ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 292, No. 1, January, pp. 128-135, 1992

Identification of Elastase in Human Eosinophils: Immunolocalization, Isolation, and Partial Characterization’ Giuseppe Lungarella,’ Renzo Menegazzi,* Concetta Gardi, Paola Spessotto,* M. Margherita Paolo Bertoncin,* Pierluigi Patriarca,* Paola Calzoni, and Giuliano Zabucchi* Institute of General Pathology, Siena University, University of Trieste, I-34127 Trieste, Italy

I-53100 Siena, Italy; and *Institute

de Santi,

of General Pathology,

Received May 13, 1991, and in revised form August 20, 1991

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation. 0 1992 Academic Press, I~C.

Elastolytic enzymes have been isolated from a wide variety of mammalian cells and tissues and have been implicated in physiological and pathological processes characterized by the destruction of structural proteins and/ or tissue remodeling (l-3). i Supported by grants from the MURST (40% and 60%) and CNR of Italy (Target Project on Biotechnology and Bioinstmmentation and Grant 89.04075. CT 04). ’ To whom correspondence should be addressed at: Institute of General Pathology, Siena University, Via de1 Laterino, 8, I-53100 Siena, Italy. Fax (577) 270642. 128

Although much information is known concerning the properties of elastases from human neutrophils (4-9), monocytes (lo), macrophages (ll), and platelets (12), no data are available about the presence of an elastolytic activity in human eosinophils. Indeed the presence of an elastolytic activity in eosinophil-rich cell fractions from mice has been reported (13), but as yet this enzyme has not been purified and characterized. Eosinophils are known to play an important role in the pathophysiology of such inflammatory and allergic conditions as helminth infections, bronchial asthma, fibrosing alveolitis, and pulmonary eosinophilia (14, 15). It is well known that many eosinophilic reactions (viz. granulomas, fibrosing alveolitis) undergo fibrous healing that is generally thought to be influenced by the degradative action of collagenolytic and other proteolytic enzymes (16). It has been recently reported that protease activity in certain granulomas diminishes with time, supporting the hypothesis that such a decrease is conducive to the development of stable tissue fibrosis (13, 17, 18). Indeed, elastase has been shown to digest not only elastin but also other matrix proteins, such as fibronectin (19), proteoglycans (20), collagen (21), and fibrin (22, 23), that are components of stromal tissues. Therefore, the availability of pure elastase from human eosinophils, as well as knowledge of its properties, is expected to facilitate studies on the possible biological role played by this enzyme under different physiological and pathological conditions. In this paper we describe some physical and biochemical properties of an elastolytic enzyme purified from human eosinophils, as well as its subcellular localization. In addition, the properties of this enzyme were compared with those of the neutrophil enzyme, and those reported in literature for elastase from other human white blood cells. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. Al1 rights of reproduction in any form reserved.

ELASTASE

MATERIALS

IN HUMAN

AND METHODS

Materials The following materials were obtained from the indicated sources: Sue-(Ala),-pNA (SAPNA),3 Methoxy-Sue-(Ala),-Pro-Val-pNA (MSAPN), elastin powder (bovine ligamentum nuchae), elastatinal, ophenanthroline, soybean trypsin inhibitor, chicken ovoinhibitor, phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), pepstatin, leupeptin, and a,-antiprotease (a,-AP) inhibitor, guaiacol and 3,3’,5,5’-tetramethylbenzidine (TMB), alkaline phosphatase-conjugated donkey anti-sheep, alkaline phosphatase-conjugated rabbit anti-mouse, from Sigma (St. Louis, MO); 3-amino-lH-1,2,4,-triazole, from Schuchardt (Miinchen, FRG); az-macroglobulin from Boehringer (Mannheim, FRG); carrier ampholytes from LKB (Sweden); molecular weight standards from Bio-Rad (Richmond, CA); monoclonal antibody anti-human neutrophil elastase (mAb NP57) from Dakopatts (Denmark); polyclonal sheep anti-human elastase from ICN Immuno Biological (Milan, Italy); protein A-gold particles (5 nm) from E-Y LABS; NaB3H4 (25 mCi), from Amersham (England); human neutrophil elastase (HNE) from Elastin Products Co. Other reagents were of the highest quality available and were used without further purification.

