Subcellular Distribution and Cytokine- and Chemokine-regulated Secretion of Leukolysin/MT6-MMP/MMP-25 in Neutrophils*

Tiebang KangDagger , Jun YiDagger , Athena Guo§, Xing WangDagger , Christopher M. Overall, Weiping Jiang||, Robert Elde§, Niels Borregaard**, and Duanqing PeiDagger DaggerDagger

From the Departments of Dagger  Pharmacology and § Neurosciences, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455, the  Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, || R&D Systems, Minneapolis, Minnesota 55413, and the ** Department of Hematology, The Finsen Center, The National University Hospital, Copenhagen, Denmark

Received for publication, August 31, 2000, and in revised form, March 22, 2001


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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Leukolysin, originally isolated from human leukocytes, is the sixth member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily with a potential glycosylphosphatidylinositol (GPI) anchor. To understand its biological functions, we screened subpopulations of leukocytes and localized the expression of leukolysin at the mRNA level to neutrophils. Polyclonal and mono-specific antisera raised against a synthetic peptide from its hinge region recognized a major protein species at 56 kDa and several minor forms between 38 and 45 kDa in neutrophil lysates. In resting neutrophils, leukolysin is distributed among specific granules (~10%), gelatinase granules (~40%), secretory vesicles (~30%), and the plasma membrane (~20%), a pattern distinct from that of neutrophil MMP-8 and MMP-9. Consistent with its membrane localization and its reported GPI anchor, leukolysin partitions into the detergent phase of Triton X-114 and can be released from intact resting neutrophils by glycosylphosphatidylinositol-specific phospholipase C. Phorbol myristate acetate stimulates neutrophils to discharge 100% of leukolysin from specific and gelatinase granules and ~50% from the secretory vesicles and plasma membrane, suggesting that leukolysin can be mobilized by physiological signals to the extracellular milieu as a soluble enzyme. Indeed, interleukin 8, a neutrophil chemoattractant, triggered a release of ~85% of cellular leukolysins by a process resistant to a mixture of proteinase inhibitors, including aprotinin, BB-94, pepstatin, and E64. Finally, purified recombinant leukolysin can degrade components of the extracellular matrix. These results not only establish leukolysin as the first neutrophil-specific MT-MMP but also implicate it as a cytokine/chemokine-regulated effector during innate immune responses or tissue injury.


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INTRODUCTION
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Neutrophils (polymorphonuclear leukocytes, or PMNs1) are the first cells to arrive at the site of infection where they play a critical role in host defense against invading microbes (1). In the process of resolving ongoing infection, they deploy a powerful arsenal of molecules to destroy the infectious agent as well as the infected cells and tissues, thus, resulting in host tissue damage (1, 2). Among proteolytic enzymes identified in the PMNs, matrix metalloproteinases (MMPs) have attracted considerable attention for their proposed role in the destruction of extracellular matrix under physiological and pathological conditions (1, 2). The first MMP identified in PMNs is MMP-8 or neutrophil collagenase that is stored in the specific granules and is capable of cleaving type I collagen into typical one-fourth and three-fourths fragments (3-6). MMP-9, originally detected in alveolar macrophages, is also known as the gelatinolytic activity discharged by PMNs (7-9). Both MMPs have been detected in many other cell types and implicated in diseases such as arthritis and tumor invasion and metastasis (5, 7, 10-12). Furthermore, MMP-9-deficient mice appear to produce fully functional neutrophils but exhibit deficiencies in angiogenesis during endochondral ossification (13, 14). Recently, Liu and colleagues (15) demonstrated that MMP-9-deficient mice are resistant to blister formation due to a deficiency in neutrophil-mediated inactivation of alpha 1-proteinase inhibitor (alpha 1-PI) in a murine bullous pemphigoid model. Given their destructive nature, neutrophils may express additional proteinases, including those from the MMP family.

We recently isolated a novel MMP from peripheral blood leukocytes by cDNA cloning (16). Named leukolysin for its specific expression in leukocytes, this novel MMP is the sixth member of the membrane-type MMP subfamily (MT6-MMP) and has a serial designation of MMP-25 (16). Interestingly, the same gene was isolated by Velasco and colleagues (17) and shown to be expressed in brain tumors, echoing a theme in MMP biology that a given MMP may be expressed under defined physiological conditions but dysregulated transcriptionally in tissues undergoing carcinogenic transformations (2, 18-20). To seek clues to its biological function, we defined the leukocyte subpopulations that express leukolysin and characterized its protein products under normal physiological conditions. We report here that PMNs are the primary source of leukolysin at the mRNA level, and its protein products are segregated in the membranes of various granules, vesicles, and cell surfaces apparently via a GPI anchor yet released to the extracellular milieu upon cytokine and chemokine stimulation.

