Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors M. Bergenfeldt, L. Axelsson & K. Ohlsson To cite this article: M. Bergenfeldt, L. Axelsson & K. Ohlsson (1992) Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors, Scandinavian Journal of Clinical and Laboratory Investigation, 52:8, 823-829, DOI: 10.3109/00365519209088387 To link to this article: http://dx.doi.org/10.3109/00365519209088387
Published online: 08 Jul 2009.
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Scand J Clin Lab Invest 1992: 52: 823-829
Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors M. B E R G E N F E L D T , * t L. A X E L S S O N t & K . O H L S S O N t
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*Department of Surgery and ?Department of Surgical Pathophysiology, Lund University, Malmo General Hospital, Malmo, Sweden
Bergenfeldt M, Axelsson L, Ohlsson K. Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors. Scand J Clin Lab Invest 1992; 52: 823-829. Neutrophil proteinase 4 (NP4) is a major neutral proteinase of the human polymorphonuclear (PMN) leukocyte, which is present in amounts similar to leukocyte elastase. NP4(3) is a potent, non-specific proteinase, which may degrade structural and soluble proteins in the tissues and body fluids, and it has been implicated as an important pathogenetic factor in lung emphysema. We have studied the release of elastase and NP4(3) in an in vitro model of phagocytosis. al-proteinase inhibitor (a,-PI) is the major plasma inhibitor of both leukocyte elastase and NP4(3). but a,-PI bound leukocyte elastase more readily than NP4(3). The basic conditions were designed so that some proteolytic activity was present in the medium. Addition of increasing amounts of Secretory leukocyte protease inhibitor (SLPI) to the incubation mixtures resulted in binding of leukocyte elastase to this inhibitor and extinction of free proteolytic activity against both natural and synthetic substrates. The progressive binding of leukocyte elastase to SLPI instead of al-PI was paralleled by an increasing binding of NP4(3) to al-PI. SLPI is a potent inhibitor of leukocyte elastase and cathepsin G, and although it lacks inhibitory effect on NP4(3), it may obviously indirectly aid in the binding and inhibition of NP4(3) to al-PI, by taking care of at least part of the leukocyte elastase. As a specific NP4(3)inhibitor is not readily available for therapeutic use. this effect may prove useful under in vivo conditions and enhance the protective effect of administered recombinant human SLPI.
Key words: leukocyte elastase; neutrophil proteinase 4; proteinase-3; phagocytosis; secretory leukocyte protease inhibitor D r M . Bergenfeldt, Department of Surgery, Malmo General Hospital, S-21401 Malrno, Sweden
Besides leukocyte elastase and cathepsin G, neutrophil proteinase 4 (NP4) is a major neutral serine proteinase in the primary (azurophil) granules of human PMN leukocytes. The
enzyme wasdescribed already in the 1970s [ 1-61, Recently, sufficient amounts of NP4 were produced by affinity chromatography on a monoclonal antibody-resin to allow a more 823
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detailed characterization including the Nterminal sequence [7]. Comparison with other studies of serine proteinases of human leukocytic origin strongly indicates that NP4 is identical to neutrophil proteinase 3 (NP-3; leukocyte proteinase 3), Wegener autoantigen and AGP7 [8-141. NP4(3) is a potent, non-specific proteinase, which has the potential to degrade structural and soluble proteins in the tissues and body fluids [7,8,14]. NP4(3) has been implicated as an important pathogenetic factor in lung emphysema [S]. Secretory leukocyte protease inhibitor (SLPI) is the quantitatively dominant protease inhibitor of the mucus secretions of the respiratory [15] and genital tracts 1161. It is a potent inhibitor of leukocyte elastase and cathepsin G, but lacks effect on NP4(3) [14, 171. The inhibitor has been purified and its amino acid and nucleotide sequences determined [17-211. Expression of the corresponding gene in E Coli has enabled large-scale production of recombinant human SLPI (rhSLPI) in E Coli, which has provided the means for antiprotease therapy in conditions of congenital (i.e. al-protease inhibitor deficiency) or acquired protease-antiprotease imbalance. The purpose of the present paper was to study the effect of SLPI on the interaction of elastase and NP4(3) with al-PI in an in vitro model of phagocytosis [22].
MATERIALS AND METHODS
Grand Island, NY, USA). Bakers yeast was prepared as previously described, opsonization was done in human serum as before [22]. Human leukocyte elastase was prepared from buffy coat as previously described (23). Dr Robert Thompson at Synergen Inc (Boulder, Colorado, USA) kindly provided rhSLPI, which was produced as described [19]. Specific monoclonal antibodies against NP4(3) were produced as recently described [7]. Radiolabelling of the monoclonal antiNP4(3) antibodies with '25iodinewas done by a lactoperoxidase method [24]. Antisera against human leukocyte elastase, al-PI, a2-M, fibronectin and complement factors C3 (C3) and C3c were prepared in the laboratory [25-281. The IgG fractions of rabbit and sheep antisera against human leukocyte elastase antisera were isolated by affinity chromatography on Protein A-Sepharose as previously described [29]. Immunochemical methods ELISA of NP41NP3 was done as described [30]. Fibronectin split products were measured by electroimmunoassay, intact fibronectin being bound in an intermediary gel strip containing gelatin 1311. Crossed immunoelectrophoresis of complement factor C3 and protease al-PI complexes was done according to Ganrot [32]. Complexation was estimated from the precipitate area on the crossed immunoelectrophoresis plate, and expressed as per cent of maximum.