Isolation

of Granulocytes

Eosinophil and neutrophil suvpensions were prepared according to the method previously described (24). Briefly, peripheral blood from normal subjects (0.4 X lo6 eosinophils/ml) was collected in ACD solution (Don Baxter Lab., Trieste, Italy). After the addition of EDTA (1 mM final concentration), the red cells were removed by dextran sedimentation (1 ml 4.5% dextran in saline was added to 5 ml of blood). Polymorphonuclear leukocytes (i.e., a mixed neutrophil-eosinophil population) were separated from mononuclear cells by centrifuging the post-dextran white cell-rich plasma for 20 min at 1OOOgon isotonic Percoll (density 1.077 g/ml) (Pharmacia). A 90-s hypotonic treatment was used to remove residual erythrocytes from the granulocyte-rich pellet. The cells were then centrifuged, washed once in phosphate-buffered saline (PBS), suspended in the same medium and counted electronically (Coulter Counter ZB I, Luton, U.K.). The percentage of neutrophils and eosinophils in the final cell suspension was determined by differential counts carried out on Wright-Giemsa-stained smears.

Eosinophil

Separation

Eosinophils were purified acco’rding to the method reported by Menegazzi et al. (24). Briefly, the granulocyte-containing pellet obtained as described above was washed once in PBS containing 13 mM sodium citrate and 0.5% bovine serum albumin (BSA, Miles Laboratoires, Goodwood, South Africa). Granulocytes were then suspended in isotonic Percoll containing 13 mM sodium citrate and 0.5% BSA. The density of the Percoll suspension was 1.0853 + 0.0002 g/ml, as measured at 2O’C by a DMA 45 density meter (A. Paar, Graz, Austria), and the Percoll osmotic value was 290 f 2 mOSM. The cell suspension was layered onto Percoll with a density higher than 1.1 g/ml and then centrifuged at 1OOOgfor 20 min at 20°C. Neutrophils (whose normal density is lower than 1.085 g/ml) were collected from the top of the gradient, washed once in PBS, and suspended in PBS. Differential counts showed that the final cell suspension usually contained only a few contaminating

3 Abbreviations used: SAPNA, Sue-(Ala),-pNA; MSAPN, methoxySue-(Ala),-Pro-Val-pNA; PMSF, phenylmethylsulfonyl fluoride; DFP, diisopropyl fluorophosphate; ol,-AP, a,-antiprotease; TMB, 3,3’,5,5’-tetramethylbenzidine; HNE, human neutrophil elastase; PBS, phosphatebuffered saline; BSA, bovine serum albumin; CTAB, cetyltrimethylammonium bromide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; TBS, Tris-buffered saline; EPO, eosinophil peroxidase; MPO, myeloperoxidase.

129

EOSINOPHILS

eosinophils (less than 1%). However, only 100% neutrophil populations were selected as starting material for the preparation of the neutrophil extract. The cell ring formed at the interface between Percoll1.085 and 1.1 was collected and treated with a hypotonic solution at 4°C to remove the contaminating red cells. As judged by differential counts carried out on Wright-Giemsa-stained smears, the resulting eosinophil suspension usually contained 85-100% eosinophils, the remaining cells being neutrophils with dense pycnotic nuclei. Unless otherwise stated, only eosinophil suspensions containing more than 99% eosinophils were used to prepare eosinophil extracts.

