Human neutrophil gelatinase: A marker for circulating blood neutrophils. Purification and quantitation bv enzvrne linked immunosoEbent assay J

Kjeldsen L, Bjerrum OW, Hovgaard D, Johnsen AH, Sehested M, Borregaard N. Human neutrophil gelatinase: A marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay. Eur J Haematol 1992: 49: 180-191. Abstract: Human neutrophil gelatinase was purified to apparent homogeneity. The N-terminal amino-acid sequence of the purified enzyme could be aligned to an internal part of the cDNA-derived amino-acid sequence of 92-kDa type IV collagenase from SV 40-transfected human lung fibroblasts and from a TPA differentiated monocytic cell line, U937. Total amino-acid composition of U937 and neutrophil gelatinases was identical. Gelatinase was susceptible to treatment with o- and n-glycanase, indicating that posttranslational addition of oligosaccharide side chains occurs. An enzyme-linked immunosorbent assay for gelatinase was developed using specific polyclonal rabbit antibodies. The assay was specific, sensitive, accurate, and reproducible. Ninety percent range for plasma gelatinase from normal subjects was 17.3 to 102.9 ng/ml. In patients treated with cytostatic agents for non-Hodgkin’s lymphoma, there was a parallel drop in plasma gelatinase and peripheral granulocyte count. This indicates that plasma gelatinase is a marker for circulating neutrophils. Plasma gelatinase does not seem to reflect bone marrow cellularity.

Introduction

A number of human cells are capable of producing proteases that are members of the metalloproteinase family. These metalloproteinases are important in the degradation and remodeling of extracellular matrix (1, 2). Among these, specific collagenase and type IV collagenase or gelatinase are secreted by human neutrophil granulocytes upon activation (35). These proteases have been shown to reside in separate subsets of granules. Specific collagenase is contained within specific granules (6) and gelatinase is located in a separate type of granules that are mobilized more easily and extensively than specific granules (7-10). Both collagenases are released in latent form ( 5 ) and subsequently activated by mechanisms that are not fully clarified. Gelatinase can be activated in vitro by trypsin or organomercurials ( 5 , 1l), probably involving what is termed a “cysteine switch” (12). Activation has been reported to be associated with a proteolytical cleavage of the amino terminus ( 13). Type IV collagenase (gelatinase) is capable of de180

J

Lars Kjeldsen, Ole Weis Bjerrum, Doris Hovgaard, Anders H. Johnsent, Maxwell Sehested * and Niels Borregaard Granulocyte Research Laboratory, Department of Hematology L, tDepartment of Clinical Chemistry, University Hospital, Copenhagen, * Department of Pathology, Sundby Hospital, Copenhagen, Denmark

Key words: neutrophils - gelatinase

-

ELISA

Correspondence: Lars Kjeldsen, M.D.. Granulocyte Research Laboratory, Department of Haematology L, University Hospital, Rigshospitalet afsnit 4041, Blegdamsvej 9, DK-2 100, Copenhagen, Denmark Accepted for publication 22 Julv 1992

grading denatured collagen as well as type IV and V collagen (4); the latter two are important components of the basement membrane of endothelium. Neutrophil gelatinase is thus potentially important for diapedesis of neutrophils (14). Neutrophil gelatinase is immunologically crossreactive with the 92 kDa gelatinase produced by macrophages, keratinocytes, SV 40-transfected human lung fibroblasts, the fibrosarcoma HT 1080 cell line, and the monocytic leukemia U 937 cell line (13). The total nucleotide sequence of 92 kDa gelatinase cDNA from SV 40-transfected human lung fibroblast and U937 cells has been determined (13). In line with the immunologic cross-reactivity there was identity between the amino acid sequences deduced from this cDNA sequence and the amino terminal of neutrophil gelatinase, apart from 8 residues missing at the amino terminal part of neutrophil gelatinase (15, 16). In order to study the content of gelatinase in various tissues, cells and body fluids, as well as the function of gelatinase, a sensitive and specific assay for gelatinase is essential. Previously, gelatinase concentrations have been determined by

Human neutrophil gelatinase measuring the gelatinolytic activity towards tritiated gelatine (17). Major drawbacks of this assay are lack of specificity, low sensitivity and susceptibility to proteases and other inhibitors present during the assay (3, 5). In this paper we present data characterizing the purified neutrophil gelatinase by amino-acid composition, N-terminal amino-acid sequence analysis, and susceptibility to treatment with various glucosidases. Furthermore, we present an ELISA for gelatinase employing polyclonal rabbit antibodies. The high sensitivity of the ELISA allows quantitative measurements of gelatinase in plasma samples. It is further examined as to whether plasma gelatinase can serve as a marker for myelopoiesis. Materials and method Preparation of neutrophils