Special materials
Biotinylation of antibodies
Dextran T500, Sephadex G-25 (pre-packed PD-10 columns), CNBr-activated Sepharose CL-4B and proteinA-Sepharose C I 4 b were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Agarose was purchased from FMC-Bioproducts (Rockland, ME, USA). Lymfoprep@ (sodium metrizoate-Ficoll) was purchased from Nyegaard (Olso, Norway). Low-molecular chromogenic substrates for leukocyte elastase (N-Suc-Ala-Ala-Ala-pNa) and NP4(3) (t-N-Boc-Ala-ONp) were purchased from Sigma Chemical Co (St Louis, MO, USA). Hanks buffered salt solution (HBSS) with CaZf and Mg2+ and phosphate buffered physiologic saline (PBS) were obtained from Gibco Laboratories (Life Technologies Inc.,
Biotinylation was doneessentially as described [33]. Briefly, 5 mg of rabbit IgG antibodies against human a,-PI in 1 ml of glycine-HCI buffer was dialysed overnight at room temperature against 0.1 mot I-' NaHC03. To the dialysate was added 120 pg N-hydroxysuccinimido-biotin dissolved in dimethylsulphoxide. The solution was incubated for 4 h at room temperature and hence dialysed against 0.01 mol I-' PBS (pH 7.2 with NaN3 0.1%) overnight at 4 "C. Preparation of leukocyte elastase a,-PI standurd Three milligrams of human a,-PI in 1 ml of 0.05 mol I-' Tris HCI-buffer (NaCI 0.15 mol I-';
Release of Neutrophil proteinuse 4(3) and elastase
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pH 7.4) was mixed with 1 mlO.15 mol I" NaCl with 0.725 mg active human leukocyte elastase, and the mixture was incubated for 30 min at 37 "C. Complexation was checked by immunoelectrophoresis [34] against al-PI and leukocyte elastase, respectively, as well as absence of proteolytic activity against chromogenic substrate. ELISA of human leukocyte elastase-a,-PI complexes Between each incubation step, the Microtitre plates were rinsed five times with PBS containing o.05"/0(w/v) Tween 20 (pH 7.4). The initial step was coating of the wells with 100 pl of specific absorbed sheep IgG-antibodies against antihuman leukocyte elastase (prepared at 7.5 mg I-' in 0.05 mol 1" carbonate buffer; 0.02% NaN3, pH 9.6) for 24 h at 4 "C. Secondly, wells were incubated with 150 pl blocking buffer (Tris-HCI 0.05 mol I-' CaCI2 0.05 mol I-', NaN3 0.02% (w/v), BSA 1% (w/v); pH 7.4) for 2 h at room temperature. Then, 100 pl of sample or standard diluted in sample buffer (Tris-HC1 0.05 mol I-', NaCl 0.5 mol I-', CaCIz 0.05 mol I-', 0.2% BSA; pH 7.4) was applied followed by overnight incubation at 4 "C. After thorough rinsing, 100 p1 of biotinylated rabbit IgG-antibodies against human al-PI (diluted 1/750 in sample buffer with 0.5% sheep serum) was added, and incubated for 2 h at room temperature. Then, avidin with conjugated alkaline phosphatase (diluted 1/1OOO in sample buffer without NaCI) was added and incubated for 2 h at room temperature. In the last step, 100 pI of phosphatase substrate (1 mg mi-') in 10% (v/v) diethanolamine-HCI buffer with 0.01 mol I-' MgCI2 (pH 9.8) was added. The absorbance at 405 nm was recorded after 30-60 min at room temperature in an automatic Titertec Multiscan photometer, supplied with Titresoft EIA software.
Characteristics of
ELISA
Standard curves and dilution curves of plasma samples were parallel, indicating a good specificity. The sensitivity was 0.05 nmol I-' (reading constantly above background 3 SD). The coefficients of intra- and inter-assay variation were 7.5% (n=22) and 10.2% (n=32),
+
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respectively. The plasma concentration of a'PI-leukocyte elastase in 18 healthy blood donors was 2.58k0.61 nmol I-' (meankSD). The range was 1.8-4.1 nmol I-'. The assay is designed for measurements in biological fluids, where leukocyte elastase is present mainly in complex with a'-PI. Free leukocyte elastase can be measured only if the sample is measured before and after saturation with al-PI. Free a'-PI does not interfere with the assay.
Isolation of PMN leukocytes PMN leucocytes were isolated as earlier described [35]. Briefly, human blood was collected in sterile, heparinized tubes and mixed with an equal volume of Dextran T500 2% in 0.9% NaCl. After sedimentation for 45 min at room temperature, 15 ml of supernatant was layered on 25 ml of Lymphoprep and centrifuged at 1350 g for 15min. After lysis of contaminating erythrocytes in 0.87% ammonium chloride, cells were washed twice in PBS, counted and resuspended in HBSS with 1.5% heat-inactivated serum. Purity and viability (trypan blue exclusion) were more than 98%.
Phagocytosis experiments Experiment 1. In this experiment incubation mixtures were prepared in siliconized reaction vials with 3.10' PMN leukocytes in 750 pl HBSS and 2% autologous serum. To the vials were added 150 pl SLPI or diluent (PBS). The final assay concentration of SLPI was 0-8 pmol I-'. After 15 min preincubation at 37 "C, opsonized yeast in 75 pl PBS (8X 10' yeast cells) or diluent was added to the mixtures. The vials were then incubated for 60 min in a shaker bath at 37 "C. The incubation experiments were repeated three times, vials without leukocytes ('cell-free controls') were included in each run. The experiments were terminated by immediate centrifugation of the vials at 1500 g. The supernatants were recovered and immediately stored at -70 "C until analysis. Experiment 2. This was done as above, but after termination of the experiment additional leukocyte elastase was added to the supernatants to check if al-PI was capable of further complexation.
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RESULTS
In cell incubations with opsonized yeast, leukocyte elastase and NP4(3) were released to the medium and free proteolytic activity against both fibronectin, complement factor C3 and lowmolecular chromogenic substrates were noted in the supernatants. The final concentrations of leukocyte elastase and NP4(3) in the supernatants were0.4 pmol I-' and 0.7 pmol I-', respectively. Cell suspensions that were incubated without opsonized yeast contained 0.08 pmol I-' and 0.02 pmol I-' of leukocyte elastase and NP4(3), respectively. When supernatants without SLPI were analysed by crossed immunoelectrophoresis against
anti-a,-PI antiserum, three peaks with different electrophoretic mobilities were present (Figure 1A). The right peak was shown to contain a,-PIleukocyte elastase complexes, and the intermediary peak contained complexes between alPI and NP4(3) as verified by radiolabelling of the complex with monoclonal antibodies against NP4(3) (Figure 1). The left peak had the same electrophoretic mobility as native al-PI, but addition of leukocyte elastase in a control experiment (experiment 2) verified that further complexation was not possible (data not shown). In vials incubated without SLPI there was proteolytic activity against the peptide substrates N-Suc-Ala-Ala-Ala-pNa and t-N-Boc-Ala-ONp (Figure 2B).