Purification

of Eosinophil

Elastolytic

Enzyme

Cytosomes derived from about 8 X 10s eosinophils and prepared according to the method described by Zabucchi et al. (25) were extracted at 0°C for 7 h by means of 0.1 M sodium acetate buffer, pH 4.7, containing bromide 0.1 M sodium sulfate and 0.075% cetyltrimethylammonium (CTAB). The procedure was repeated twice and, after a 30-min centrifugation at ZO,OOOg,the supernatants were concentrated in an ultrafiltration chamber (Amicon) equipped with a YM-10 diaflo membrane (Amicon). The cytosome extract was then fractionated by gel-filtration chromatography on Ultrogel AcA-44 (LKB). Fractions (2 ml each) were collected in 0.025 M sodium acetate buffer, pH 4.7, containing 0.3 M NaCl and 0.02% CTAB. Pools of fractions 55-61 containing elastolytic activity were dialyzed against 0.025 M phosphate buffer, pH 7.4 (buffer A), centrifuged for 20 min at ZO,OOOg,and fractionated by ion-exchange chromatography using a middle pressure FPLC system (Pharmacia) equipped with a Mono-S HR 5/5 (strong cationic exchanger) column. Fractions (0.5 ml each) were eluted applying a linear gradient (0 to 100%) of 0.025 M phosphate buffer, pH 7.4, containing 2 M NaCl (buffer B), followed by 3 ml of 100% buffer B. The buffer flux was adjusted to 0.5 ml/min and the pressure was 15 bar.

Enzyme Assays Elastase activity was measured using tritium-labeled elastin prepared by modification of a NaBH, reduction procedure as previously described by Banda and Werb (26). The labeled elastin suspension prepared by us showed a specific activity of 5.8 X lo5 dmp/mg. Elastase activity was measured by determining the amount of radioactivity released after incubation of enzyme preparations, at various steps of purification, with 250 gg of [3H]elastin in 0.1 M Hepes, pH 7.5, containing 0.5 M NaCl, 0.1% Brij. Final reaction volume was 130 ~1. The reactions were carried out in Eppendorf tubes at 37°C for 12-36 h. At the end of incubation, assay tubes were centrifuged for 20 min at 20,OOOg in an Eppendorf microfuge. The radioactivity released into the supernatant of the reaction mixture was used as a measure of the enzyme activity. The radioactivity was determined by dissolving 100 ~1 of the supernatant in 18 ml of Hionic Fluor (Packard) and counted in a Packard liquid-scintillation counter (Tricarb 2000 CA). Elastase-like activity against synthetic substrates was determined using SAPNA (27) and MSAPN (28). Peroxidase activity was tested by measuring the oxidation of either guaiacol(29) or TMB (30). Inhibition studies were performed using 3-amino-lH-1,2,4-triazole in the guaiacol assay (31).

Kinetic

Studies

Hydrolysis of SAPNA and MSAPN at 410 nm was employed for K, studies. All assays were performed at 25°C. Substrates were used at a minimal range of concentration of 0.1-3 times K,,,. Purified enzymes were employed at a final concentration of 0.2, 0.4, or 0.6 pg/ml. The data were plotted according to method of Lineweaver and Burk (32) and the K,,, was estimated by an interactive least-squares fit to the Michaelis-Menten equation. The K,,, for hydrolysis of [3H]elastin was expressed in milligrams per milliliter because of the insolubility of this substrate and therefore its unknown molecular weight (33).

LUNGARELLA

130 Inhibitor

Studies

Ten proteinase inhibitors were used to characterize the enzyme activity. These included elastatinal, o-phenanthroline, soybean trypsin inhibitor, chicken ovoinhibitor, PMSF, DFP, leupeptin, pepstatin, criAP, and a,-macroglobulin. The enzymes were preincubated with each inhibitor in 0.1 M Hepes buffer, pH 7.5, 0.5 M NaCl, 0.1% Brij at 37’C for 30 min before assaying. The residue enzyme activities were measured on [sH]elastin substrate.

AC&amide

Electrophoresis

SDS-PAGE was carried out according to Laemmli (34). For the molecular weight determination of purified enzyme, bovine serum albumin (M, 66,500), ovalbumin (M, 45,000), carbonic anhydrase (M, 30,000), soybean trypsin inhibitor (M, 21,500), and lysozyme (M, 14,400) were used as marker proteins. IEF was performed in an LKB flatplate apparatus (LKB 2117) using 5% polyacrylamide gels, pH 7.9-10.0, containing 2% carrier ampholytes. Acid gel electrophoresis was performed as described by Sweetman and Ornstein (8) except that slab gels were used instead of tube gels.