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Chemicals, Cells, Cell Culture, and Immunological Reagents-- General laboratory chemicals and proteinase inhibitors were from Sigma Chemical Co. (St. Louis, MO). Cell culture reagents and fetal bovine sera were from Life Technologies (Rockville, MD). Cell lines, including COS, MDCK, and MDCK derivatives, were obtained and maintained as described previously (16, 21). M2 anti-FLAG monoclonal antibody was purchased from Sigma Chemical Co. Anti-leukolysin antisera were raised in guinea pig against synthetic peptides as shown in Fig. 2 as described previously (16). IL-1alpha , IL-1beta , IL-8, and anti-MMP-9 antibody were from R&D Systems (Minneapolis, MN).

Analysis of MMP25/Leukolysin Expression-- Monocytes, lymphocytes, and PMNs were obtained from R&D Systems with ~98% purity. Total RNA was isolated from these cells with TRI reagent as suggested by the manufacturer (MRC, Cincinnati, OH). For RT-PCR analysis, 2 µg of total RNAs were reverse-transcribed with Superscript II (Life Technologies, MD) in 20-µl reactions. PCR reactions were carried out with 2 µl of RT reactions for both leukolysin and the internal reference glyceradehyde-3-phosphate dehydrogenase as described previously (16). For Northern analysis, total RNA (20 µg) from PMNs was denatured with glyoxal and Me2SO, fractionated on a 1% agarose gel in 10 mM phosphate buffer (55 V for 5 h), and then transferred to a nylon membrane overnight. The membrane was prehybridized at room temperature for 30 min, hybridized at 62 °C for 16-24 h with 32P-labeled leukolysin cDNA (~700 bp), washed, exposed to an ABI screen, and scanned on a imager (ABI).

Characterization of Anti-leukolysin Antisera against Recombinant MMPs-- pCR3.1-MT6, pREP9MT1(1-528), and pCR3.1MT5(1-539)F were described previously (16, 22, 23). pCR3.1MT6(1-509)F was constructed by isolating the cDNA fragment coding 1-509 residues of leukolysin with high fidelity PCR and inserting it into the EcoRV site of pCR3.1. pCR3.1MT3(1-556)F, containing the coding region for residues 1-556 with a FLAG tag at its C terminus, was constructed by isolating the corresponding fragment by PCR and inserting it into a pCR3.1 vector as described above. pCR3.1ST3/MT4(129-525)F, harboring a hybrid molecule with the pro-domain of MMP-11 followed by the catalytic, hinge, and pexin domains of MT4-MMP, was obtained by inserting a chimera DNA fragment generated through sequential PCR to combine the pro-domain of MMP-11 and the mature portion of MT4-MMP as described (24). pCR3.1ST3/CA-MMP(80-391)F, also a chimera between the pro-domain of MMP-11 and a fragment from CA-MMP containing predicted matured protein with residues 80-391, was constructed by combining the first 80 residues of MMP-11 and a fragment of CA-MMP coding for 80-391 residues. All recombinant constructs were confirmed by sequencing double-stranded DNAs with nested primers. DNA transfection was carried out with LipofectAMINE (Life Technologies) as described (16). Stable lines were derived from MDCK cells with G418 (400 µg/ml) selection and screened by Western blotting as described (25). The recombinant proteins were extracted in 1% Triton X-100 (in PBS). Western blot analyses were carried out as described using either anti-FLAG antibody M2 or anti-MT1-MMP and G280e (16, 22). The specificity of antisera was confirmed by pre-absorbing them with the corresponding peptide used for immunization (1 µg/ml).

Detection of Leukolysin in PMN Lysates-- PMN cells (107) were extracted with 1 ml of 1% Triton X-100 in PBS (phosphate-buffered saline) supplemented with a mixture of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µM BB-94). Cell debris was removed by centrifugation at 15,000 × g for 15 min at 4 °C, and the resulting extracts (10 µl/lane, ~105 cells) were analyzed by SDS-PAGE and Western blotting.