0
2
4
6
8
6
8
SLPI ~ o l l - ' )
0
2
4 SLPI @mol I-')
FIG. I. Crossed immunoelectrophoresis of supernatants from phagocytosis experiments. A mixture of anti-al-PI-antiserum and radiolabelled monoclonal antibodies against NP4(3) was used in the second dimension. Figure 1A demonstrates the pattern without SLPI in the medium and Figure IB with 4 pmol I-' SLPI present. Three precipitates with different electrophoretic mobilities were obtained. The left and right peaks contained as al-PI and leukocyte elastase-al-PI-complexes, respectively. The intermediary peak contained NP4(3)-al-PI complexes, as verified by autoradiography (shown below the corresponding immunoelectrophoretic gelstrips).
FIG. 2. (a) Complexation of a,-PI with NP4(3) (.-.-.-*) and leukocyte elastase (0-0-0-0) in supernatants from phagocytosis experiments. With increasing concentrations of SLPI in the incubation medium, NP4(3)-a,-PI complexes increased simultaneously with a decrease of leukocyte elastase-a,PI complexes on crossed immunoelectrophoresis. Leukocyte elastase-a,-PI complexes as measured by ELISA also decreased (- - - -), (b) Catalytic activity in supernatants from phagocytosis experiments. With increasing concentrations of SLPI in the incubation medium, the catalytic activity against fibronectin (- - - -) and low-molecular chromogenic substrates and -. N-Suc-Ala) Alat-N-Boc-Ala-ONp (.-.-. Ala-pNa (0-0-0-0) decreased. Results of typical experiment shown (reproduced three times).
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Release of Neutrophil proteinuse 4(3) and cJla.stuse
Increasing concentrations of SLPI in the reaction mixtures resulted in a progressive decrease of leukocyte elastase al-PI- complexes, with only a small amount of complexes present at concentrations exceeding 4 pmoi I-' of SLPI. This decrease was obvious from the crossed immunoelectrophoretic pattern, but could also be measured with the ELISA (Figures 1 and 2a). A simultaneous increase of NP4(3) al-PIcomplexes on the crossed immunoelectrophoresis was noted, which seemed to reach maximum at approximately 8 pmol I-' of SLPI (Figure 1 and 2a). The ELISA of NP4(3) measures both free and complexed enzyme, and the amount of NP4(3) in the vials was the same irrespective of the SLPI-concentration. Also with increasing concentrations of SLPI, the proteolytic activity against N-Suc-AlaAla-Ala-pNA progressively decreased and approached zero at 4 pg I - ' of SLPI. A progressive decrease in activity against t-N-
FIG. 3. Crossed immunoelectrophoresis of supernatants from phagocytosis experiments with rabbit anti-C3 antiserum in the second dimension. When no SLPI was present in the incubation medium a complete conversion of C3 was seen (Figure 3 : 2 ) . With 4 pmol I-' SLPI (Figure 3 : 3 ) the pattern was restored to that seen in the cell-free incubation mixtures containing opsonised yeast (Figure 3: I ) .
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Boc-Ala-ONp was also noted, with some activity left also at 8 pg I-' of SLPI (Figure 2B). Reaction mixtures without SLPI showed proteolytic activity against fibronectin, which gradually decreased with increasing concentrations of SLPI (Figure 2b). In the absence of SLPI, complement factor C3 showed a precipitate pattern on crossed immunoelectrophoresis indicating complete degradation. (Fig 3:2). At 4 pmol I-' of SLPI (Figure 3 : 3 ) , the precipitate pattern was identical to control experiments with opsonized yeast but without leukocytes and SLPI (Figure 3:l).
DISCUSSION Both leukocyte elastase and NP4(3) were released to the supernatants by phagocytosing PMN leukocytes. Only minute amounts were present in controls incubated without yeast, which indicates that the proteases were not released as the result of cell death or during harvesting of the supernatants. Leukocyte elastase and NP4(3) have been found in similar amounts in PMN leukocytes [3, 121 and inflammatory exudates [30,36]. In this study, the level of leukocyte elastase in the medium was lower than that of NP4(3); but to this may be added free elastase (evident from the substrate activity), which is not measured by the ELISA. Free leukocyte elastase is exceptional in biological fluids, and this should not ordinarily limit the utility of the assay. However, when measurements are done in mucus secretions, it must be noted that the ELISA does not measure SLPIbound leukocyte elastase. In this study, this made it possible to measure leukocyte elastase a I-PI-complexes separately in the supernatants. In good agreement with the crossed immunoelectrophoresis, it was found that the amount of leukocyte elastase-al-PI-complexes decreased with increasing SLPI-concentration (Figure 2a). However, the level of NP4(3) in the vials, as measured by ELISA, was the same irrespective of the SLPI-concentration which indicates that SLPI does not affect the release process per se. Similar to leukocyte elastase and cathepsin G, NP4(3) is a potent, non-specific proteinase, with the potential to degrade structural and soluble proteins in the tissues and body fluids [7, 8, 141. The main inhibitors of leukocyte
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elastase and NP4(3) are a2-M and al-PI, the latter inhibitor being quantitatively dominant. Imbalance between released leukocyte proteases and available inhibitors may result in pathologic proteolysis. This mechanism has been considered in lung emphysema, and leukocyte elastase as well as NP4(3) have been implicated as important in the pathogenesis [8, 371. The in-vitro model used may be a good representation of an inflammatory exudate with proteases in excess of available proteinase inhibitors. In this situation, al-PI bound leukocyte elastase in preference to NP4(3) as shown by the pattern on crossed immunoelectrophoresis (Figure 1). This is consistent with earlier estimations of their dissociation constants [14]. Addition of increasing amounts of SLPI to the incubation mixtures resulted in binding of excess leukocyte elastase and extinction of free