Human neutrophils were isolated either from buffy coats supplied by the blood bank or from blood donated by healthy volunteers. Whole blood was anti-coagulated in 25 mmol/l sodium citrate, 126 mmol/l glucose. Erythrocytes were sedimented by adding an equal volume of 2% Dextran T-500 (Pharmacia LKB, Uppsala, Sweden) in 0.9% NaCI. The resulting leukocyte-rich supernatant was aspirated, and the cells pelleted at 200 g for 10 min. Cells were resuspended in saline and neutrophils separated by centrifugation through Lymphoprep (18) (Nygaard, Oslo, Norway) at 400 g for 30 min. Remaining erythrocytes were removed by hypotonic lysis in ice-cold H,O for 30 s. Tonicity was restored with an equal volume of 1.8% NaCl. The neutrophils were then washed once in 0.9% NaCl and subsequently resuspended in the desired buffer. All steps except for dextran sedimentation (room temperature) were performed at 4' C. Purification of gelatinase

Neutrophil gelatinase was purified essentially as described by Hibbs et al. (19). In short, neutrophils were prepared from buffy coats as described, and cells stimulated with PMA (Sigma Chemical Co., St. Louis, MO, USA) 2 pg/ml. The resulting supernatant was collected and subjected to ion exchange chromatography on a D E 52 column (Whatman International, Ltd. Maidstone) followed by affinity chromatography on a CNBr-Sepharose 4b column (Pharmacia) to which heat-denatured type I collagen had been coupled. SDS-PAGE

SDS-PAGE was performed essentially as described by Laemmli (20).

Amino-acid analysis

The purified protein was hydrolysed for 20 h in 6 N HCI gas phase at 110°C under argon in pyrolysed tapered microvials (1 00 p1, Hewlett-Packard, Waldbronn, Germany). The hydrolysate was dried and redissolved in 60 p1 of 0.4 mol/l sodium borate, pH 10.4. Amino-acid analysis was performed on 6 pl using a Hewlett-Packard Aminoquant analyzer (21) (precolumn derivatization with o-phtaldialdehyde followed by 9-flourenylmethylchloroformate, both reagents supplied by Hewlett-Packard). Amino terminal sequence analysis

The amino-acid sequence of the purified protein was determined using an automatic protein sequencer (475A, Applied Biosystems, Foster City, CA, USA) equipped with an on-line high-performance liquid chromatography (HPLC) system for detection of the amino-acid phenylthiohydantoin (PTH) derivatives. The PTH-derivatives were separated on a C18-DB column (5-7943, Supelco, PA, USA). All chemicals and solvents were sequence- or HPLC-grade and delivered by Applied Biosystems. Endoglucosidase treatment

Samples of the purified gelatinase (1 5 pg) were dried in a Speed-Vac vacuum centrifuge (Savant, NY, USA) and redissolved in 0.5% SDS and 1% P-mercaptoethanol, boiled for 3 min, and diluted 5-fold into either 0.2 mol/l NaHPO,/NaH,PO,, pH 8.6, 10 mmol/l 1,lO-phenantroline, 1.25 % Nonidet P-40 (Sigma, St. Louis, MO, USA) or 20mmol/l Tris-maleat, pH 6.0,lO mmol/l D-galactono-lactone, 1.25 "/, Nonidet P-40 and incubated with either 20 U/ ml of N-glycanase (Genzyme, Boston, MA USA) or 50 mU/ml 0-glycanase (Genzyme) respectively for 16 h at 37°C (13). For treatment with endoglucosidase H (Genzyme) the dried gelatinase was redissolved in SDS and mercaptoethanol as described above. The redissolved protein was diluted 5-fold in 0.1 mol/l NaH,PO,, pH 6.1, containing 50 mmol/l EDTA and 0.5% Nonidet P-40 and incubated with 0.25 U/ml of endoglucosidase H (Genzyme) for 10 h (22). Samples were diluted in SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blot analysis. Activation of the latent gelatinase

The purified preparation was dialyzed against 5 mmol/l CaCl,, 50 mmol/l NaC1, 50 mmol/l TrisHC1, pH 7.6, 0.01 % Brij-35 and activated by incubation at 37 " C with either aniinophenylmercuric acid (1 nimol/l) or trypsin ( 15 pg/ml) at the indicated 181

Kjeldsen et al. intervals of time. Trypsin treatment was terminated by addition of soybean trypsin inhibitor (60 pg/ml). These samples were diluted in sample buffer and subsequently subjected to SDS-PAGE and Western blotting under reducing conditions. The gelatinase content of the activated samples was measured by ELISA. Antibody preparation