Triton X-114 Phase Partitioning-- This procedure was carried out essentially as described previously (26). Briefly, a stock solution of Triton X-114 was prepared by mixing a 3% (v/v) aqueous solution at 4 °C to dissolve the detergent and then incubated overnight at 37 °C to induce phase transition. The upper aqueous phase was discarded, and the lower detergent phase was mixed with 0.1 volume of 10× TNE (0.5 M Tris, pH 7.5, 1.5 M NaCl, 50 mM EDTA) to create a Triton X-114 stock. Neutrophils (4 × 106) were washed with cold PBS (3×), resuspended in 200 µl of 1× TNE containing 0.25 volume of Triton X-114 stock and extracted at 4 °C for 10 min before being centrifuged at 3000 × g for 10 min at 4 °C to remove any insoluble aggregates (pellets, Fig. 4). The supernatant (lysate) was transferred to a new microcentrifuge tube, which was heated at 37 °C for 10 min to induce phase transition, then centrifuged at 3000 × g for 10 min to collect the detergent-protein micelles (detergent phase, Fig. 4). The upper aqueous phase was re-extracted three more times with 0.25 volume of Triton X-114 stock as described above. The collected detergent-protein micelles were pooled together. All samples were then precipitated by adding 10× volumes of methanol, incubating overnight at -20 °C, and then centrifuging at 3000 × g for 30 min at 4 °C. The resulting samples were resuspended in 1× SDS-PAGE buffer and analyzed by Western blotting as described in the previous sections.

Subcellular Fractionations of PMNs-- PMNs (4.5 × 108) isolated from peripheral blood were resuspended in Krebs-Ringer phosphate (KRP) buffer (130 mM NaCl, 5 mM KCl, 1.27 mM MgCl2, 0.95 mM CaCl2, 10 mM NaH2PO4/Na2HPO4, pH 7.4) supplemented with 5 mM glucose at 3 × 107 cells/ml and incubated with dilsopropyl fluorophosphate (5 mM) for 15 min. Cells were pelleted by centrifugation and resuspended in 14 ml of relaxation buffer and cavitated (27). 10 ml of the post-nuclear supernatant (S1) was put on a 3-layer Percoll gradient (generated in relaxation buffer containing 1 mM phenylmethylsulfonyl fluoride) and centrifuged (28). Samples were collected in fractions of 1 ml. Fractions were pooled as alpha -band (fractions 1-6), beta 1-band (fractions 7-12), beta 2-band (fractions 13-18), and gamma -band (fractions 19-24) (28). These materials were centrifuged to remove Percoll, and the pellets were resuspended in 1000 µl of PBS. Markers for various granules were assayed as quality control for the fractionation process according to a previous study (28). For SDS-PAGE and Western blotting, the samples were boiled in 1× Laemmli sample buffer and stored frozen until assays were performed. Free-flow electrophoresis was carried out to separate the gamma -band into plasma membranes and secretory vesicles as described (29). The markers for plasma membranes and secretory vesicles were HLA and latent alkaline phosphatase, respectively, and these were assayed as described (29). For PMA (phorbol myristate acetate) stimulations, freshly isolated neutrophils were resuspended in KRP + 5 mM glucose at 3 × 107 cells/ml and divided into two equal portions (11 ml each). One was kept on ice as a control; the other was stimulated at 37 °C for 15 min with PMA (2 µg/ml). The stimulation was stopped by adding 2 volumes of ice-cold buffer (KRP + glucose). Both the resting as well as the activated portions were pelleted by centrifugation, and the resulting supernatants were saved. The pellets from both control and stimulated cells were resuspended in 11 ml of buffer and fractionated (see above). The supernatants and fractions were assayed for markers and leukolysin as described above.

Glycosylphosphatidylinositol Phospholipase-C Treatment of Neutrophils-- Resting neutrophils (~2 × 107/ml) were incubated with GPI-PLC (0.5 unit) in a 100-µl volume for 2 h at 37 °C as suggested by the supplier (Glykomed, Oxford, UK) (30). The supernatants were analyzed by Western blotting as described above.

Treatment of Neutrophils with IL-1alpha , IL-1beta , and IL-8-- Freshly isolated neutrophils were divided into equal portions (~4 × 106 in 100 µl) and treated with cytokines IL-1alpha (100 ng/ml) and IL-1beta (100 ng/ml) and chemokine IL-8 (100 ng/ml) for 15 min at 37 °C. The cells were then separated from supernatants by centrifugation (5000 rpm, 5 min) before being lysed with equal volume (100 µl) of 1% Triton X-100 in PBS (see above). Both the supernatants and cell lysates were analyzed for leukolysin and MMP-9 by Western blotting as described above. For analyzing the effects of proteinase inhibitors, a mixture of E64 (10 µg/ml), pepstatin A (10 µg/ml), aprotinin (100 µg/ml), and BB-94 (5 µM) was added to the cell aliquots before being stimulated with IL-8 as described above.