elastolytic activity (Figure 1, 2a, 2b). Moreover, with increased binding of leukocyte elastase of SLPI, more a,-PI obviously became available to bind NP4(3) as shown by the increase in complexes between NP4(3) and al-PI (Figure 1). Proteolytic activity on the natural substrates C3 and fibronectin was markedly reduced (Figure 2b, 3). The cleavage of t-N-Boc-Ala-ONp was also markedly reduced. The residual activity on t-N-Boc-AlaONp at an 8 pmol I-‘ of SLPI may be explained by the fact that NP4(3) retains some activity against low molecular substrates also when complexed with az-M. The concentrations of SLPI used in this study are similar to the levels in the mucus secretions of the airways and genital tract [15,16, 381, and the model may thus mimick a mucus secretion with a low al-PI concentration as in congenital a,-PI deficiency. Large-scale production of recombinant human SLPI in E Coli has provided a means of anti-protease therapy in situations of congenital or acquired protease-antiprotease imbalance. The findings in this study may be of therapeutic interest, as t h e used SLPI-concentrations should be possible to reach locally and probably also in the circulation on intravenous infusion. Recombinant human SLPI is identical to the native inhibitor also in that both lack carbohydrate, which may eliminate the risk of allergic manifestations and make it superior to hitherto suggested agents. Moreover, SLPI is a potent inhibitor of leukocyte elastase and cathepsin G, but lacks inhibitory effect on
NP4(3) in vitro [14, 171. In spite of this, SLPI may obviously indirectly aid in the binding and inhibition of NP4(3) to al-PI, by taking care of at least part of the leukocyte elastase. As a specific NP4(3)-inhibitor is not readily available for therapeutic use, this effect may prove useful under in-vivo conditions and enhance the protective effect of administered SLPI. ACKNOWLEDGMENTS This project was supported by grants from ‘Forenade Liv’ Mutual Group Life Insurance Company, the Swedish Medical Research Council (grant no 3910), the Swedish Society for Medical Science, the Swedish Medical Society, the Swedish Foundation against Heart and Lung Diseases and the Medical Faculty at Lund University. REFERENCES 1 Ohlsson K, Olsson I. The neutral proteases of human granulocytes. Isolation and partial characterisation of two collagenases. Eur J Biochem 1973; 36: 473-81. 2 Ohlsson K, Olsson I. The extracellular release of granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Scand J Haematol 1977; 19: 145-52. 3 Ohlsson K,Olsson I. Neutral proteases of human granulocytes. IV Interaction between human granulocytes collagenase and plasma protease inhibitors. J Lab Clin Med 1977; 89: 269-77. 4 Ohlsson K, Olsson I, Spitznagel JK. Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes. Hoppe-Seyler’s Z Physiol Chem 1977; 358: 361 -6. 5 Ohlsson K. Polymorphonuclear leukocyte collagenase. In: Woolley DE, Evanson JM, eds. Collagenase in Normal and Pathological Connective Tissues. John Wiley & Sons Ltd, London 1980: 209-22. 6 Barrett AJ, McDonald JK. Mammalian proteases: a glossary and bibliography. Academic Press, London 1980: 189-90. 7 Ohlsson K , Linder C, Rosengren M. Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme. Biol Chem Hoppe-Seyler 1990; 371: 549-55. 8 Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal J R . Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963-73. 9 Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger M, Nathan CF. Antibiotic proteins of human polymorphonuclear
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Release of Neutrophil proteinuse 4(3) and elustuse leukocytes. Proc Nat Acad Sci USA 1989; 86: 5610- 14. 10 Niles JL, McCluskey RT, Ahmad MF, Arnaout MA. Wegener’s granulomatosis antigen is a novel serine protease. Blood 1989; 74: 1888-93. 1 1 Ludemann J , Utecht B, Gross WL. Anti-neutrophi1 cytoplasm antibodies in Wegener’s granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990; 171: 357-62. 12 Wilde CG, Snable JL, Griffith JE, Scott RW. Characterization of two azurophil granule proteinases with active-site homology to neutrophi1 elastase. J Biol Chem 1990; 265: 2038-41. 13 Jenne DE, Tshopp J, Ludemann J, Utecht B, Gross WL. Wegener’s antigen decoded. Nature 1990; 346: 520. 14 Rao NV, Wehner NG, Marshall BC, Gray WR, Gray BH, Hoidal JR. Characterization of proteinase 3 (PR-3), a neutrophil serine proteinase. Structural and functional properties. J Biol Chem 1991; 266: 9540-8. 15 Tegner H . Quantitation of human granulocyte protease inhibitors in non-purulent bronchial lavage fluids. Acta Otolaryngol 1978; 85: 282-9. 16 Cassltn B, Rosengren M, Ohlsson K. Localization and quantitation of a low molecular weight proteinase inhibitor, antileukoprotease, in the human uterus. Hoppe-Seyler’s Z Physiol Chem 1981; 362: 953-61. 17 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. 18 Ohlsson K, Rosengren M, Stetler G, Brewer MT, Hale KK, Thompson RC. Structure, genomic organisation, and tissue distribution of human secretory leukocyte-protease inhibitor (SLPI): a potent inhibitor of neutrophil elastase. In: Taylor J C, Mittman C, eds. Pulmonary emphysema and Proteolysis. London: Academic Press 1986: 30722. 19 Stetler G , Brewer MT, Thompson RC. Isolation and sequence of a gene encoding a potent inhibitor of leukocyte proteases. Nucleic Acids Res 1986; 20: 14: 7883-95. 20 Heinzel R, Appelhans H, Gassen H G , SeemQller U, Arnhold M, Fritz H, Lottspeich F, Wiedenmann K, Machleidt W. The neutrophil elastasecathepsin G inhibitor of human mucus tissues and secretions (antileukoprotease, HUSI-I): complete primary structure as revealed by protein and DNA sequencing. In: Taylor JC, Mittman C, eds. Pulmonary emphysema and proteolysis. London: Academic Press, 1986: 297-306. 21 SeemQller U, Arnhold M, Fritz H , Wiedenmann K, Machleidt W, Heinzel R , Appelhans H, Gassen HG, Lottspeich F. The acid-stable proteinase inhibitor of human mucous secretions (HUSI-I, antileukoprotease). FEBS Lett 1986; 199: 43-8.
