtduit de moitie le taux de mortalite precocc observe apr~s instillation de HLE. Apr~s 8 semaines, les animaux survivants ont ete soumis a un examen bistologique et morphometrique du poumon, important notamment !'intercept lineaire moyen (MLI) et la surface alveolaire interne (ISA). Les fragments d'heparine CY 222 exercent un effet protecteur contre l'emphys~me induit par HLE, en diminuant de 70% le MLI. Ces fragments d'heparine n'exercent pas d'effet sur l'emphys~me induit par l'tlastase pancreatique cbez les hamsters ou les souris.

Ces dtrives de l'Mparine representent une nouvelle classe d'inhibiteurs pbysiologiques de HLE a poids moleculaire bas et soot susceptibles de prevenir l'emphys~me induit par HLE. Eur Respir J., 1991, 4, 1004-1009.

Note added in proofs: During the time of revision of this manuscript Rea et al Am. Rev. Resp. Dis. 1990; 142: 407--412 also presented evidence of the potential value of other glycosaminoglycan in pre­venting leucocyte elastase induced emphysema.