Immunostaining of Leukolysin-- PMNs were attached to coverslips for 5 min and then fixed in 4% paraformaldehyde solution with or without X-100. The coverslips were blocked with 1% normal donkey serum, then stained with G280e followed by fluorescein isothiocyanate-labeled goat anti-guinea pig antibody (1 h each, Jackson Laboratories, Bar Harbor, ME). The slides were then scanned on a Bio-Rad confocal system at the University of Minnesota Bioimaging Laboratory.

Purification and Characterization of Recombinant Leukolysin-- The conditioned media were collected from MDCK cells stably expressing leukolysin-(1-280) (16). Recombinant leukolysin was purified by affinity chromatography carried out essentially as described (25). Briefly, the conditioned media were loaded onto an anti-FLAG M2 antibody column (1 × 1 cm). After washing, the bound materials were eluted by antigen competition using a 2 molar excess of FLAG peptide (Sigma). Fractions were then analyzed by zymography, Western blotting, and SDS-PAGE-stained with Coomassie Brilliant Blue R-250 as reported previously (25). The purified materials were transferred to PVDF membrane and sequenced for 5 cycles as described (22). To assay its activity in solution, various ECM components were incubated with leukolysin in the activity buffer (150 mM NaCl, 50 mM Tris HCl, 5 mM CaCl2, 1 µM ZnCl2, 0.1% Brij 35) for 8-24 h at 37 °C with or without MMP inhibitors (22). The reaction mixtures were analyzed by SDS-PAGE as described (22).

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INTRODUCTION
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RESULTS
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Leukolysin Is PMN-specific-- Previously, we determined that leukolysin is expressed by pooled peripheral blood leukocytes among 26 different human tissues and cell types tested (16), in contrast to the subsequent description of apparently the same gene being expressed by brain tumor cells (17). In this report, we chose to focus on defining the leukocyte populations that express leukolysin. Toward this end, we separated leukocytes into three main categories of cells, namely lymphocytes, monocytes, and polymorphonuclear leukocytes (PMNs) and extracted total RNAs for RT-PCR analysis (16). As shown in Fig. 1A, PMNs are the only cells positive for leukolysin, whereas both lymphocytes and monocytes, known to share MMP profiles with the PMNs, are virtually negative, even with 35 cycles of amplification (lanes 3 and 5 versus lane 4). To confirm its presence as mRNA, we performed Northern blot analysis and demonstrated that leukolysin is expressed as a single ~4.0-kilonucleotides mRNA species (Fig. 1B). This mRNA is detectable in as little as 5 µg of total PMN RNA with a 3-h exposure (data not shown). Based on these experiments, we concluded that PMNs are the primary source of leukolysin in peripheral blood.


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Fig. 1.   Leukolysin/MT6-MMP/MMP-25 in PMNs. A, RT-PCR analysis of leukolysin (MMP-25) among subpopulations of leukocytes. Total RNAs (5 µg) isolated from lymphocytes (lymph, lanes 3), PMNs (lane 4), and monocytes (mono, lane 5) were reverse-transcribed and PCR-amplified as described previously (16) for leukolysin (35 cycles, upper panel; ~550 bp) or the internal reference gene glyceradehyde-3-phosphate dehydrogenase (25 cycles, lower panel; ~1 kb). Lane 1 is the molecular weight standard, and lane 2 is a control without any RNA added. B, Northern blot. Total RNA (20 µg) from PMNs was fractionated on a 1.0% agarose gel, blotted onto Nylon membrane, and probed with 32P-labeled leukolysin-specific probe for 16 h. After washing, the filter was exposed to a phosphorimaging screen and scanned on an ABI imager. Note that the size of leukolysin mRNA is ~4 kb, estimated from the sizes of 18 S and 28 S rRNA shown on the left.