Purified gelatinase (0.5 mg/ml) was added to an equal volume of incomplete Freunds adjuvant. Five rabbits were immunized by injection of 50 pg protein every 14 d. The rabbits were initially bled at 2-w intervals but only once a month after 3 months of immunization. Immunoglobulins were purified as described by Harboe et al. (23). The antiserum was affinity-purified on a CNBr-activated Sepharose4B column (Pharmacia) to which 8 mg of purified gelatinase had been coupled. The column was equilibrated in PBS. Ten ml of antiserum was dialyzed against PBS and subsequently applied to the gelatinase column. Bound material was eluted in 3 mol/l KSCN in PBS and dialyzed against PBS. The specificity of the antibodies was tested by immunoblotting of a postnuclear supernatant of disrupted human neutrophils. This showed that the antibodies reacted with both gelatinase and with a 25 kDa protein. To remove antibody with specificity for the 25 kDa protein, neutrophils were stimulated with PMA (2 pglml) to exocytose granule proteins. The 25 kDa protein, present in the exocytosed material, was separated from gelatinase by gelfiltration on a Sephadex G-200 column (Pharmacia) and coupled to CNBr-activated sepharose. The antigelatinase antibodies (affinity-purified as well as IgG fraction) were depleted of antibodies against the 25 kDa protein by passage through this column. The affinity-purified anti-gelatinase antibody was biotinylated essentially as described (24), and dialyzed twice against saline and finally against PBS containing 0.1 % NaN,.

affinity-purified anti-gelatinase antibody diluted 1:9000 and by addition of avidin-peroxidase (Dakopatts (P347), Glostrup, Denmark) diluted 1:4000. All incubations were carried out with 100 pl/well for 1 h unless otherwise stated. Color was developed during a40-min incubation in 0.1 mol/l sodium phosphate, 0.1 mol/l citric acid buffer, pH 5.0, containing 0.04% o-phenylenediamine and 0.03 % H,02 (100 pl/well), and stopped by addition of 100 pl 1 mol/l H,SO,. The plates were washed 3 times in buffer A between all steps, unless otherwise stated. Prior to color development, an additional wash in sodium phosphate citric acid buffer was included. Absorbance was read at 492 nm in a Multiscan Plus ELISA-reader (Labsystems, Helsinki, Finland). A standard of purified gelatinase ranging from 0.3 1 to 5 ng/ml was used. Plasma samples were diluted from 20- to 100-fold, whereas neutrophil samples were diluted from 10- to 5000-fold. All dilutions were in buffer B. All steps were performed at room temperature. lmmunoblotting

Protein was transferred from SDS-PAGE slabs to 0.2 pm nitrocellulose filters (BioRad Laboratories, Richmond, CA) essentially as described by Towbin (25). Transfer buffer was 192 mmol/l glycine, 25 mmol/l Tris, pH 8.3, 20% (v/v) methanol, and proteins were transferred in a Biorad trans-blot vertical system at 60 V, 210 mA for 4 h. Additional binding sites were blocked by incubating the nitrocellulose filters in 2% Tween-20 in PBS for 30 min. After 3 washes in PBS, 0.05% Tween, the blots were incubated with the primary antibody overnight. Primary antibodies were labeled with peroxidaseconjugated swine anti-rabbit antibody (Dakopatts (P217)) diluted 1:lOOO and incubated for 2 h. The filters were then washed 3 times in PBS, 0.05% Tween and once in 50 mmol/l Tris pH 7.6 and developed in 50 mmol/l Tris, pH 7.6 containing diaminobenzidine tetrahydrochloride (DAB) chromogen (Dakopatts) 0.20 mg/ml and 0.03% H,O,.

Gelatinase-ELISA

Gelatinase was assayed by an ELISA using 96-well flat-bottom immunoplates (NUNC, Roskilde, Denmark). The plates were coated overnight with antigelatinase IgG, diluted 1:5000 in carbonate buffer (50 mmol/l Na,CO,/NaHCO,, pH 9.6) and washed once in buffer A (0.5 mol/l NaC1, 3 mmol/l KC1, 8 mmol/l Na,HPO,/KH,PO,, p H 7.2, 1% Triton X- 100). Additional binding sites were blocked by incubation with 200 pl/well of buffer B (0.5 mol/l NaCl, 3 rnmol/l KCl, 8 mmol/l Na,HPO,/KH,PO,, pH 7.2, 1% BSA, 1% Triton X-100). Samples were then applied followed by addition of biotinylated 182

lmmunocytochemistry

Ten-minute methanol fixation was used for blood and bone marrow smears and 4% phosphatebuffered formaldehyde for fixation prior to paraffin embedding. The intensity of the staining reaction decreased markedly with increasing length of formalin fixation and was lost at 24-48 h. The antigen was otherwise robust as demonstrated by preservation of staining in spleens removed > 24 h post-mortem. Subsequently, 30-min formalin fixation was used throughout. A two-step indirect immunoperoxidase method was used: After deparaffinization, slides