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22 Axelsson L, Linder C, Ohlsson K, Rosengren M. The effect of the secretory leukocyte protease inhibitor on leukocyte proteases released during phagocytosis. Biol Chem Hoppe-Seyler 1988; 369: 89-93. 23 Baugh RJ, Travis J. Human leukocyte granule elastase: rapid isolation and characterisation. J Biochem 1976; 15: 437-45. 24 Thorell JI, Johansson BG. Enzymatic iodination of polypeptides with ‘251to high specific activity. Biochim Biophys Acta 1971; 251: 363-9. 25 Ohlsson K, Olsson I. Neutral proteases of human granulocytes. 111. Interactions between human granulocyte elastase and plasma protease inhibitors. Scand J Clin Lab Invest 1974; 34: 349-55. 26 Ohlsson K, Olsson A-S. Immunoreactive granulocyte elastase in human serum. Hoppe-Seyler’s Z Physiol Chem 1978; 359: 1531-9. 27 Johnson U, Ohlsson K , Olsson I. Effects of granulocyte neutral proteases on complement components. Scand J lmmunol 1976; 5: 421-6. 28 Bjork P, Bergenfeldt M, Axelsson L, Ohlsson K. Influence of plasma proteinase inhibitors and the secretory leukocyte protease inhibitor on leukocyte elastase-induced consumption of selected plasma proteins in-vitro in man. Scand J CIin Lab Invest 1988; 48: 205-11. 29 Bjiirk P, Axelsson L, Ohlsson K. Release of dog polymorphonuclear leukocyte cathepsin G, normally and in endotoxin and pancreatic shock. Biol Chem Hoppe-Seyler 1901; 372: 419-26. 30 Lundberg E, Bergenfeldt M, Ohlsson K. Release of immunoreactive human neutrophil proteinase 4, normally and in peritonitis. Scand J Clin Lab Invest 1991; 51: 23-9. 31 Laurel1 CB. Electroimmuno Assay. Scand J Clin Lab Invest 1972; suppl 124: 21-37. 32 Ganrot PO. Crossed lmmunoelectrophoresis. Scand J Clin Lab Invest 1972; suppl 124: 39-41. 33 Bayer E A, Wilchek M. Protein biotinylation. Methods Enzymol 1990; 184: 138-60. 34 Scheidegger JJ. Une micro-methode de I’immunotlectrophorese. Int Arch Allergy 1955;7: 103- 110. 35 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968; 21 (suppl 97 : 77-89. 36 Delshammar M, Lasson , Ohlsson K. Proteases and protease inhibitor balance in peritonitis with different causes. Surgery 1990; 106: 555-62. 37 Senior RM, Tegner H, Kuhn C, Ohlsson K , Starcher BC, Pierce JA. The induction of pulmonary emphysema with human leukocyte elastase. Am Rev Respir Dis 1977; 116: 469-75. 38 Fryksmark U, Jannert M, Ohlsson K, Tegner H, Wihl J-A. Secretory leukocyte protease inhibitor in normal, allergic and virus induced nasal secretions. Rhinology 1989; 27: 97- 103.
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Received 28 February 1992 Accepted 9 July 1992
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors M. Bergenfeldt, L. Axelsson & K. Ohlsson To cite this article: M. Bergenfeldt, L. Axelsson & K. Ohlsson (1992) Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors, Scandinavian Journal of Clinical and Laboratory Investigation, 52:8, 823-829, DOI: 10.3109/00365519209088387 To link to this article: http://dx.doi.org/10.3109/00365519209088387
Published online: 08 Jul 2009.
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Scand J Clin Lab Invest 1992: 52: 823-829
Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors M. B E R G E N F E L D T , * t L. A X E L S S O N t & K . O H L S S O N t
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*Department of Surgery and ?Department of Surgical Pathophysiology, Lund University, Malmo General Hospital, Malmo, Sweden
Bergenfeldt M, Axelsson L, Ohlsson K. Release of neutrophil proteinase 4(3) and leukocyte elastase during phagocytosis and their interaction with proteinase inhibitors. Scand J Clin Lab Invest 1992; 52: 823-829. Neutrophil proteinase 4 (NP4) is a major neutral proteinase of the human polymorphonuclear (PMN) leukocyte, which is present in amounts similar to leukocyte elastase. NP4(3) is a potent, non-specific proteinase, which may degrade structural and soluble proteins in the tissues and body fluids, and it has been implicated as an important pathogenetic factor in lung emphysema. We have studied the release of elastase and NP4(3) in an in vitro model of phagocytosis. al-proteinase inhibitor (a,-PI) is the major plasma inhibitor of both leukocyte elastase and NP4(3). but a,-PI bound leukocyte elastase more readily than NP4(3). The basic conditions were designed so that some proteolytic activity was present in the medium. Addition of increasing amounts of Secretory leukocyte protease inhibitor (SLPI) to the incubation mixtures resulted in binding of leukocyte elastase to this inhibitor and extinction of free proteolytic activity against both natural and synthetic substrates. The progressive binding of leukocyte elastase to SLPI instead of al-PI was paralleled by an increasing binding of NP4(3) to al-PI. SLPI is a potent inhibitor of leukocyte elastase and cathepsin G, and although it lacks inhibitory effect on NP4(3), it may obviously indirectly aid in the binding and inhibition of NP4(3) to al-PI, by taking care of at least part of the leukocyte elastase. As a specific NP4(3)inhibitor is not readily available for therapeutic use. this effect may prove useful under in vivo conditions and enhance the protective effect of administered recombinant human SLPI.
