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(Journal of Leukocyte Biology. 2002;72:462-469.)
© 2002 by Society for Leukocyte Biology

Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils

Lene Udby^*, Jero Calafat, Ole E. Sørensen^*, Niels Borregaard^* and Lars Kjeldsen^*

^* Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, Copenhagen, Denmark; and
Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam

Correspondence: Lene Udby, M.D., Granulocyte Research Laboratory, Department of Hematology L-9322, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen Ø, Denmark. E-mail: l.udby{at}rh.dk

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense.

Key Words: specific granules • gelatinase granules • SGP28 • immunogold electron microscopy • subcellular fractionation • granulocytes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The neutrophilic granulocyte plays an important role in innate immunity and in inflammatory reactions in human disease. Neutrophils circulate in a quiescent state, but become activated and leave the bloodstream during the encounter with an inflammatory stimulus. In the tissue, they exert their primary function: killing and digesting invading microorganisms. Neutrophils are equipped with four types of exocytosable storage organelle: azurophil, specific, and gelatinase granules and secretory vesicles [¹ ]. These organelles are formed sequentially during myelopoiesis in the bone marrow and contain different matrix and membrane proteins, which are important for the functional characteristics of the various granules and secretory vesicles. The current classification of granules into three different subsets is based on the content of only a few granule proteins. Granules containing myeloperoxidase (MPO) are designated azurophil or peroxidase-positive granules, which appear at the promyelocytic stage of differentiation. Peroxidase-negative granules are arbitrarily separated into specific granules identified by their content of lactoferrin, and gelatinase granules, which contain the majority of gelatinase. Rather than being separate entities, peroxidase-negative granules should be envisioned as a continuum from granules containing lactoferrin but lacking gelatinase, over granules containing lactoferrin and gelatinase to granules containing the majority of gelatinase but very little lactoferrin. This granule heterogeneity is explained by differences in the biosynthetic window of these granule proteins, with lactoferrin mainly being synthesized at the myelocyte/metamyelocyte stage, whereas gelatinase is synthesized later at the metamyelocyte/band neutrophil stage [^{1 2 3} ].

Mobilization of granules and vesicles upon stimulation follows a strict hierarchy allowing a gradual activation of the neutrophil [⁴ , ⁵ ]. The secretory vesicles are released initially, possibly already during the rolling of the neutrophil along inflamed, selectin-expressing, postcapillary venules. Secretory vesicle mobilization causes an up-regulation of receptors and adhesion proteins, which enable the neutrophil to adhere to the endothelium [⁶ ]. The next steps involve exocytosis of gelatinase granules first and specific granules second, presumably facilitating diapedesis and migration of the neutrophil through the release of matrix-degrading proteins [⁷ , ⁸ ]. Normally, only very small amounts of the azurophil granules are released to the exterior. When the neutrophils ingest microorganisms by phagocytosis, the azurophil granules fuse with the phagosomes, whereby their content of proteolytic and bactericidal substances is released into the phagolysosome [¹ , ⁹ ].

The cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was discovered in human neutrophils in 1996, and its cDNA was cloned from a human bone marrow cDNA library [¹⁰ ]. CRISP-3 belongs to a family of cysteine-rich secretory proteins [¹¹ ], where two other members (CRISP-1 and CRISP-2) are believed to play important roles in sperm maturation and fertilization [^{12 13 14} ]. CRISPs are characterized by their content of 16 highly conserved cysteine residues in the C-terminal 2/3, which form intramolecular disulfide bonds [¹⁵ ]. CRISP-3 contains a consensus sequence for N-linked glycosylation, and recently we have shown that CRISP-3 exists in a 29 kDa-glycosylated and a 27 kDa-nonglycosylated form with identical amino acid sequences [¹⁶ ].