Human neutrophil gelatinase were pretreated with 0.5% H,02 and 10% filtered swine serum, with washes in between with phosphatebuffered saline, pH 7.2, before incubation with the primary antibody followed by 3 washes in phosphatebuffered saline and incubation with peroxidaseconjugated swine anti-rabbit (DAKO P 164). Primary antibody was diluted from a stock solution of 0.5 mg/ml for anti-gelatinase. Anti-lactoferrin was obtained from Dakopatts (DAKO A186) at a titre of 750 pg/ml. Following 3 washes with phosphatebuffered saline, the slide was developed with 3-amino ethyl carbazole. Negative controls were substitution of the primary antibody with non-immune rabbit serum and, for anti-gelatinase, specific blocking of the staining reaction with antibody at a 1:2000 dilution preincubated for 30 min at 24 O C with purified antigen at equimolar and 5- and 10-fold molar antigen excess before application for 1 h at 24" C. Absorption at equimolar concentration showed near complete inhibition and, at 5-fold molar excess, total inhibition of the staining reaction.

Assays

Gelatinase activity was measured by the method described by Harris and Krane with slight modifications (17). Plasma lactoferrin was measured by an ELISA, using goat antibody against human lactoferrin (Nordic Immunology, Tilsburg, Netherlands) diluted 1:500 as catching antibody. Rabbit antibody against human lactoferrin (Dakopatts (A186)) was used as detecting antibody in a dilution of 1:2000. Peroxidase-conjugated affinity-purified goat antirabbit IgG antibody was diluted 1: 1000 (Dakopatts (P448)). Purified human milk lactoferrin was used as standard in concentrations ranging from 6.25400 ng/ml. Buffers and all other procedures were as described for the gelatinase ELISA. Total protein was measured by a commercial kit from Biorad employing Commassie Brilliant Blue using catalase as a standard.

Results and discussion Subcellular fractionation

Subcellular fractionation of human neutrophils was performed as described (26). In short, neutrophils were disrupted by nitrogen cavitation, the cavitate centrifuged and the resulting postnuclear supernatant layered on top of a two-layer Percoll (Pharmacia) gradient. The gradient was centrifuged at 37000 g for 30 min and fractions collected by aspiration from the bottom of the tube.

Purification and characterization of gelatinase

Neutrophil gelatinase was purified to apparent homogeneity as evidenced by SDS-PAGE (Fig. 1). This revealed 3 bands of 220 kDa, 135 kDa and 92 kDa respectively under non-reducing conditions and 1 major band of 92 kDa under reducing conditions. This is in accordance with the results found by Hibbs et al. (19). In this respect, neutrophil gelatinase differs from gelatinases from other sources. An

Blood samples

Serum or plasma (using EDTA 3.9 mmol/l as anticoagulant) was isolated from 3.5 ml whole blood. The serum or plasma was removed at room temperature after centrifugation at 1000 g for 10 min. Centrifugation was performed within 7 h after venipuncture. The samples were stored immediately at - 20" C. For determination of reference interval, plasma samples were collected from 28 males and 3 5 females, 18-62 yr old, all subjectively healthy. Simultaneous determination of circulating neutrophil count was performed. The correlation between plasma gelatinase and neutrophil count was investigated using the Spearman test. P-values less than 0.05 were considered significant. To further characterize the relationship between plasma gelatinase and neutrophil count in peripheral blood, plasma samples were obtained from patients treated for non-Hodgkin's lymphoma with CHOP (adriamycin (50 mg/m2),cyclophosphamide (750 mg/m2), vincristine (1.4 rngim') and prednisone (100 mg daily for 5 d)).

x

rma

200 >

97.4 69 > 46

30

>

21.5

14.3 > 1

2

Fig. 1. SDS-PAGE of purified gelatinase: 5 pg purified sample was subjected to SDS-PAGE under non-reducing (lane 1) and reducing conditions (lane 2) in a 5-20"/, gradient gel.