Key words: leukocyte elastase; neutrophil proteinase 4; proteinase-3; phagocytosis; secretory leukocyte protease inhibitor D r M . Bergenfeldt, Department of Surgery, Malmo General Hospital, S-21401 Malrno, Sweden
Besides leukocyte elastase and cathepsin G, neutrophil proteinase 4 (NP4) is a major neutral serine proteinase in the primary (azurophil) granules of human PMN leukocytes. The
enzyme wasdescribed already in the 1970s [ 1-61, Recently, sufficient amounts of NP4 were produced by affinity chromatography on a monoclonal antibody-resin to allow a more 823
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detailed characterization including the Nterminal sequence [7]. Comparison with other studies of serine proteinases of human leukocytic origin strongly indicates that NP4 is identical to neutrophil proteinase 3 (NP-3; leukocyte proteinase 3), Wegener autoantigen and AGP7 [8-141. NP4(3) is a potent, non-specific proteinase, which has the potential to degrade structural and soluble proteins in the tissues and body fluids [7,8,14]. NP4(3) has been implicated as an important pathogenetic factor in lung emphysema [S]. Secretory leukocyte protease inhibitor (SLPI) is the quantitatively dominant protease inhibitor of the mucus secretions of the respiratory [15] and genital tracts 1161. It is a potent inhibitor of leukocyte elastase and cathepsin G, but lacks effect on NP4(3) [14, 171. The inhibitor has been purified and its amino acid and nucleotide sequences determined [17-211. Expression of the corresponding gene in E Coli has enabled large-scale production of recombinant human SLPI (rhSLPI) in E Coli, which has provided the means for antiprotease therapy in conditions of congenital (i.e. al-protease inhibitor deficiency) or acquired protease-antiprotease imbalance. The purpose of the present paper was to study the effect of SLPI on the interaction of elastase and NP4(3) with al-PI in an in vitro model of phagocytosis [22].
MATERIALS AND METHODS
Grand Island, NY, USA). Bakers yeast was prepared as previously described, opsonization was done in human serum as before [22]. Human leukocyte elastase was prepared from buffy coat as previously described (23). Dr Robert Thompson at Synergen Inc (Boulder, Colorado, USA) kindly provided rhSLPI, which was produced as described [19]. Specific monoclonal antibodies against NP4(3) were produced as recently described [7]. Radiolabelling of the monoclonal antiNP4(3) antibodies with '25iodinewas done by a lactoperoxidase method [24]. Antisera against human leukocyte elastase, al-PI, a2-M, fibronectin and complement factors C3 (C3) and C3c were prepared in the laboratory [25-281. The IgG fractions of rabbit and sheep antisera against human leukocyte elastase antisera were isolated by affinity chromatography on Protein A-Sepharose as previously described [29]. Immunochemical methods ELISA of NP41NP3 was done as described [30]. Fibronectin split products were measured by electroimmunoassay, intact fibronectin being bound in an intermediary gel strip containing gelatin 1311. Crossed immunoelectrophoresis of complement factor C3 and protease al-PI complexes was done according to Ganrot [32]. Complexation was estimated from the precipitate area on the crossed immunoelectrophoresis plate, and expressed as per cent of maximum.
Special materials
Biotinylation of antibodies
Dextran T500, Sephadex G-25 (pre-packed PD-10 columns), CNBr-activated Sepharose CL-4B and proteinA-Sepharose C I 4 b were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Agarose was purchased from FMC-Bioproducts (Rockland, ME, USA). Lymfoprep@ (sodium metrizoate-Ficoll) was purchased from Nyegaard (Olso, Norway). Low-molecular chromogenic substrates for leukocyte elastase (N-Suc-Ala-Ala-Ala-pNa) and NP4(3) (t-N-Boc-Ala-ONp) were purchased from Sigma Chemical Co (St Louis, MO, USA). Hanks buffered salt solution (HBSS) with CaZf and Mg2+ and phosphate buffered physiologic saline (PBS) were obtained from Gibco Laboratories (Life Technologies Inc.,
Biotinylation was doneessentially as described [33]. Briefly, 5 mg of rabbit IgG antibodies against human a,-PI in 1 ml of glycine-HCI buffer was dialysed overnight at room temperature against 0.1 mot I-' NaHC03. To the dialysate was added 120 pg N-hydroxysuccinimido-biotin dissolved in dimethylsulphoxide. The solution was incubated for 4 h at room temperature and hence dialysed against 0.01 mol I-' PBS (pH 7.2 with NaN3 0.1%) overnight at 4 "C. Preparation of leukocyte elastase a,-PI standurd Three milligrams of human a,-PI in 1 ml of 0.05 mol I-' Tris HCI-buffer (NaCI 0.15 mol I-';
Release of Neutrophil proteinuse 4(3) and elastase
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pH 7.4) was mixed with 1 mlO.15 mol I" NaCl with 0.725 mg active human leukocyte elastase, and the mixture was incubated for 30 min at 37 "C. Complexation was checked by immunoelectrophoresis [34] against al-PI and leukocyte elastase, respectively, as well as absence of proteolytic activity against chromogenic substrate. ELISA of human leukocyte elastase-a,-PI complexes Between each incubation step, the Microtitre plates were rinsed five times with PBS containing o.05"/0(w/v) Tween 20 (pH 7.4). The initial step was coating of the wells with 100 pl of specific absorbed sheep IgG-antibodies against antihuman leukocyte elastase (prepared at 7.5 mg I-' in 0.05 mol 1" carbonate buffer; 0.02% NaN3, pH 9.6) for 24 h at 4 "C. Secondly, wells were incubated with 150 pl blocking buffer (Tris-HCI 0.05 mol I-' CaCI2 0.05 mol I-', NaN3 0.02% (w/v), BSA 1% (w/v); pH 7.4) for 2 h at room temperature. Then, 100 pl of sample or standard diluted in sample buffer (Tris-HC1 0.05 mol I-', NaCl 0.5 mol I-', CaCIz 0.05 mol I-', 0.2% BSA; pH 7.4) was applied followed by overnight incubation at 4 "C. After thorough rinsing, 100 p1 of biotinylated rabbit IgG-antibodies against human al-PI (diluted 1/750 in sample buffer with 0.5% sheep serum) was added, and incubated for 2 h at room temperature. Then, avidin with conjugated alkaline phosphatase (diluted 1/1OOO in sample buffer without NaCI) was added and incubated for 2 h at room temperature. In the last step, 100 pI of phosphatase substrate (1 mg mi-') in 10% (v/v) diethanolamine-HCI buffer with 0.01 mol I-' MgCI2 (pH 9.8) was added. The absorbance at 405 nm was recorded after 30-60 min at room temperature in an automatic Titertec Multiscan photometer, supplied with Titresoft EIA software.