CRISP-3 has sequence similarity to pathogenesis-related proteins involved in the host defense of plants [¹⁷ , ¹⁸ ] and is present in a variety of exocrine secretions [¹⁶ ]. This, together with the presence in neutrophils, indicates a possible role of CRISP-3 in the innate immune system. Preliminary studies using nonquantitative methods suggested CRISP-3 to be localized in specific granules in human neutrophils [¹⁰ ], but firm conclusions could not be drawn because of the low affinity of the antipeptide antibodies used. We have recently raised specific polyclonal antibodies against recombinant C-terminally truncated CRISP-3 suitable for immunoblotting and immunocytochemistry and have also developed an accurate, specific, and sensitive enzyme-linked immunosorbent assay (ELISA) for the quantification of CRISP-3 in fluids and cell lysates [¹⁶ ]. Using these new methods, we wanted to investigate the subcellular localization and mobilization of CRISP-3 in resting and activated human neutrophils by subcellular fractionation and double-labeling immunogold electron microscopy.

	MATERIALS AND METHODS

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Isolation of neutrophils
Human neutrophils were isolated from freshly prepared buffy coats supplied by the blood bank. Erythrocytes were sedimented by addition of an equal volume of 2% dextran T-500 (Amersham Pharmacia Biotech, Upsala, Sweden) in 0.9% saline. The leukocyte-rich supernatant was aspirated, and the cells were pelleted by centrifugation at 200 g for 10 min. Cells were resuspended in saline, and neutrophils were separated by centrifugation through Lymphoprep (Nycomed Pharma, Oslo, Norway) at 400 g for 30 min. Remaining erythrocytes were lysed by hypotonic shock in ice-cold water for 30 s. Tonicity was restored by addition of an equal volume of 1.8% NaCl. The neutrophils were then washed once in saline and resuspended in the desired buffer. All steps except the dextran sedimentation (room temperature) were performed at 4°C.

Isolation of eosinophils
Eosinophils were purified from two buffy coats by magnetic cell sorting using the magnetic cell sorter (MACS) system (Miltenyi Biotec, Germany) following the instructions given by the manufacturer. In short, granulocytes were isolated as described above and labeled with mouse monoclonal anti-CD49d antibodies (Pharmingen, San Diego, CA), which label eosinophils but not neutrophils [¹⁹ ]. Following incubation with MACS rat anti-mouse immunoglobulin G (IgG)₁ microbeads, the eosinophils were isolated by positive selection on a MACS BS column placed in the magnetic field of a VarioMACS separator. After removal of the column from the magnetic field, the eosinophils were eluted, washed, and counted (yield: 1.6x10⁷ cells). All steps were performed at 4°C.

Cytospin preparations of the positively and negatively selected cell fractions (and also of the mononuclear cells harvested from the intermediate layer following Lymphoprep centrifugation of leukocytes) were stained with May-Grünwald Giemsa and evaluated by light microscopy. The concentration of CRISP-3 in the cell populations was assayed by ELISA and immunoblotting.

Release studies
Neutrophils were resuspended at 3 x 10⁷ cells/ml in Krebs-Ringer phosphate buffer plus glucose (KRG; 130 mM NaCl, 5 mM KCl, 1.27 mM MgCl₂, 0.95 mM CaCl₂, 10 mM NaH₂PO₄/Na₂HPO₄, pH 7.4, 5 mM glucose). For stimulation, 1 ml cell suspension was preincubated at 37°C for 5 min followed by addition of the stimulus and then incubated for 15 min. The stimulus was 10 nM formyl-Met-Leu-Phe (fMLP; Sigma Chemical Co., St. Louis, MO), 2 µg/ml phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.), 1 µM ionomycin (Sigma Chemical Co.), or 1 mg/ml serum-treated zymosan (STZ). STZ was prepared as follows: Zymosan A (Sigma Chemical Co.) was homogenized in H₂O. After pelleting, the zymosan particles were incubated in serum for 30 min at 37°C in a concentration of 4 mg/ml, washed three times in KRG, resuspended in the same buffer at a concentration of 10 mg/ml, and stored at 20°C until further use. As the concentration of CRISP-3 in serum is relatively high [¹⁶ ], the amount of CRISP-3 in the final preparation of STZ was measured by ELISA and was found to be below the detection limit.