Immunological

Studies

Immunologic cross-reactivity between eosinDot immunobinding. ophilic and neutrophil elastases was studied with dot immunobinding immunoassay (dot blot) according to Renner (35). In brief, samples of neutrophil and eosinophil elastase (starting dilution 2.3 mg/ml) were dotted in aliquots of 3 ~1 onto nitrocellulose paper. Free protein binding sites were blocked by incubation in 3% bovine serum albumin for 20 min. The paper was then incubated with the mAb NP57 and stained with a solution of protein A-gold particles. Immunocytochemistry. Neutrophil and eosinophil-mixed cell suspensions, deposited onto slides by means of a cytocentrifuge, were fixed in 0.125% glutaraldehyde in TBS for 15 min. The slides were incubated for 20 min with 2.5% human serum in TBS and then treated with 0.5% Triton X-100 in TBS for 10 min. After the slides were washed three times with TBS, they were incubated with 50 U/ml heparin for 15 min in order to prevent eventual aspecific reactions between the cationic proteins of the eosinophils and the alkaline phosphatase or the substrate employed for the staining reaction. After this treatment the slides were rinsed three times in TBS, incubated for 30 min with heterologous serum, and then washed three more times in TBS. The slides were incubated with specific antibodies against human elastase, either polyclonal or monoclonal. Polyclonal sheep anti-human elastase (ICN, ImmunoBiological) (diluted 1:50 in TBS) or mAb NP57 (Dakopatts) (diluted 1:lOO in TBS) were then applied onto the cell smears for 30 min. After three rinses in TBS, the slides were incubated for 30 min with either alkaline phosphatase-conjugated donkey anti-sheep antibodies (Sigma) (diluted 1:200 in TBS) or alkaline phosphatase-conjugated rabbit anti-mouse antibodies (Sigma) (diluted 1:lOO in TBS). After three more washes in TBS, alkaline phosphatase reaction was carried out using a solution of Tris-HCl 0.1 M, pH 8.2, containing 0.2 mg/ml Naphthol AS-MX phosphate sodium salt (Sigma), 2% dimethylformamide, 1 mM levamisole, 1 mM MgCl,, and 1 mg/ml Fast Red (Sigma), added just before use. The cell smears were finally counterstained with hemallume for 3 min. The percentage of positive cells was calculated by counting at least 200 cells for each sample. The immunogold method (postembedZmmunoelectron microscopy. ding technique) was used to localize elastase in white blood cells from normal and hypereosinophilic subjects. Blood cells were pelletted onto silicon-coated plastic tubes using a cytocentrifuge, then fixed for 1-2 h in 2.5% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 1% (wt/vol) sucrose. The pellets were washed with cacodylate buffer, dehydrated in acetone, and embedded in epoxy resin (Araldite), without postfixation in 1% osmium tetroxide. Ultrathin sections (about

ET AL. 600 A thick) were picked up on nickel grids and pretreated with PBS containing 1% albumin for 5 min. The grids were then floated on a drop of the diluted anti-elastase mAb (1:2600) for 24 h at 4°C; the grids were thoroughly rinsed for 10 min with a mild spray of PBS and then distilled water, and transferred onto 15.~1 drops of a protein A-gold colloidal particles (5 nm) (E-Y LABS) solution diluted 1:8 in PBS. The sections were then washed, dried, stained with uranyl acetate-lead citrate, and examined in a Zeiss EM 109 electron microscope. Additional control samples from the same specimens were processed in a similar manner, but the antibodies were substituted with nonimmune mouse serum or PBS.

RESULTS

Purification and Biochemical of Elastase from Eosinophils

Characterization

In preliminary experiments we found that a pool of highly pure populations of human eosinophils (>99.5%) and neutrophils (>90%) contained an elastolytic activity (as judged by [3H]elastin cleavage) of 2410 k 330 and 10,700 + 1760 cpm/h/106 cells, respectively (mean of five experiments + SD). The enzyme activity in the eosinophil population could not be accounted for by neutrophil contamination, since the neutrophil:eosinophil ratio was found to be 1:200-300. This elastolytic activity was indeed recovered in fractions obtained by gel-filtration chromatography of eosinophilic cytosome extracts (Fig. 1A). As can be seen, the enzyme activity was associated with eosinophil peroxidase (EPO) activity in fractions 55-61. The absence of any myeloperoxidase (MPO) activity in the chromatographic profile (as judged by both spectrophotometric analysis and inhibition studies using the guaiacol oxidation assay (31)) was an additional support that our elastase activity was not due to neutrophil contamination. Pooled fractions 55-61 were further processed by ion-exchange chromatography. This procedure allows us to separate EPO activity from the elastolytic activity (Fig. 1B).