Generation of Polyclonal and Mono-specific Antisera against Leukolysin-- PMNs mediate innate immunity and tissue damage by releasing various enzymes and anti-microbial polypeptides from their intracellular granules (1). Thus, we hypothesized that leukolysin is a part of the proteolytic arsenal stored in intracellular granules. To test this, we developed antisera against leukolysin that can detect its protein products. First, two peptides, RYALSGSVWKKRTLT (Arg107) and GKAPQTDYDKPTRKPLA (Gly280), were designed. The Arg107 peptide is from the beginning of the catalytic domain and we should be able to generate antibodies specific for the processed and active leukolysin with a predicted N terminus at Tyr108. The second peptide, Gly280, is derived from the hinge region of leukolysin with minimal homology to all other MMPs and may be used to generate a leukolysin-specific antibody. These two peptides were synthesized with an extra cysteine residue at each N terminus for conjugation to keyhole limpets hemocyanin (Pierce, IL). The immunogenic peptides were injected into a total of five guinea pigs (animals A to E), and the immune responses were monitored by collecting blood samples for testing against recombinant leukolysin protein expressed in stable MDCK cell lines (see "Materials and Methods"). As shown in Fig. 2B, Gly280 was able to generate immunoreactive antibody in all three guinea pigs injected (arrows, lanes 4, 8, and 12), whereas Arg107 was less immunogenic (data not shown). The antisera from these animals detected leukolysin, albeit with different specificity and titers. Sera from animals C and D were not suitable due to the nonspecific bands detected from MDCK lysates that were not blocked by the immunizing peptides (Fig. 2B, lanes 3, 4 versus 1, 2, and lanes 7, 8 versus 5, 6). However, the sera from animal E appeared to be specific for leukolysin and did not react with any of the MDCK proteins (Fig. 2, lanes 9-12). Designated G280e, its reactivity toward leukolysin (MT6) can be blocked by the immunizing peptides (Fig. 2, lanes 12 versus 10), a desirable property for the detection of leukolysin protein products in PMN lysates.


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Fig. 2.   Generation of anti-leukolysin antisera. A, schematic presentation of leukolysin domain structure and the regions from which two peptides were synthesized as indicated. S, signal peptide; Pro, pro-domain; R, furin cleavage site; CAT, catalytic domain; H, hinge region; Pexin, hemopexin-like domain; G, putative GPI anchor. B, screening for anti-leukolysin antisera. Sera samples from three different guinea pigs from C to E were prepared and used for immunoblotting against cell lysates from MDCK (lanes 1, 3, 5, 7, 9, 11) or MDCK transfected with a full-length leukolysin expression construct, pCR3.1MT6 (16) (lanes 2, 4, 6, 8, 10, 12) either alone (lanes 3, 4, 7, 8, 11, 12) or pre-absorbed with the immunizing peptides (lanes 1, 2, 5, 6, 9, 10). The major leukolysin species are marked with arrows. C, specificity of anti-leukolysin antisera. MDCK cells transfected with either control vector (lanes 1, 8) or MT1-MMP (lanes 2, 9), MT3-MMP (lanes 3, 10), MT4-MMP (lanes 4, 11), MT5-MMP (lanes 5, 12), CA-MMP (lanes 6, 13), or MT6-MMP (lanes 7, 14) were extracted with 1% Triton in PBS and analyzed by Western blot analyses with anti-FLAG antibody (lanes 1, 3, 4, 5, 6, 7) or anti-MT1 antibody (lane 2), or anti-leukolysin antisera (lanes 8-14). The immunoreactive species were detected using alkaline phosphatase-conjugated secondary antibody as described (25). The asterisk indicates that anti-MT1-MMP antibody was used for MT1 protein in lane 2. The leukolysin product is indicated by an arrow.

Because MMPs, especially MT-MMPs, are highly homologous at the amino acid level, antisera generated for a given member may potentially cross-react with other members. Thus, G280e may potentially react with other MT-MMPs with similar hinge regions, especially MT4-MMP, which shares 56% amino acid identity with leukolysin (16). To test this possibility, we produced recombinant proteins in MDCK cells for MT1-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and CA-MMP and tested their reactivity toward G280e (see "Materials and Methods"). As shown in Fig. 2C, G280e can only detect recombinant leukolysin (lane 14). MT4-MMP, a MMP with the highest homology to leukolysin, did not react with G280e (Fig. 2C, lane 11). Furthermore, G280e does not cross-react with recombinant MMP-8 or MMP-9, two MMPs found in neutrophils, under identical conditions (data not shown). Therefore, we conclude that the antisera G280e is mono-specific for leukolysin.