Characteristics of
ELISA
Standard curves and dilution curves of plasma samples were parallel, indicating a good specificity. The sensitivity was 0.05 nmol I-' (reading constantly above background 3 SD). The coefficients of intra- and inter-assay variation were 7.5% (n=22) and 10.2% (n=32),
+
825
respectively. The plasma concentration of a'PI-leukocyte elastase in 18 healthy blood donors was 2.58k0.61 nmol I-' (meankSD). The range was 1.8-4.1 nmol I-'. The assay is designed for measurements in biological fluids, where leukocyte elastase is present mainly in complex with a'-PI. Free leukocyte elastase can be measured only if the sample is measured before and after saturation with al-PI. Free a'-PI does not interfere with the assay.
Isolation of PMN leukocytes PMN leucocytes were isolated as earlier described [35]. Briefly, human blood was collected in sterile, heparinized tubes and mixed with an equal volume of Dextran T500 2% in 0.9% NaCl. After sedimentation for 45 min at room temperature, 15 ml of supernatant was layered on 25 ml of Lymphoprep and centrifuged at 1350 g for 15min. After lysis of contaminating erythrocytes in 0.87% ammonium chloride, cells were washed twice in PBS, counted and resuspended in HBSS with 1.5% heat-inactivated serum. Purity and viability (trypan blue exclusion) were more than 98%.
Phagocytosis experiments Experiment 1. In this experiment incubation mixtures were prepared in siliconized reaction vials with 3.10' PMN leukocytes in 750 pl HBSS and 2% autologous serum. To the vials were added 150 pl SLPI or diluent (PBS). The final assay concentration of SLPI was 0-8 pmol I-'. After 15 min preincubation at 37 "C, opsonized yeast in 75 pl PBS (8X 10' yeast cells) or diluent was added to the mixtures. The vials were then incubated for 60 min in a shaker bath at 37 "C. The incubation experiments were repeated three times, vials without leukocytes ('cell-free controls') were included in each run. The experiments were terminated by immediate centrifugation of the vials at 1500 g. The supernatants were recovered and immediately stored at -70 "C until analysis. Experiment 2. This was done as above, but after termination of the experiment additional leukocyte elastase was added to the supernatants to check if al-PI was capable of further complexation.
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RESULTS
In cell incubations with opsonized yeast, leukocyte elastase and NP4(3) were released to the medium and free proteolytic activity against both fibronectin, complement factor C3 and lowmolecular chromogenic substrates were noted in the supernatants. The final concentrations of leukocyte elastase and NP4(3) in the supernatants were0.4 pmol I-' and 0.7 pmol I-', respectively. Cell suspensions that were incubated without opsonized yeast contained 0.08 pmol I-' and 0.02 pmol I-' of leukocyte elastase and NP4(3), respectively. When supernatants without SLPI were analysed by crossed immunoelectrophoresis against
anti-a,-PI antiserum, three peaks with different electrophoretic mobilities were present (Figure 1A). The right peak was shown to contain a,-PIleukocyte elastase complexes, and the intermediary peak contained complexes between alPI and NP4(3) as verified by radiolabelling of the complex with monoclonal antibodies against NP4(3) (Figure 1). The left peak had the same electrophoretic mobility as native al-PI, but addition of leukocyte elastase in a control experiment (experiment 2) verified that further complexation was not possible (data not shown). In vials incubated without SLPI there was proteolytic activity against the peptide substrates N-Suc-Ala-Ala-Ala-pNa and t-N-Boc-Ala-ONp (Figure 2B).
0
2
4
6
8
6
8
SLPI ~ o l l - ' )
0
2
4 SLPI @mol I-')
FIG. I. Crossed immunoelectrophoresis of supernatants from phagocytosis experiments. A mixture of anti-al-PI-antiserum and radiolabelled monoclonal antibodies against NP4(3) was used in the second dimension. Figure 1A demonstrates the pattern without SLPI in the medium and Figure IB with 4 pmol I-' SLPI present. Three precipitates with different electrophoretic mobilities were obtained. The left and right peaks contained as al-PI and leukocyte elastase-al-PI-complexes, respectively. The intermediary peak contained NP4(3)-al-PI complexes, as verified by autoradiography (shown below the corresponding immunoelectrophoretic gelstrips).
FIG. 2. (a) Complexation of a,-PI with NP4(3) (.-.-.-*) and leukocyte elastase (0-0-0-0) in supernatants from phagocytosis experiments. With increasing concentrations of SLPI in the incubation medium, NP4(3)-a,-PI complexes increased simultaneously with a decrease of leukocyte elastase-a,PI complexes on crossed immunoelectrophoresis. Leukocyte elastase-a,-PI complexes as measured by ELISA also decreased (- - - -), (b) Catalytic activity in supernatants from phagocytosis experiments. With increasing concentrations of SLPI in the incubation medium, the catalytic activity against fibronectin (- - - -) and low-molecular chromogenic substrates and -. N-Suc-Ala) Alat-N-Boc-Ala-ONp (.-.-. Ala-pNa (0-0-0-0) decreased. Results of typical experiment shown (reproduced three times).
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Release of Neutrophil proteinuse 4(3) and cJla.stuse
Increasing concentrations of SLPI in the reaction mixtures resulted in a progressive decrease of leukocyte elastase al-PI- complexes, with only a small amount of complexes present at concentrations exceeding 4 pmoi I-' of SLPI. This decrease was obvious from the crossed immunoelectrophoretic pattern, but could also be measured with the ELISA (Figures 1 and 2a). A simultaneous increase of NP4(3) al-PIcomplexes on the crossed immunoelectrophoresis was noted, which seemed to reach maximum at approximately 8 pmol I-' of SLPI (Figure 1 and 2a). The ELISA of NP4(3) measures both free and complexed enzyme, and the amount of NP4(3) in the vials was the same irrespective of the SLPI-concentration. Also with increasing concentrations of SLPI, the proteolytic activity against N-Suc-AlaAla-Ala-pNA progressively decreased and approached zero at 4 pg I - ' of SLPI. A progressive decrease in activity against t-N-
FIG. 3. Crossed immunoelectrophoresis of supernatants from phagocytosis experiments with rabbit anti-C3 antiserum in the second dimension. When no SLPI was present in the incubation medium a complete conversion of C3 was seen (Figure 3 : 2 ) . With 4 pmol I-' SLPI (Figure 3 : 3 ) the pattern was restored to that seen in the cell-free incubation mixtures containing opsonised yeast (Figure 3: I ) .