Control cells were kept on ice or incubated at 37°C for 20 min. Stimulation was stopped by addition of 1 vol ice-cold KRG and immediate sedimentation of the cells by centrifugation (200 g for 6 min). The supernatant, termed S₀, was aspirated. The cell pellet was resuspended in 1 ml KRG and immediately diluted further in ELISA dilution buffer (0.5 M NaCl, 3 mM KCl, 8 mM Na₂HPO₄/KH₂PO₄, 1% bovine serum albumin, 1% Triton X-100, pH 7.2). Release of MPO, lactoferrin, gelatinase, and CRISP-3 was calculated as the content in the supernatant in terms of percentage of the total content (pellet+supernatant). Recovery of CRISP-3 was calculated for each condition and expressed in percentage in relation to the amount of CRISP-3 measured in control cells kept at 4°C.

Subcellular fractionation
Neutrophils (control cells kept on ice or cells stimulated with fMLP or PMA as described above) were resuspended in KRG and incubated with 5 mM diisopropyl fluorophosphate (Aldrich Chemical Co., Milwaukee, WI) for 5 min on ice. Cells were then pelleted by centrifugation at 200 g for 6 min and resuspended at 3 x 10⁷ cells/ml in disruption buffer (100 mM KCl, 3 mM NaCl, 1 mM ATPNa₂, 3.5 mM MgCl₂, 10 mM Pipes, pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by nitrogen cavitation at 600 psi as described [²⁰ ]. Nuclei and intact cells were pelleted by centrifugation at 400 g for 15 min (P₁). The postnuclear supernatant (S₁; 10 ml) was carefully applied on top of a three-layer Percoll gradient (1.05/1.09/1.12 g/ml) containing 9 ml of each density of Percoll in disruption buffer supplemented with 0.5 mM PMSF [²⁰ ]. The gradient was centrifuged at 37,000 g for 30 min and collected in fractions of 1 ml each by aspiration from the bottom of the tube. All fractions were assayed for markers as described below. Recovery of CRISP-3 was calculated as total amount measured in the fractions expressed as percentage of the amount measured in the postnuclear supernatant applied on the gradient.

Samples (450 µl) of each fraction from a gradient of unstimulated control cells were centrifuged 20 min at 28 psi in an Airfuge (Beckmann, Palo Alto, CA) to sediment out the Percoll. The biological material was resuspended in 450 µl phosphate-buffered saline (PBS) and mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer.

Marker assays
CRISP-3 was quantified with a novel sandwich ELISA using polyclonal antibodies against recombinant, C-terminally truncated CRISP-3, as described [¹⁶ ]. Purified, native CRISP-3 was used as standard.

MPO (marker for azurophil granules), lactoferrin (marker for specific granules), gelatinase (marker for gelatinase granules), human leukocyte antigen (HLA) class I (marker for plasma membrane), and albumin (marker for secretory vesicles) were measured by ELISA as previously described [²⁰ ].

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting
Samples of 20 µl were applied to SDS-PAGE under reducing conditions using Bio-Rad Mini-PROTEAN 3 Cell (Bio-Rad Laboratories, Hercules, CA) [²¹ ]. Protein was transferred from the 14% polyacrylamide gels in 10 mM CAPS, pH 11.0, 10% methanol using Bio-Rad Mini Trans-Blot Module (Bio-Rad Laboratories) [²² ]. Additional binding sites were blocked by incubation of the nitrocellulose blots in 5% skim milk in PBS for 1 h. The blots were incubated overnight with anti-CRISP-3 antiserum diluted 1/1000, followed by a 2 h incubation with peroxidase-conjugated swine anti-rabbit Ig [Dako (P217), Glostrup, Denmark] diluted 1/1000. Color was developed using 3'-diaminobenzidine tetrahydrochloride/metal concentrate and stable peroxide substrate buffer (Pierce, Rockford, IL).

