Peptides. Vol. 11, pp. 869-871. © Pergamon Press plc, 1990. Printed in the U.S.A.

0196-9781/90 $3.00 + .00


Somatostatin Inhibits Neutrophil Elastase Release In Vitro E. O. A D E Y E M I , A. P. S A V A G E , S. R. B L O O M A N D H. J. F. H O D G S O N

Department of Medicine, Royal Postgraduate Medical School Hammersmith Hospital, Ducane Road, London W12 ONN R e c e i v e d 14 D e c e m b e r 1989

ADEYEMI, E. O., A. P. SAVAGE, S. R. BLOOM AND H. J. F. HODGSON. Somatostatin inhibits neutrophil elastase release in vitro. PEPTIDES 11(4) 869-871, 1990.--The influence of the long-acting somatostatin analogue, SMS 201-995, on FMLP-induced neutrophil elastase release in vitro has been investigated. Doses from 150 ng/ml upwards inhibited elastase release, with 100% inhibition by 2500 ng/ml. Inhibition was demonstrated both by an assay measuring elastase immunometrically and by an assay based on its enzyme activity. The demonstration that SMS 201-995 inhibits protease release from polymorphonuclear leukocytes may have implications for the long-term clinical use of this somatostatin analogue. Somatostatin

Neutrophil elastase release

SMS 201-995

ELASTASE is a serine proteinase, stored in the azurophil granules of polymorphonuclear leucocytes (PMNL), released during phagocytosis and capable of degrading connective tissue at physiological pH (1). It causes tissue damage in diseases in which PMNLs are involved (1). The tetradecapeptide somatostatin (SST) has been shown to inhibit various secretory processes in the central nervous system, endocrine system, and gastrointestinal tract (4). The mechanism of action of SST in inhibiting secretory processes is not fully understood, but two inhibitory mechanisms among others have been identified, inhibition of cyclic adenosine monophosphate (cAMP) and blockade of cytosolic calcium increases (8). As neutrophil enzyme release is influenced by intracellular cAMP levels (7), we investigated the effect of a somatostatin analogue, SMS 201-995, on elastase release from neutrophils in vitro (7).

In vitro study

raacetic acid (EDTA), 20 U/ml aprotinin (provided by Prof. Hoffmeister, Bayer Ag, West Germany), 0.2 mg/ml sodium azide and 1 mg/ml BSA, pH 7.4. Cytochalasin B was dissolved in 1% dimethyl sulphoxide (DMSO) (BDH Ltd., Poole, England) to form a 100 ixg/ml stock solution. SAAV-PNA was stocked at 0.001 M concentration in straight DMSO and FMLP at 0.001 M in dimethyl formamide containing 1% triethylamine. Elastase was purified from granulocytes harvested from patients with chronic granulocytic leukemia (1).

Radioiodination of Protein A One mg protein A (PA) was radioiodinated with 1 ixCi sodium iodide-125 (Amersham International UK Ltd.) per microgram PA using the iodogen radioiodination technique (3). After passing the free radioiodine and labeled PA down the PD-10 column (Pharmacia UK Ltd.), 75% of the total radioactivity was trichloroacetate (TCA)-precipitable PA-bound radioiodine.

METHOD Synthetic somatostatin 14 (SST 14) was obtained from Bachem Inc., USA and somatostatin analogue, SMS 201-995, was a kind gift from Sandos UK Ltd. Cytochalasin B was purchased from Aldrich Chemicals UK Ltd., N-formyl-methionyl-leucyl-phenylalanine (FMLP) from Bioproducts, Belgium and Suc-Ala-AlaVal-pNa (SAAV-pNA) from Cambridge Research Biochemicals, UK Ltd. The cell washing solution contained 0.01 M Tris, 0.14 M NaC1, 0.004 M KC1 and 1 mg/ml D-glucose at pH 7.4. The peptide diluent contained in addition 2.5 mg/ml bovine serum albumin (BSA). Cell suspension buffer contained 0.01 M Tris, 0.14 M NaC1, 0.004M KC1, 0.001 M CaCI 2, 0.0005 M MgC12, 0.0004 M MgSO 4, 1 mg/ml of D-glucose and 2.5 mg/ml BSA pH 7.4. Radioimmunometric assay (RIMA) buffer was phosphatebuffered saline (PBS) containing 0.002 M ethylene-diaminetet-

Isolation of Neutrophils Blood from ten donors (six females, four males, mean age 36.7 years) was drawn into tubes containing 0.1 M EDTA. PMNLs were isolated by hydroxyethylstarch (HES) sedimentation, centrifugation on hypaque/ficoll gradient and hypotonic lysis of erythrocytes Boyum (2). Giemsa stain confirmed that 99% were PMNLs. Unstimulated cells excluded trypan blue after 2 hr (each experiment lasted 30 minutes) at RT and a random LDH measurement during the study was negative.

Elastase Secretion Studies Cells [2.5


× 10 6

(in 760 Ixl)] preincubated at 37°C in two



successive steps of 5 minutes each with 4 Ixg cytochalasin B (in 40 I~1) and varying concentrations of SMS 201-995 or SST 14 in 100 Ixl were incubated for 20 min at RT with 10 - 6 M FMLP. FMLP ( 1 0 6 M ) stimulated maximal elastase release in preliminary experiments. The cells were separated at 1000 x g at 4°C and elastase measured in supernatants (vide infra). Negative controls consisted of cells in plain buffer, and buffer solutions containing 1 and 10% DMSO without exposure to FMLP. DMSO at these concentrations was demonstrated not to affect elastase release. SMS 201-995 was omitted in positive controls.