Detection of Leukolysin Products in Neutrophils-- Given the specificity G280e exhibited in Fig. 2, it may identify leukolysin products from its natural source, i.e. PMNs. In a preliminary experiment, different numbers of PMNs ranging from 9 × 103, 9 × 104, 3 × 105, to 9 × 105 were lysed in SDS sample buffer and analyzed directly by Western blotting. Clear signals were observed in as few as 9 × 104 cells per lane (data not shown). Given recent reports that leukolysin may be a GPI-anchored protein (31), we extracted freshly isolated PMNs with 1% Triton X-100 in PBS (with proteinase inhibitors) for 30 min to harvest all cellular proteins, including those from membranes. The total proteome from ~105 PMNs was fractionated by SDS-PAGE and detected with Coomassie Brilliant Blue R-250 staining (Fig. 3A, lane 1). The same amount of proteins was transferred to PVDF membrane and probed with G280e. A major 56-kDa species and a few smaller ones were detected (Fig. 3A, lane 2). When G280e was preincubated with the immunizing peptide, the signals were completely blocked as expected (Fig. 3A, lane 3). It is concluded that neutrophils produce leukolysin protein products with the 56-kDa species as the full-length molecule and the smaller ones as processed products from the full-length species similar to those reported for MMP-11 (32).


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Fig. 3.   Identification and characterization of leukolysin in neutrophil lysates. A, neutrophils (105 cells/lane) were lysed in 1% Triton X-100 (PBS) and analyzed by SDS-PAGE (lane 1), and Western blotting without (lanes 2) or with (lane 3) the blocking peptide. The arrow indicates the full-length leukolysin and the arrowheads depict its degraded products. B, Triton X-114 partitioning of leukolysin products. Neutrophils were lysed in Triton X-114 lysis buffer as described under "Materials and Methods." The residual cell debris (lane 1), the detergent (det., lane 2) and the aqueous (aq., lane 3) phases were analyzed by Western blotting as described in A.

Partitioning of Leukolysin into X-114 Detergent Phase-- Based on its sequence homology to MT4-MMP, leukolysin/MT6-MMP appears to be a GPI-anchored protein (16, 30). Indeed, Kojima and colleagues (31) have reported that recombinant MT6-MMP is anchored on cell membrane-like MT4-MMP in transfected cells. Should leukolysin/MT6-MMP be a GPI-anchored protein in neutrophils, it would partition into the detergent phase as demonstrated for many other GPI-anchored proteins (26, 33). Indeed, when leukolysin extracted in Triton X-114 was allowed to partition at 37 °C, it exclusively segregated into the detergent phase as expected (Fig. 3B, lane 2), arguing that it does contain a hydrophobic moiety presumably provided by the GPI anchor (also see below, Fig. 5D).

Distribution of Leukolysin in PMN Granules-- One of the defining features for the PMNs is a series of intracellular granules formed during maturation that store various enzyme systems and anti-microbial agents important for destroying microbial cells and destroying infected tissues (34, 35). MMP-8, the first MMP discovered in neutrophils, is stored in the specific granules (4), whereas MMP-9 is present largely in the gelatinase granules (35). To define the subcellular distribution of leukolysin, PMNs were fractionated into four distinct fractions on a three-layer Percoll gradient, namely alpha , beta 1, beta 2, and gamma , potentially representing the azurophil, specific, gelatinase granules, the secretory vesicles, and plasma membranes (28). Indeed, both the classification and fractionation process were validated by assaying several known markers established for these granules and vesicles: myeloperoxidase (MPO) for azurophil granules (Fig. 4A); lactoferrin and NGAL (neutrophil gelatinase-associated lipocalin) for specific granules (Fig. 4, B and C); MMP-9 for gelatinase granules (Fig. 4D); and human serum albumin for secretory vesicles and HLA (human leukocyte antigen) for plasma membranes (Fig. 4, E and F) (27, 28, 35). The distribution of leukolysin among these fractions was subsequently determined by Western blot analysis (panel I) and quantified by densitometry (panel H) (Fig. 4). S1 represents the postnuclear fractions prior to being loaded onto the Percoll gradient, thus, the total leukolysin proteins (Fig. 4I, lanes 1 and 6). Upon fractionation, it became apparent that the bulk of leukolysin appears to be in gelatinase granules, secretory vesicles, and plasma membrane (Fig. 4I, lanes 9 and 10). A substantial amount of leukolysin is also present in the specific granules where MMP-8 is localized (Fig. 4I, lane 8), whereas the azurophil granules are negative (lane 7). The fractionation process preserved the migratory patterns for leukolysin (Fig. 4I, lanes 6 versus 8, 9, and 10), and the signals were specific as judged by the complete blockade with the immunizing peptide on Western blots (Fig. 4I, lanes 1-5 versus 6-10). The broader distribution than those of MMP-8 and MMP-9 argues strongly that leukolysin may play different roles from these two previously identified neutrophil MMPs.