22 -$ ELASTASE ---*--.

EPO

IO

20

30

40

Fractions

50

60

70

80

(n)

FIG. 1A. Elution profile of cytosome extract fractionated by Ultrogel AcA-44. Each fraction was analyzed for peroxidatic (OD at 413 nm) and elastolytic activity (dpm/h/protein). Peroxidase activity was measured by the guaiacol oxidation assay (29).

ELASTASE

IN HUMAN

EOSINOPHILS

131

FIG. 3. Acid gel electrophoresis of leukocyte elastases according to Sweetman and Ornstein (8). HNE migrates as four individual protein components (E, - EJ; eosinophil elastase shows a relative mobility similar to Ed. The slab gel was stained with Coomassie brilliant blue.

a0 % of

2 M

NaCl

FIG. 1B. Elution profile of pooled fractions 55-61 on Mono-S HR 5/ 5 column in a linear gradient (0 to 100%) of 2 M NaCl. Each fraction was analyzed for protein (OD at 280 nm) and peroxidatic and elastolytic activity.

The purification procedure detailed above yielded a very pure elastase preparation as revealed by electrophoretic studies. The protein was judged pure by the presence of only a single band on acrylamide gels (Figs. 2 and 3) and analytical IEF. The molecular weight and the isoelectric point of the purified enzyme were reported in Table I. As can be seen, eosinophil-derived elastase shares with the major isoenzyme of HNE a common molecular weight (Fig. 2) and isoelectric point, as well as a similar relative mobility in acidic gel (Fig. 3). The K, for SAPNA and MSAPN, as well as the proteolytic activity on tritiumlabeled elastin by the isolated enzyme, is also given in Table I. The biochemical and biophysical properties reported above suggest that a common enzyme activity is

present in human neutrophils and eosinophils. This conclusion is supported by the results obtained with a variety of inhibitors as well as by immunological observations (see below). The inhibition profile observed for the enzyme isolated by us is very similar to that of HNE and provides evidence that eosinophil elastase is a serinoproteinase (Table II). The enzyme activity was, in fact, greatly inhibited by PMSF and DFP, potent inhibitors of serinoproteinases, whereas it was insensitive to o-phenanthroline, a chelator of bivalent cations. In addition, like HNE, it is greatly inhibited by oil-AP, soybean trypsin inhibitor, and (Yemacroglobulin. Immunological

Observations

On the basis of the biochemical above, we tested the immunological

TABLE

similarities detailed reactivity of the eo-

I

Comparison of Some Properties of Elastases from Human Eosinophils and Neutrophils

45K

Eosinophil Molecular weight Isoelectric point K, (mM) for hydrolysis of SAPNA” K, (mM) for hydrolysis of MSAPN* Km (mg/ml) for hydrolysis of [3H]elastinc

a

b

C

FIG. 2. Analytical SDS-PAGE of the purified enzyme in a 10% gel. Lane a, molecular weight markers; lane b, isoforms of HNE; lane c, eosinophil elastase. The slab gel was stained with Coomassie brilliant blue.

28,000 9.0

Neutrophil 27,500-28,000-31,000 8.2-9.0

0.53 f 0.08

0.45 f 0.07

0.058 f 0.007

0.052 f 0.006

3.8

3.4

Note. The molecular weight and isoelectric point were given for the different forms of granulocyte elastase detectable in SDS-PAGE and IEF gels. ’ 0.2 M Tris-HCl, pH 8.0, at 25°C. * 0.1 M Hepes, pH 7.5, 0.5 M NaCl, 10% Me*SO at 25°C. ‘0.1 M Hepes, pH 7.5, 0.5 M NaCl, 0.1% Brij at 37°C; the Km for elastolysis ([“Hlelastin) is expressed in mg/ml because of the insolubility of this substrate and, therefore, its unknown molecular weight.