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Boc-Ala-ONp was also noted, with some activity left also at 8 pg I-' of SLPI (Figure 2B). Reaction mixtures without SLPI showed proteolytic activity against fibronectin, which gradually decreased with increasing concentrations of SLPI (Figure 2b). In the absence of SLPI, complement factor C3 showed a precipitate pattern on crossed immunoelectrophoresis indicating complete degradation. (Fig 3:2). At 4 pmol I-' of SLPI (Figure 3 : 3 ) , the precipitate pattern was identical to control experiments with opsonized yeast but without leukocytes and SLPI (Figure 3:l).
DISCUSSION Both leukocyte elastase and NP4(3) were released to the supernatants by phagocytosing PMN leukocytes. Only minute amounts were present in controls incubated without yeast, which indicates that the proteases were not released as the result of cell death or during harvesting of the supernatants. Leukocyte elastase and NP4(3) have been found in similar amounts in PMN leukocytes [3, 121 and inflammatory exudates [30,36]. In this study, the level of leukocyte elastase in the medium was lower than that of NP4(3); but to this may be added free elastase (evident from the substrate activity), which is not measured by the ELISA. Free leukocyte elastase is exceptional in biological fluids, and this should not ordinarily limit the utility of the assay. However, when measurements are done in mucus secretions, it must be noted that the ELISA does not measure SLPIbound leukocyte elastase. In this study, this made it possible to measure leukocyte elastase a I-PI-complexes separately in the supernatants. In good agreement with the crossed immunoelectrophoresis, it was found that the amount of leukocyte elastase-al-PI-complexes decreased with increasing SLPI-concentration (Figure 2a). However, the level of NP4(3) in the vials, as measured by ELISA, was the same irrespective of the SLPI-concentration which indicates that SLPI does not affect the release process per se. Similar to leukocyte elastase and cathepsin G, NP4(3) is a potent, non-specific proteinase, with the potential to degrade structural and soluble proteins in the tissues and body fluids [7, 8, 141. The main inhibitors of leukocyte
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elastase and NP4(3) are a2-M and al-PI, the latter inhibitor being quantitatively dominant. Imbalance between released leukocyte proteases and available inhibitors may result in pathologic proteolysis. This mechanism has been considered in lung emphysema, and leukocyte elastase as well as NP4(3) have been implicated as important in the pathogenesis [8, 371. The in-vitro model used may be a good representation of an inflammatory exudate with proteases in excess of available proteinase inhibitors. In this situation, al-PI bound leukocyte elastase in preference to NP4(3) as shown by the pattern on crossed immunoelectrophoresis (Figure 1). This is consistent with earlier estimations of their dissociation constants [14]. Addition of increasing amounts of SLPI to the incubation mixtures resulted in binding of excess leukocyte elastase and extinction of free elastolytic activity (Figure 1, 2a, 2b). Moreover, with increased binding of leukocyte elastase of SLPI, more a,-PI obviously became available to bind NP4(3) as shown by the increase in complexes between NP4(3) and al-PI (Figure 1). Proteolytic activity on the natural substrates C3 and fibronectin was markedly reduced (Figure 2b, 3). The cleavage of t-N-Boc-Ala-ONp was also markedly reduced. The residual activity on t-N-Boc-AlaONp at an 8 pmol I-‘ of SLPI may be explained by the fact that NP4(3) retains some activity against low molecular substrates also when complexed with az-M. The concentrations of SLPI used in this study are similar to the levels in the mucus secretions of the airways and genital tract [15,16, 381, and the model may thus mimick a mucus secretion with a low al-PI concentration as in congenital a,-PI deficiency. Large-scale production of recombinant human SLPI in E Coli has provided a means of anti-protease therapy in situations of congenital or acquired protease-antiprotease imbalance. The findings in this study may be of therapeutic interest, as t h e used SLPI-concentrations should be possible to reach locally and probably also in the circulation on intravenous infusion. Recombinant human SLPI is identical to the native inhibitor also in that both lack carbohydrate, which may eliminate the risk of allergic manifestations and make it superior to hitherto suggested agents. Moreover, SLPI is a potent inhibitor of leukocyte elastase and cathepsin G, but lacks inhibitory effect on
NP4(3) in vitro [14, 171. In spite of this, SLPI may obviously indirectly aid in the binding and inhibition of NP4(3) to al-PI, by taking care of at least part of the leukocyte elastase. As a specific NP4(3)-inhibitor is not readily available for therapeutic use, this effect may prove useful under in-vivo conditions and enhance the protective effect of administered SLPI. ACKNOWLEDGMENTS This project was supported by grants from ‘Forenade Liv’ Mutual Group Life Insurance Company, the Swedish Medical Research Council (grant no 3910), the Swedish Society for Medical Science, the Swedish Medical Society, the Swedish Foundation against Heart and Lung Diseases and the Medical Faculty at Lund University. REFERENCES 1 Ohlsson K, Olsson I. The neutral proteases of human granulocytes. Isolation and partial characterisation of two collagenases. Eur J Biochem 1973; 36: 473-81. 2 Ohlsson K, Olsson I. The extracellular release of granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Scand J Haematol 1977; 19: 145-52. 3 Ohlsson K,Olsson I. Neutral proteases of human granulocytes. IV Interaction between human granulocytes collagenase and plasma protease inhibitors. J Lab Clin Med 1977; 89: 269-77. 4 Ohlsson K, Olsson I, Spitznagel JK. Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes. Hoppe-Seyler’s Z Physiol Chem 1977; 358: 361 -6. 5 Ohlsson K. Polymorphonuclear leukocyte collagenase. In: Woolley DE, Evanson JM, eds. Collagenase in Normal and Pathological Connective Tissues. John Wiley & Sons Ltd, London 1980: 209-22. 6 Barrett AJ, McDonald JK. Mammalian proteases: a glossary and bibliography. Academic Press, London 1980: 189-90. 7 Ohlsson K , Linder C, Rosengren M. Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme. Biol Chem Hoppe-Seyler 1990; 371: 549-55. 8 Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal J R . Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963-73. 9 Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger M, Nathan CF. Antibiotic proteins of human polymorphonuclear
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Received 28 February 1992 Accepted 9 July 1992