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Fig. 4.   Subcellular localization of leukolysin in PMNs. Resting PMNs were treated with proteinase inhibitors, disrupted, and fractionated on a Percoll gradient as described under "Materials and Methods" into four fractions alpha , beta 1, beta 2, and gamma . Each fraction was assayed for markers in the azurophil granules (myeloperoxidase, MPO; panel A), specific granules (neutrophil gelatinase-associated lipocalin, NGAL; and lactoferrin, B and C), gelatinase granules (MMP-9, D), secretory vesicles and plasma membranes (human serum albumin, HSA; human leukocyte antigen, HLA; E and F) and total protein contents (G). H, quantification of leukolysins detected in panel I and expressed as percentages of leukolysin in each band over that of the whole cells. I, detection of leukolysin in subcellular fractions. Equivalent proportions of proteins from the postnuclear supernatants (S1, lanes 1, 6) as well as the fractionated bands, alpha  (lanes 2 and 7), beta 1 (lanes 3 and 8), beta 2 (lanes 4 and 9), and gamma  (lanes 5 and 10) were loaded onto SDS-PAGE, separated by electrophoresis, transblotted onto PVDF membrane, and developed with G280e alone (lanes 6-10) or G280e preincubated with the immunizing peptide (lanes 1-5). Note the complete blockade of anti-leukolysin antibody with the immunizing peptide (lanes 1-5 versus 6-10).

Leukolysin Is Present on Plasma Membrane-- The unexpected identification of leukolysin in the gamma  band (composed of secretory vesicles and plasma membrane) raises the possibility that it may be on the plasma membrane and displayed on the cell surface, a novel location for neutrophil MMPs. To confirm this possibility, we took advantage of a technique, free-flow electrophoresis, which can separate plasma membrane from secretory vesicles (29). As shown in Fig. 5A, pure plasma membranes were obtained by separating and removing the secretory vesicles as confirmed by the absence of latent alkaline phosphatase, a marker for secretory vesicles (Fig. 5A, middle column). Western blot analysis of these fractions confirmed the presence of leukolysin in plasma membrane (Fig. 5B, lane 1), representing ~36% of the leukolysin in gamma  band (Fig. 5C, left column). Considering that the amount of protein in the plasma membrane fraction is considerably less than that of the secretory vesicles (Fig. 5A, right column), it is possible that the relative level of leukolysin is similar in both secretory vesicles and the plasma membrane. Indeed, when normalized against protein levels, leukolysin appears to be slightly more concentrated on plasma membrane than in the secretory vesicles (Fig. 5C, right column). Throughout the fractionation process, all the fractions were kept proportional to the total number of cells analyzed (per cell). Thus, we can estimate the distribution profile of leukolysin in resting PMNs as follows: azurophil granules (0%), specific granules (~10%), gelatinase granules (~40%), secretory vesicles (~30%), and plasma membranes (~20%). This pattern suggests that leukolysin overlaps significantly with MMP-9 in the gelatinase granules, but has a dramatically broader distribution than both MMP-8 and MMP-9 by extending to the secretory vesicles and the plasma membrane (~50%). One surprising feature is that nearly 20% of leukolysin molecules appear to be displayed as surface molecules in resting neutrophils (Fig. 5C). To confirm that leukolysin is anchored on the neutrophil surface via a putative GPI-anchored protein, as suggested by its partition into the detergent phase of Triton X-114 (Fig. 3B), we treated intact neutrophils with GPI-PLC. As expected, leukolysin was released into supernatants by GPI-PLC (Fig. 5D, lane 2). Based on signals on the Western blots, we estimated that GPI-PLC released ~5% of the total cellular leukolysin, which is about 25% of the cell surface-associated leukolysin. Surprisingly, the smaller fragments of <40 kDa escaped GPI-PLC-mediated release. To further characterize the localization of leukolysin in PMNs, we performed immunofluorescent stainings of PMNs with or without permeabilization with Triton X-100. As shown in Fig. 5E, leukolysins were detected primarily in intracellular granules when PMNs were permeabilized (panel a), but clearly detectable on PMN surface without permeabilization (panel b). Together, these data establish leukolysin as a membrane-associated MMP on the neutrophil cell surface.