Immunoelectron microscopy
Isolated human neutrophils from peripheral blood were fixed for 24 h in 4% paraformaldehyde in 0.1 M PHEM buffer (pH 6.9) and then processed for ultrathin cryosectioning as previously described [²³ ]. Cryosections (45 nm) were cut at -125°C using diamond knives (Drukker Cuijk, The Netherlands) in an ultracryomicrotome (Leica Aktiengesellschaft, Vienna, Austria) and were transferred with a mixture of sucrose and methylcellulose onto formvar-coated copper grids [²⁴ ]. The grids were placed on 35-nm petri dishes containing 2% gelatine. For single immunolabeling, the sections were incubated with rabbit anti-CRISP-3 antibody for 45 min, followed by 30 min incubation with 10 nm protein A-conjugated colloidal gold (EM Lab., Utrecht University, The Netherlands). For double immunolabeling, the procedure described by Slot et al. [²⁵ ] was followed with 10- and 15-nm protein A-conjugated colloidal gold probes. After immunolabeling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands). For the controls, the primary antibody was replaced by a nonrelevant rabbit antibody.

The antibodies used were anti-CRISP-3 [¹⁶ ], antigelatinase [²⁶ ], rabbit anti-human lactoferrin (Cappel Laboratories, Cochranville, PA), and mouse monoclonal antieosinophil peroxidase (anti-EPO; Pharmacia Biotech).

Statistical analysis
Differences among the release of lactoferrin, gelatinase, and CRISP-3 were tested by the paired t-test (two-tailed) using Microsoft Excel 2000. P < 0.05 was considered significant.

	RESULTS

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To establish the subcellular localization of CRISP-3 in human neutrophils, subcellular fractionation of disrupted neutrophils was performed on a three-layer Percoll density gradient, which resolves all the known, mobilizable organelles (azurophil, specific, and gelatinase granules and secretory vesicles) [²⁰ ]. The content in each fraction of CRISP-3 and of five marker proteins was measured by ELISAs. From Figure 1 , it is readily observed that CRISP-3 was located in the same fractions as peroxidase-negative granules. However, the distribution profile of CRISP-3 was different from the profile of lactoferrin and gelatinase, as it showed a broader peak, overlapping the entire peak of lactoferrin and extending into the peak of gelatinase. This was reproduced in all of three independent experiments (Fig. 2 ) and indicates a localization of CRISP-3 in specific granules and also in the most dense subset of gelatinase granules. As seen from the immunoblotting of subcellular fractions (Fig. 2) , CRISP-3 coexists in two different forms of 31 and 29 kDa (29 and 27 kDa under nonreducing conditions), which represent a glycosylated and a nonglycosylated form of the protein [¹⁶ ]. The subcellular distribution of the two forms was identical.

View larger version (26K): [in this window] [in a new window]   Figure 1. Subcellular fractionation of human neutrophils. Isolated neutrophils were disrupted by nitrogen cavitation, centrifuged on a three-layer Percoll density gradient, and fractionated by aspiration from the bottom of the tube. The gradient was collected in 36 fractions of 1 ml. Each fraction was assayed by ELISA for the following marker proteins: MPO (azurophil granules), lactoferrin (specific granules), gelatinase (gelatinase granules), CRISP-3, albumin (secretory vesicles), and HLA (plasma membranes). Data from one representative experiment are shown. Concentrations for each protein are given as measured concentration in the fraction relative to the maximal concentration. Recovery of CRISP-3 was 88.2%.