183

Kjeldsen et a]. Table 1. Amino acid analysis of the purified neutrophil gelatinase compared with amino acid composition of gelatinase from the monocytic cell line U937. The latter is deduced from the cDNA sequence. The hydrolysis was performed on 20 pmol of which 1/10 was analyzed. Mole % is expressed as % of all amino acids except Cys and Trp, which were not determined mole % Amino acid Asx Glx Ser His

G~Y Thr Ala Arg TYr Val Met Ile Phe Leu LYS

Pro

*

u937*

Neutrophil gelatinase

10.3 8.1 6.5 2.1 10.0 7.6 7.6 7.4 4.1 5.5 1.4 2.1 6.5 8.5 3.3 9.0

10.0 9.2 6.2 2.5 11.1 6.5 1.3 1.5 3.6 5.2 1.2 2.5 6.2 8.5 3.7 8.8

ref. 13.

explanation for the 135 and 220 kDa forms is still lacking. The minor 25 kDa band in the SDS-Page of the purified gelatinase (Fig. 1) is only visible under reducing conditions, and is thus presumably copurified with gelatinase. This also seemed to be the case in a previous study (4). In agreement with previous reports ( 5 , 11, 19) the purified gelatinase was found to be latent and the molecular weight on gelfiltration on an AcA 34 column was estimated to be 165000 to 170000 kDa using the gelatinolytic assay (not shown). The total amino acid composition of the purified gelatinase was determined and agreed with the composition derived from the deduced amino-acid sequence of 92 kDa gelatinase from the phorbol esterdifferentiated monocytic cell line U 937 and from SV40-transfected human lung fibroblasts (Table 1) (deduced from the known cDNA sequence). Furthermore, the sequence of the first 20 N-terminal amino acids was determined (Table 2) and aligned with the known sequence of the corresponding part of U 937 gelatinase. This shows identity. In contrast to our findings, previous reports on neutrophil gelatinase (15, 16) claim that neutrophil gelatinase lacks eight residues at the amino terminus. To characterize the posttranslational glycosylation of gelatinase, the purified enzyme was subjected to treatment with various glucosidases followed by SDS-PAGE and Western blotting. This showed that the molecular weight of gelatinase was reduced to approximately 80 kDa and 89 kDa upon treatment with Nand O-glycanase respectively, whereas Endoglucosidase H had no apparent effect (Fig. 2). This confirms 184

Table 2 Sequence analysis of the N-terminus of neutrophil gelatinase compared to the known sequence of gelatinase from the monocytic cell line U937. The analysis was performed on 200 pmol protein Amino Acid

#

u937**

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Ala Pro Arg Gln Arg Gln Ser Thr Leu Val Leu Phe Pro Glv ASP Leu Ar9 Thr Asn Leu

Neutrophil

Pro Arg Gln Arg

Gln Ser Thr Leu Val Leu Phe Pro

GlY

Ar9 Thr Leu

"-": Could not be determined ref. 13.

*

that neutrophil gelatinase has a protein backbone of 80 kDa, which is modified by posttranslational addition of several oligosaccharide side chains including o-linked and complex n-linked carbohydrates. These findings are in agreement with those on 92 kDa gelatinase of the U 937 cell line (13) and further confirm the similarities between the 92 kDa gelatinases produced by different cell types including neutrophils and tumor cell lines. Purified gelatinase was activated by treatment with organomercurials or trypsin and subsequently subjected to SDS-PAGE and Western blot analysis. This revealed that activation resulted in a drop in molecular weight of approximately 15 kDa after trypsin treatment (Fig. 3). Upon treatment with paraaminomercuric acetate for various intervals of time a successive drop to 85 kDa, 75 kDa, and 68 kDa forms resulted (Fig. 3). The activation of gelatinase seemed to reduce the reactivity of gelatinase towards the antibodies as measured by ELISA. This was most pronounced for paraaminomercuric acetate activation and the reactivity dropped successively with time to approximately 20% after 4 h. The activation studies indicate that the activation of gelatinase is accompanied by proteolytic cleavage of the enzyme as has previously been shown for neutrophil(4, 5 ) and macrophage gelatinase (28) and for gelatinase from H T 1080 fibrosarcoma cells (29). This proteolytic cleavage might result in conformational changes of the molecule allowing interaction

Human neutrophil gelatinase

A

200 >

A

97.4 > 69 >

200 >

46 >

97.4 > 69 >

30 >

46 >

21.5 > 21.5 >

14.3 > 1

2

3

14.3 >

4 1 2 3 4 5 6

B

200 >

B M r (lo3)

46 >

200 >

30

>

97.4 ' 69 >

21.5 >

46 >

14.3 >

30 >

1

2

3

4

Fig.2. Posttranslational glycosylation of neutrophil gelatinase: Samples of purified gelatinase were incubated with either buffer (lane I), N-glycanase (lane 2), 0-glycanase (lane 3), or Endoglucosidase H (lane 4) as described in methods. Samples were subsequently subjected to SDS-PAGE and Western blotting under reducing conditions in a 5-20?" gradient gel. For SDS-PAGE (panel A) 6 pg of gelatinase was applied. For Western blotting (panel B) 0.75 pg was applied. Affinity purified anti-gelatinase (diluted 1000-fold) was used as primary antibody.

between collagen/gelatin and the active site of gelatinase, although it remains to be elucidated whether proteolytic cleavage is necessary for activation rather than being a result of activation followed by autoproteolysis. The proteolytic cleavage and the potential conformational changes might explain the reduced reactivity of activated gelatinase towards the gelatinase antibodies.