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Functional Assay


One hundred i~1 SAAV-pNA (stocked at 0.001 M in 100% DMSO) was added to 400 ixl each of peptide diluent and supernatant. Eight hundred ILl diluent and 100 txl SAAV-pNA served as negative control. The reaction was stopped after incubating at 37°C for 30 min with 100 ILl of 10 Ixg/ml soybean trypsin inhibitor, and absorbance values measured at 405 nm. SMS 201-995 inhibitory capacity (SIC) was calculated as the percentage decrease of absorbance value from the positive control.











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SMS 201-995 (ug/ml) ° FIG. 1. Effect of different concentrations of SMS 201-995 (abscissa, Ixg/ml) on FMLP-induced elastase release from neutrophils (ordinate, ng/ml). Each bar shows mean elastase concentration_ +s.e.m. from ten individuals.

Radioimmunometric Assay Elastase standards and proteins from the supernatants were bound to microtitre plate wells (Dynatech Lab Inc., USA) precoated with polylysine A and glutaraldehyde at 37°C at 1 hr. Antiserum (1:1000) and radiolabeled PA (1:500) were added in successive steps of 1 hr at RT, washing thrice with peptide diluent between steps. Radioactivity was determined in a gamma counter. To compare the SIC values obtained with both methods, the SIC was calculated as the percentage decrease of radioactivity from the positive control. The calibration RIMA curve was linear from 6.25-100 ng/ml of elastase. The within-run coefficient of variation (CV) was 5% for eighteen measurements, whilst the interassay CV was 8% for ten separate measurements.

profound inhibition of FMLP-stimulated elastase release from neutrophils at 0.15 txg/ml SMS 201-995 concentration upwards. This suggests a potential role for SMS 201-995 as an adjunct to therapy in inflammatory conditions involving migration, aggregation and degranulation of granulocytes. The in vitro SMS 201-995 levels in this study are considerably higher than those obtained in vivo (5), when 50 Ixg SMS 201-995 was injected subcutaneously in healthy volunteers. However, doses 20-fold higher have now been used for the treatment of acromegaly (6). A reduced release of PMNL proteolytic enzymes in response to inflammatory stimuli might be anticipated in these individuals.


In the functional assay, there was near total inhibition of elastase release at 2.5 ixg/ml SMS 201-995 concentration. At 1.25 ixg/ml, the inhibitory capacity was 84___5.4% s.e.m. Similarly high inhibition of elastase release was observed when the actual elastase quantity released into the supernatant was determined with RIMA (Fig. 1). The bars of the SIC values obtained with both functional assay and RIMA are identical and these are displayed in Fig. 2. The calculated in vitro concentration of SMS 201-995 causing half-maximal inhibition of neutrophil elastase release in each of both assays was ca. 1.0 p,g/ml. At concentrations below 0.15 txg/ml, SMS 201-995 showed no inhibition of elastase release. SST 14 had an inhibitory capacity of only 20% at 1 txg/ml. At the same concentration, 6 0 ± 7 . 1 % s.e.m, inhibition occurred with SMS 201-995. DISCUSSION

The results of this study indicate that SMS 201-995 causes

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40 20

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2.5 1.25 0.62 0.31 SMS 201-995 (ug/ml)


FIG. 2. Comparison of inhibition of neutrophil elastase release using a functional (heavily hatched bars) and a radioimmunometric assay (lightly hatched bars). Each bar represents mean ---s.e.m, of measurements in ten individuals.

REFERENCES 1. Barrett, A. J. Leucocyte elastase. Methods Enzymol. 80:581-588; 1981. 2. Boyum, A. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21(97):77-89; 1968.

3. Fraker, P. J.; Speck, J. C., Jr. Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80:849-857; 1978. 4. Hall, R.; Page, M. D.; Dieguez, C.; Scanlon, M. F. Somatostatin: A



historical perspective. Horm. Res. 29:50-53; 1988. 5. Kraenzlin, M. E.; Wood, S. M.; Neufeld, M.; Adrian, T. E.; Bloom, S. R. Effect of long acting somatostatin-analogue, SMS 201-995, on gut hormone secretion in normal subjects. Experientia 41:738-740; 1985. 6. Sandier, L. M.; Burrin, J. M.; Joplin, G. F.; Bloom, S. R. Effect of high dose somatostatin-analogue on growth hormone concentrations in

acromegaly. Br. Med. J. 296:751-752; 1988. 7. Smolen, J. E.; Korchak, H. M.; Weissmann, G. Increased levels of cyclic adenosine-3(1)5(1)-mono phosphate in human polymorphonuclear leucocytes after surface stimulation. J. Clin. Invest. 65:10771085; 1980. 8. Toro, M. J.; Birnbaumer, L.; Redon, M. C.; Montoya, E. Mechanism of action of somatostatin. Horm. Res. 29:59-64; 1988.

Somatostatin inhibits neutrophil elastase release in vitro.

The influence of the long-acting somatostatin analogue, SMS 201-995, on FMLP-induced neutrophil elastase release in vitro has been investigated. Doses...
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