View larger version (35K): [in this window] [in a new window]   Figure 2. Distribution profiles of CRISP-3 and peroxidase-negative granule markers. Subcellular fractions of disrupted human neutrophils on three-layer Percoll density gradients were assayed by ELISA for CRISP-3 and the marker proteins lactoferrin (specific granules) and gelatinase (gelatinase granules), as shown in the upper panel. Data are mean from three independent experiments. Concentrations for each protein are given as mean concentration in the fraction relative to the mean concentration in the peak fraction. Average recovery of CRISP-3 was 83.4% ± 13% (±SD, n=3). The lower panel shows immunoblotting of subcellular fractions from one experiment with anti-CRISP-3 antibodies. No reactivity was observed in fractions 27–35 (not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3. Subcellular fractionation of resting and fMLP- or PMA-stimulated human neutrophils on three-layer Percoll density gradients. Isolated human neutrophils were stimulated, disrupted by nitrogen cavitation, and fractionated, and each fraction was assayed for lactoferrin, CRISP-3, and gelatinase by ELISA. Concentrations are given as µg/ml. The subcellular distribution of lactoferrin, CRISP-3, and gelatinase in control (nonstimulated, kept on ice) and fMLP- and PMA-stimulated human neutrophils is shown. Recovery of CRISP-3 was 88.2%, 83.4%, and 102.1%, respectively.

View larger version (158K): [in this window] [in a new window]   Figure 4. Ultrastructural localization of CRISP-3 in neutrophils by immunogold electron microscopy. (A) Cryosections of neutrophils were labeled with anti-CRISP-3 antibodies followed by protein A-gold (10 nm). An area of a neutrophil shows CRISP-3 labeling localized on the matrix of granules (arrows). No labeling is seen on the cytosol, mitochondria (m), or nucleus (n). (B and C) Double-labeling immunogold electron microscopy with antibodies against CRISP-3 and gelatinase (B) or CRISP-3 and lactoferrin (C). Cryosections were labeled with anti-CRISP-3 antibodies followed by protein A gold (10 nm). Subsequently, sections were labeled with antigelatinase antibodies or antilactoferrin antibodies followed by protein A gold (15 nm). CRISP-3 labeling is observed in gelatinase- and lactoferrin-positive granules (arrows). Original bars, 200 nm.

View larger version (140K): [in this window] [in a new window]   Figure 5. Ultrastructural localization of CRISP-3 in eosinophils by immunogold electron microscopy. Cryosections of eosinophils were labeled with anti-CRISP-3 antibodies followed by protein A gold (10 nm; A). (B) Sections were labeled with anti-EPO antibody followed by rabbit anti-mouse IgG and protein A gold (10 nm) and then with anti-CRISP-3 antibodies followed by protein A gold (15 nm). (A) An area of an eosinophil demonstrates CRISP-3 labeling localized on the matrix of granules (arrows). (B) It is observed that all CRISP-3-positive granules (15 nm gold) are also EPO-positive (10 nm gold; arrows). Original bars, 200 nm.

	DISCUSSION

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The subcellular fractionation data do not completely match the findings obtained by immunogold electron microscopy. Looking at the distribution profiles of lactoferrin and CRISP-3 (Figs. 1 and 2) , we expected a higher degree of colocalization between lactoferrin and CRISP-3 than the observed 62% (Table 2) . One possible explanation for this observation could be that lactoferrin is far more abundant and of higher molecular weight than CRISP-3 and therefore is more easily detected by immunogold labeling using a polyclonal antibody, which is likely to recognize several epitopes along the protein [²⁹ ]. Considering the low concentration of CRISP-3 measured in eosinophils compared with neutrophils, there is a surprisingly high CRISP-3 labeling of eosinophils by immunogold electron microscopy. The explanation for this is not known, but it may be caused by differences in labeling efficiency between eosinophil and neutrophil granules. Another explanation could be that the number of CRISP-3 containing granules in eosinophils is considerably lower than in neutrophils.