21.5 14.3 > 1 2 3 4 5 6 Fig. 3. Activation of purified gelatinase with paraaniinomercuric acetate or trypsin: Samples of purified gelatinase (15 pg) were incubated.at 37°C with either buffer (lane l), with paraaminomercuric acetate (1 rnmol/l) for 30 min (lane 2), 60 min (lane 3), 120 min (lane 4), or 240 min (lane 5 ) respectively or with trypsin (15 pg/ml) for 1 h (lane 6). Samples were subsequently subjected to SDS-PAGE (8.2 pg gelatinase applied) (panel A) and Western blotting (5.5 pg gelatinase applied) (panel B) under reducing conditions.

Gelatinase antibodies

Western blotting of a postnuclear supernatant from nitrogen cavitated neutrophils showed that the antibodies were specific. Three bands of 220 kDa, 130 kDa, and 92 kDa were detected under non185

Kjeldsen et al. Absorbance

M r ( lo3)

2.57 -

200

--

>

0

1

2

3

4

5

6

Concentration (ng/ml)

46 >

Fig. 5. Standard curve for the gelatinase ELISA and serial 2-fold dilutions of plasma and exocytosed material from neutrophil granulocytes: The plasma sample was diluted from 10- to 160-fold whereas the exocytosed material was diluted from 32000- to 512000-fold. Gelatinase standard; * Plasma; + Exocytosed material.

30 > 21.5 >

reducing conditions and one 92 kDa band was detected under reducing conditions (Fig. 4). Western blotting of plasma samples from healthy persons was not possible due to the very sparse amounts present in plasma.

nase, as mentioned earlier. This may interfere with detection of proteolytically modified gelatinase in plasma. The intra-assay variation was 3 % (180 4.9 pg/ ml, n = 24) for a sample of exocytosed material from a PMA-stimulated neutrophil suspension, whereas the intra-assay variation of a plasma sample was 2% (59.7 1.0 ng/ml, n = 24). The reproducibility of the ELISA was determined for another neutrophil sample and for 3 different plasma samples. Inter-assay variation for the first was 10% (139 f 13 pg/ml, n = 14), and for the plasma samples 7% (18 1.3 ng/ml, n = lo), 7 % (35 k 2.6 ng/ml, n = 9), and 11 % (31 & 3.3 ng/ml, n = 9) respectively. The accuracy of the ELISA was investigated by adding 15 ng of purified gelatinase to 3 different plasma samples, with subsequent measurement of the gelatinase content. In these experiments recovery was 99, 99 and 91 % respectively.

Gelatinase ELISA

Gelatinase in neutrophil extracts

The standard curve for the gelatinase-ELISA is shown in Fig. 5 . Near linearity between absorbance and dilutions in the range from 0.3 to 5ng/ml of gelatinase is observed. Fig. 5 also demonstrates the parallelism between the standard curve on the one hand, and serial dilutions of plasma or exocytosed material from neutrophils stimulated with PMA on the other. This indicates that irrelevant plasma proteins or proteins present in neutrophil samples beside gelatinase do no interfere with the assay. The assay is sensitive with a detection limit of 0.25 ng/ml and detects gelatinase in its latent form in contrast to the gelatinolytic assay. A drawback of the assay is the diminished reactivity towards activated gelati-

The content of gelatinase in freshly isolated neutrophils was found to be 10.0 f 2.7 pg per 3 x lo7 cells (k SD, n = 13). The stability of the assay was investigated in samples from neutrophils which contain large amounts of proteases that are potential inhibitors of this assay. Dilutions of intact neutrophil suspensions were either immediately cooled at 4 C, incubated for 1 h at 37 C, or left at room temperature for variable intervals of time. These dilutions proved stable under all circumstances without loss of immunological reactivity. To investigate the susceptibility of the assay to proteolysis, purified gelatinase was exposed to either

14.3 >

1

2

Fig. 4 . Western blotting of postnuclear supernatant from neutrophi1 granulocytes: 10 pl of postnuclear supernatant was added to 90 pl saline and 100 p1 sample buffer. 150 p1 of sample was subjected to electrophoresis under reducing (lane 1) and nonreducing conditions (lane 2). Blotting was performed as described in materials and methods. Primary antibody was IgG-anti-gelatinase (diluted 5000-fold).