132

LUNGARELLA TABLE

TABLE

II Immunocytochemical (NEU)

Comparison of Effects of Proteinase Inhibitors on Eosinophil and Neutrophil Elastases Enzyme activity (% of control value)”

Inhibitor PMSF’ DFPd o-Phenanthroline Soybean trypsin inhibitor Chicken ovoinhibitor Leupeptin Pepstatind Elastinal oc,-Antiproteinase oc,-Macroglobulin

ET AL.

Concentration

Eosinophil elastase

Neutrophil elastase* 30 3

1.0 mM 1.0 mM 1.0 mM

27.9 0 98.4

98.3

100 pg/ml 100 pg/ml 10 fig/ml 10 bez/ml 100 wg/ml 12 PM 12 ELM

16.1 19.5 0 21.3 31 15 13.3

13 15 0 n.d. 25 6 9

Note. The enzymes were preincubated with each inhibitor in 0.1 M Hepes, pH 7.5, 0.5 M NaCl, Brij at 37’C. The activity was determined on [aH]elastin and was expressed as a percentage of the control activity in the presence of each solvent. ’ Values obtained from three different measurements. * Values obtained for pooled forms of neutrophil elastases. ’ Isopropanol (4%, vol/vol) present in preincubation mixture. d Dimethyl sulfoxide (4%, vol/vol) present in preincubation mixture.

sinophil-derived elastase to a mAb raised to HNE. The results obtained by dot immunobinding are reported in Fig. 4. In particular, equivalent amounts of HNE and the pure eosinophilic enzyme gave similar heavy precipitation dots when treated with the same amounts of mAb. For this reason, an attempt was made to demonstrate elastase in the intact eosinophils, by immunocytochemistry using monoclonal or polyclonal antibodies against HNE. As can be seen in Table III a positivity was observed in about 50% of subjects, even if to a different extent (20100% of the eosinophils scored). The immunoelectron-

FIG. 4. Reactivity of mAb NPB7 with neutrophil (neu) and eosinophil (eos) elastases (a). There is no nonspecific binding of an unrelated mAb to purified elastases (b).

Subject

Fixative”

1

A/M

2

9

G G G G A/M A/M G G

10 11

G G

3 4 5 6 7 8

Detection

III of Elastase

in Neutrophils

and Eosinophils (EOS)

Type of antibody*

NEU

EOS

PolY PolY

+++ +++

+ ++

% Positive EOS 100 50

mono

+++

+

60

PolY

+++

++

90

mono

+++

+

20

PolY PolY

+++ +++

-

mmlo mono

t+t ++t

~ -

POlY POlY

+++ +++

-

Type of positivity’ d d : d&s

-

Note. In all experiments, but No. 4, eosinophils were isolated from peripheral blood of noneosinophilic subjects. a Fixative agent: A/M = acetone/methanol (l:l), G = 0.125% glutaraldehyde. ’ +++, highly positive; ++, positive; +, weakly positive; -, negative. ’ d, diffuse; s, spot.

localization of eosinophilic elastase in subcellular compartments was carried out on specimens of subjects 3 (normal donor) and 4 (eosinophilic donor) (Table III). Eosinophils and neutrophils (Figs. 5 and 6) exposed to mAb NP57 against HNE revealed a positive immunogold reaction in their granules. In particular, we observed that elastase was localized in both cytoplasm structures that are considered to be two different maturation stages of the same cytoplasmic structure. On the contrary, a negative reaction in eosinophil and neutrophil granules was seen when samples were treated with normal mouse serum or PBS. This pattern of immunological stain was observed in cells from both normal (Fig. 6) and hypereosinophilic donors (Fig. 5). DISCUSSION An elastolytic enzyme from eosinophil cytosomes of human blood was purified to apparent electrophoretic homogeneity by FPLC. The results reported in the present study demonstrate that human eosinophils, like other white blood cells (4-12), do contain an enzyme that will degrade elastin. The results obtained with the various tested inhibitors provide evidence that eosinophil elastase (like neutrophil, monocyte, and platelet elastases) is a serinoproteinase. The activity of the eosinophil enzyme toward [3H]elastin is, in fact, greatly inhibited by DFP (a potent inhibitor of serinoproteinases), whereas it is unaffected by o-phenanthroline (a potent inhibitor of metalloproteinases). The enzyme activity associated with eosinophils is due to a serinoproteinase that shares with neutrophil, monocyte, and platelet elastases the properties of hydrolyzing