We have previously investigated the timing of protein synthesis during myelopoiesis and have shown that the synthesis of MPO, lactoferrin, gelatinase, and other granule proteins [² , ³⁰ ] reflects the pattern of mRNA expression [³¹ ]. This is described in the so-called targeting-by-timing hypothesis, which implies that proteins expressed and synthesized at the same time will end up together in the same granule subset. The protein distribution of CRISP-3 in specific granules and in the most dense subset of gelatinase granules is also completely in line with the mRNA distribution profiles found in normal myeloid progenitors [³¹ ], as the mRNA expression of CRISP-3 starts in parallel with lactoferrin but continues at the metamyelocytic and band stage where lactoferrin expression has diminished considerably. Thus, the findings presented herein further corroborate the targeting-by-timing hypothesis [¹ ] at the protein and functional level.

In peroxidase-positive granules, proteases are stored in the active state following proteolytic processing after arrival to the granules. In contrast, peroxidase-negative granule proteins are generally stored in granules without any proteolytic processing, and some proteins (gelatinase, collagenase, and cathelicidin) require activation by N-terminal trimming after release to the exterior or to the phagolysosome [¹ , ⁹ , ³² ]. We have previously proposed that CRISP-3 was N-terminally trimmed prior to storage in granules [¹ , ¹⁰ ], as the form of CRISP-3 originally purified from neutrophils lacked 12 amino acids in the N-terminus, compared with the expected sequence after removal of the signal peptide [¹⁰ ]. A recent study, however, ruled out this possibility, as we demonstrated by mass spectrometry that although two forms of CRISP-3 are present in neutrophils (and elsewhere), they both represent the mature protein with identical amino acid sequences [¹⁶ ]. Differences in glycosylation account for the existence of the two forms, as treatment with N-glycanase, which removes N-linked carbohydrate residues, transformed the one form into the other [¹⁶ ]. The truncated form of CRISP-3 (lacking the N-terminal 12 amino acids) was found to be a result of proteolytic cleavage, when the protein was exocytosed upon PMA stimulation (in a concentrated cell suspension, i.e., 4–5x10⁸ cells/ml) without the presence of protease inhibitors [¹⁶ ]. We do not expect this cleavage to serve physiological functions, as PMA is a very strong and nonphysiological stimulant. Furthermore, we have never experienced the truncated form in biological fluids such as plasma, saliva, seminal plasma, or sweat [¹⁶ ].

The function of CRISP-3 remains to be established. The localization in specific and gelatinase granules suggests a matrix degradative or antimicrobial role in line with functions of other proteins localized in these granules (collagenase, gelatinase, hCAP-18, lysozyme) [³⁰ , ³³ ]. The presence in exocrine secretions [¹⁶ ] that cover mucous membranes and the resemblance to pathogenesis-related proteins in plants [¹⁷ , ¹⁸ ] support the potential antimicrobial function. The presence in eosinophils could account for a role against helminthic infestations, in which the eosinophils are believed to be of importance [³⁴ ]. Other members of the CRISP family (CRISP-1 and CRISP-2) apparently function as adhesion or fusogenic proteins [^{12 13 14} ], and a novel and closely related protein, Allurin, has a chemotactic function [³⁵ ]. A 25 kDa protein, P25TI, which is secreted by a human glioblastoma cell line, also has significant similarities to CRISP-3 and has been shown to be a weak inhibitor of the serine protease, trypsin [³⁶ ]. However, except for the high content of cysteines and the resulting compact structure, neither P25TI nor CRISP-3 has any similarities to known protease inhibitors (e.g., tissue inhibitors of metalloproteases or cystatins) [³⁷ , ³⁸ ]. A variety of possible functions are thus open for investigation.

	ACKNOWLEDGEMENTS

 
This work was supported by The Danish Medical Research Council and The John and Birthe Meyer Foundation. The expert technical assistance of Charlotte Horn is greatly appreciated. We thank Hans Janssen and Nico Ong for their expert technical assistance with electron microscopy.

Received February 12, 2002; revised April 22, 2002; accepted April 24, 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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