186

Human neutrophil gelatinase protease V8 from staphylococcus aureus or solubilized azurophil granules, obtained by subcellular fractionation (26). SDS-PAGE, Western blotting and quantitative determination of gelatinase content were subsequently performed. From Fig. 6 it is seen that purified gelatinase was proteolysed as expected by protease V8 and also degraded upon incubation with azurophil granules (A, lane 2 and 3). The proteolysis resulted in concomitant loss of detectable 92 kDa gelatinase in Western blot analysis and detection of bands with lower molecular weight (B, lane 2 and 3). In accordance with this the gelatinase content measured with the ELISA dropped to 53% and 73% respectively. In line with these findings, gelatinase content fell to 70-80 % when a subcellular fraction of neutrophils containing

Mr (lo3)

46 >

30 > 21.5 > 2

Fig. 7. Distribution of gelatinase in human EDTA plasma from healthy donors (n = 63).

the major part of gelatinase, specific granules and contaminating azurophil granules was incubated for 1 h at 37°C in the presence of a detergent. These data indicate that assays of neutrophil samples involving incubation at 37"C, as is necessary for activation of the enzyme, might result in underestimation of the gelatinase content since proteolysis and thus potential inactivation may occur. Furthermore, incomplete activation of gelatinase may also be a problem using the gelatinolytic assay. An immunologically based assay should therefore be employed in preference to a functional assay for quantitation of gelatinase in neutrophil extracts.

200 > 97.4 > 69 >

1

Gelatinase (ng/ml)

3

B

Gelatinase in plasma

The high sensitivity of the ELISA permits the measurement of gelatinase in plasma. The presence of

200 > 97.4 > 69 > 46 >

30 > 21.5 >

CT,

c W Q)

150

41

100

cn

1

2

3

Fig. 6. Susceptibility to proteolysis of gelatinase upon exposure to protease V8 and solubilized azurophil granules from neutrophil granulocytes: Purified gelatinase was dialyzed against 75 mmol/l NH,HCO,, pH 7.4 and 30 pg incubated with either buffer for 1 h (A and B, lane I), 160 U/ml of V8 staphylococcus aureus protease for 4 h (A and B, lane 2), or with solubilized neutrophil azurophil granules (1,5 mg protein/ml) for 1 h (A and B, lane 3). Azurophil granules were obtained by subcellular fractionation as described. Samples were subjected to SDS-PAGE in a 10% homogenous gel (A) as well as Western blotting in a 5-20"/, gradient gel (B), in both cases under reducing conditions. Primary antibody for Western blotting was affinity-purified anti-gelatinase diluted 1000-fold.

200

.4 u -

.

rn

I

:.. I

I

I

4

6

8

10

8

50

Q)

c3

0 0

2

-

12

N e u t r o p h i l c o u n t ( 1 09/1) Fig. 8. Correlation between neutrophil number and plasma gelatinase in 56 healthy donors. The data were correlated by the Spearmann test and no correlation could be demonstrated

(R = 0.11, p = 0.4).

187

Kjeldsen et al.

b'in.. Y. Irnmunohistochemical demonstration of ncutrophil gelatinasc (a. b) and loctoferrin (c. d ) i n a normoplastic bone niarron (a, c ) and i n a patient with P N H (b, d). Concentration o f primary antibody was 1:500 for gclatinasc and 1:8000 for lactofcrrin. both incttbated at 2 4 - C for 1 h .

gelatinase in plasma has previously been demonstrated by gelatinase zymography (29). In contrast to the ELISA, this method is not quantitative and it depends on the isolation of gelatinolytic enzymes from plasma by affinity chromatography prior to zymography . The results of measurement of gelatinase in 63 healthy persons is shown in Fig. 7. The 90",, range 188

for gelatinase was between 17.2 and 102.9 ng/ml and the median concentration was 34.1 ng/ml, whereas the arithmetic mean was 38.9 ng/nil. This is in accordance with a previous report (30). The content of gelatinase in serum exceeded that of EDTAstabilized plasma (in 2 persons gelatinasc in plasma was 50 and 58 ng/ml respectively versus 238 and 246 ng/ml in serum) probably due to activation of

Human neutrophil gelatinase neutrophils during coagulation, leading to secretion of gelatinase. In line with this, plasma gelatinase from a blood sample drawn directly into a precooled tube was, on average, 16% lower than plasma gelatinase in the same blood sample handled at room temperature. Whether kept unseparated at 4' C or at room temperature the gelatinase content in plasma was stable during the first 7 h after sampling. After this point the content tended to rise, presumably due to cell death. No correlation between plasma gelatinase and neutrophil count in peripheral blood could be demonstrated in the healthy donors (Fig. 8). There are several possible explanations of this observation. First, it is possible that plasma gelatinase not only reflects circulating neutrophils but also neutrophils present in the bone marrow and that lack of correlation should be explained by differences in the cellularity of the bone marrow. This does not seem to be the case since plasma gelatinase was found to be very low (1 1 ngirnl) in a patient with severe paroxystic nocturnal hemoglobinuria and neutropenia (1.0 x lo9 neutrophils/l). This patient had a hyperplastic bone marrow with fully differentiated myelopoiesis and neutrophils containing normal