a

donor showing immunogold localization in eosinophil cytoplasmic granules (arrows). FIG. 5. (a) Electron microgralph from hypereosinophilic Colloidal binding is also present in lysosomes of an adjacent neutrophil (arrowheads). X21,500. (b) Higher magnification of (a) showing immunogoldlabeled elastase in the different cytoplasmic granules from eosinophil X38,000. 133

134

LUNGARELLA

FIG. 6. (a) Immunogold localization of elastase in eosinophil granules from a normal donor (arrows). Azurophil granules of an adjacent neutrophil show a marked colloidal gold binding (arrowheads) X9300. (b) Higher magnification of (a). X19,700.

some synthetic elastase substrates (i.e., SAPNA or MSAPN) and of being inhibited by PMSF and soybean trypsin inhibitor (10, 12, 36, 37). Unlike platelet elastase (12), however, eosinophil enzyme is strongly inhibited by a,-AP. In addition, the eosinophilic enzyme shares some electrophoretic properties (viz. molecular weight and isoelectric point) with monocyte elastase (10) and the major isoenzyme of HNE (38, 39). Furthermore, like monocyte elastase, which shows immunologic similarities to HNE (lo), the eosinophilic enzyme reacts with a mAb against HNE.

ET AL.

The data reported by us suggest that human eosinophils and neutrophils may contain the same enzyme activity. One could believe that the enzyme activity purified from the eosinophilic cytosome population is due to a neutrophi1 contamination. However, the absence of any MPO activity in our chromatographic profile and the presence of only a single band with enzyme activity exclude such a possibility. In addition, the immunolocalization findings strongly suggest that eosinophils and neutrophils express a common elastase isoenzyme. Like neutrophils, in fact, eosinophils contain immunoreactive material in their granules that strongly reacts with a mAb against HNE. It is still possible that eosinophil does not express an elastase but internalizes elastase from lysed neutrophils. However, this seems unlikely because we failed to detect in eosinophil extracts the various isoforms of neutrophil elastase. It is generally thought that neutrophils and eosinophils derive from the same progenitor cell (40). It has been recently reported that in poorly differentiated cells, from acute leukaemia, elastase is expressed at a relative later stage of myeloid differentiation than myeloperoxidase (41, 42). In light of the available data, it is conceivable to suppose that white blood cells undergo changes in their enzyme profile during maturation and differentiation processes. Thus, the various isoforms of elastase, detectable in the different mature white blood cells, may be the result of a different gene expression throughout the differentiation steps. However, further study (via molecular biology techniques) would conclusively show that eosinophils express the same elastase that is present in neutrophils. Supportive evidence for this hypothesis is the fact that also monocytes present an elastase with catalytic and immunologic properties similar to the major isoenzyme of neutrophil elastase (10). However, during the transition from monocytes to macrophages, the expression of this elastase (serinoproteinase) is suppressed, and a different elastase (metalloproteinase) appears (10, 43). Another interesting observation is the presence of gold particles in both types of dense cytoplasmic granules from human eosinophils. This observation supports the hypothesis that the different types of eosinophilic dense granules may represent successive morphological stages of maturation of the same cytoplasmic structure (for reference see (44)). It has recently been reported that the eosinophil is not an endstage effector cell, but it is able to synthesize proteins during maturation in response to various exogenous stimuli (45). In light of these findings it is not possible to exclude that the heterogeneity of the elastase content we have reported can reflect different stages of differentiation and/ or activation of these cells. Further studies in this field should be fruitful. ACKNOWLEDGMENTS We thank Miss Patrizia Anselmi and Miss Alessandra Poggi for their friendly secretarial assistance. The authors thank the blood banks of

ELASTASE

IN HUMAN

the Hospital of Trieste, Monfalcone, and Udine for supplying blood. The skillful technical assistance of Dr. Alessandra Knowles and Dr. Patrizia Zuccato is gratefully acknowledged.

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