Count

amounts of gelatinase (Fig. 9). Furthermore, concomitant determinations of plasma gelatinase and neutrophil count in peripheral blood showed a strict correlation between gelatinase and neutrophil number in patients with non-Hodgkin's lymphoma receiving cytostatics (CHOP) (Fig. 10). It is readily observed that gelatinase content is lowest when the neutrophil count is maximally suppressed approximately 10 to 14 d after chemotherapy. The rise in plasma gelatinase does not precede the rise in neutrophil number. This would have been expected if gelatinase had been released from the regenerating bone marrow. In addition a parallel rise in plasma gelatinase and neutrophil count was observed after administration of methylprednisolon to 3 healthy subjects (Fig. 11). Thus plasma gelatinase solely reflects circulating neutrophils and cannot be used as a parameter of myelopoietic activity. Recently, a similar observation has been made regarding neutrophil lactoferrin present in plasma (3 l), although earlier reports have shown plasma lactoferrin to partially reflect bone marrow cellularity (32,33,34). The patient with paroxystic nocturnal hemoglobinuria even had a plasma lactoferrin content below normal (82 ng/ml, normal range 169-588 ng/ml) in spite of

Gelatinase (nglml)

Neutrophil Count 110'11)

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Fig. 10. Concomitant determination of plasma gelatinase and neutrophil count in 4 patients treated with CHOP for non-Hodgkin's lymphoma. Day zero is initiation of intravenous chemotherapy. + Plasma gelatinase; * Neutrophil count.

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Kjeldsen et al. Plasmagelatinase (ng/ml)

Neutrophil count (109/1)

300 r

110

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(Fig. 9). This would hardly have been the case if plasma lactoferrin reflected myelopoietic activity, although further documentation is needed to confirm this hypothesis. The lack of correlation between neutrophil number and plasma gelatinase is then best explained by inter-individual differences in neutrophil gelatinase content or differences in the degree of activation of neutrophils during circulation. The average content of gelatinase in plasma (38.9ng/ml) comprises 3.9% of the content in the neutrophils present in the corresponding blood Sample (3 x lo6 neutrophils/ml). It is thus possible that gelatinase measured in plasma is liberated after venipuncture from disintegrated neutrophils or liberated by discrete exocytosis from neutrophils before and during plasma preparation. In line with this theory is the fact that handling of the blood sample at 4 O C rather than at room temperature results in lower plasma gelatinase values. However, differences in centrifugation speed did not affect the content of gelatinase in plasma (not shown). Conclusion

'0 0

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The characterization of purified neutrophil gelatinase is presented. It is concluded that neutrophil gelatinase is similar to the gelatinase of macrophages and 92 kDa gelatinase of various tumor cell lines concerning amino-acid sequence and composition as well as posttranslational glucosylation and activation. A sensitive, accurate, and reproducible ELISA for gelatinase is described. The presence of gelatinase in plasma is demonstrated, and it is concluded that plasma gelatinase reflects circulating neutrophils and does not reflect myelopoietic activity in the bone marrow. Acknowledgements

0' 0 0 . 6

'0

I

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2

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24

Time (hours)

Fig. 1I. Concomitant determination of plasma gelatinase and neutrophil count in 3 healthy subjects before and after administration of methylprednisolon. Blood samples were drawn before and at the indicated times after intravenous administration of 40 mg methylprednisolon. + Plasma gelatinase; * Neutrophil count.

normal content of lactoferrin in bone marrow neutrophils, as assessed by immunocytochemistry

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The excellent technical assistance of Mrs. Charlotte Horn, Mrs. Mathilde Brandt and Mr. Ronnie Danielsen is greatly appreciated. Purified human milk lactoferrin was kindly provided by Dr. Henrik Birgens, Department of Hematology L, Rigshospitalet. This work was supported by grants from Danish Cancer Society, Anders Hasselbachs Fund, Emil C. Hertz Fund, Leo Nielsen Fund, Novo-Nordisk Fund, Amalie Jsrgensen Fund, The Gangsted Fund, Lundbecks Fund and P. Carl Petersen Fund, The Danish Medical Association, The Danish Medical Research Council, The Fund for The Advancement of Medical Science, Skovgaards Fund. NB is the recipient of a Neye Research Professorship.

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