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Fig. 5.   Leukolysin on plasma membrane. A, separation of secretory vesicles from plasma membranes. The gamma  band was further fractionated into secretory vesicles (filled bars) and plasma membrane (open bars) by free-flow electrophoresis, and analyzed for HLA (left), latent alkaline phosphatase (LAP, middle) and total protein (right). Note that the plasma fraction is free of latent AP, a marker for the secretory vesicles. HLA should be in both the plasma membrane and secretory vesicle as a membrane protein. PM, plasma membrane; SV, secretory vesicles. B and C, equal volumes (20 µl) from the PM (lane 1) and SV (lane 2) were analyzed by Western blot for leukolysin as described in Fig. 2. The blot in B was quantified by densitometry and presented in C as percentages of total leukolysin in gamma  band without (left column) or with (right column) normalization against the amount of proteins in each fraction shown in A. D, release of leukolysin by GPI-PLC from resting PMNs. Resting PMNs were divided into two equal aliquots (~2 million each in 100 µl of PBS) and treated with either buffer alone (lanes 1, 3) or 0.5 unit of GPI-PLC (lanes 2, 4) at 37 °C for 2 h. The reactions were terminated by pelleting down the cells. The supernatants were analyzed by Western blotting without (lanes 1, 2) or with blocking with the immunizing peptide (lanes 3 and 4). Note leukolysin as a main species at ~55 kDa and minor 50 kDa species indicated by the arrow and arrowhead, respectively. E, immunofluorescent detection of leukolysin on the cell surface. Neutrophils were allowed to attach to glass coverslips for 5 min and then fixed in 4% paraformaldehyde in the presence (a) or absence (b) of 1% Triton X-100. Cells were stained with G280e and fluorescein isothiocyanate-conjugated Goat anti-guinea pig antibody. The images were obtained by confocal microscopy to show one optical plane.

Discharge of Leukolysin by Stimulated Neutrophils-- In response to infection, PMNs are activated to release the contents of their intracellular granules for host defense (1, 2, 35). To implicate leukolysin as part of the released armament, we monitored its distribution in neutrophils treated with PMA for 15 min by subcellular fractionations. As shown in Fig. 6, PMA triggered the expected degranulation as indicated by the discharge of markers from azurophil granules, specific granules, gelatinase granules, and secretory vesicles (panels A to G). Specifically, close to 50% of MPO was discharged from azurophil granules (Fig. 6A). Both NGAL and lactoferrin in the specific granules were discharged by more than 80% (Fig. 6, B and C). Similarly, MMP-9 and albumin were released by PMA stimulation (Fig. 6, D and E). However, HLA, a marker for plasma membrane, was decreased only slightly due to PMA stimulation (Fig. 6F). Consistently, almost all leukolysins in specific and gelatinase granules were discharged (Fig. 6, panel H, lanes beta 1 and beta 2; panel I, lanes 4 versus 3, 6 versus 5). As a GPI-anchored protein (Fig. 5) (31), leukolysin should be translocated onto the cell surface because of the fusion of granules and vesicles with the plasma membranes. Surprisingly, the expected accumulation of leukolysin on the plasma membrane did not occur. On the contrary, the gamma  band, made of secretory vesicles and plasma membrane, from stimulated neutrophils actually lost ~50% of leukolysin compared with resting neutrophils (Fig. 6, panel H, lane gamma ; panel I, lanes 8 versus 7). Together, a total of ~75% of cellular leukolysin proteins was discharged in response to PMN treatment. Indeed, when the supernatants were examined, leukolysin products were detected and accounted for most of the discharged leukolysin from the granules (Fig. 6I, lane 10). These data suggest that neutrophils shed leukolysin from its membrane in response to stimulation.


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Fig. 6.   PMA-stimulated release of leukolysin by PMNs. Equal numbers of PMNs were either treated with KRP alone (resting, open bars) or with PMA for 15 min (activated, filled bars). Both samples were then fractionated and analyzed as described in Fig. 5. Panels A to G were measurements for various markers, MPO (A), NGAL (B), lactoferrin (C), MMP-9 (D), albumin (E), HLA (F), and total proteins (G) for the fractionation process as described under "Materials and Methods" (28). Panel H shows the distributions of leukolysin as quantified from gels in panel I. I, subcellular distribution of leukolysin in activated PMNs. The fractions, alpha  (lanes 1 and 2), beta 1 (lanes 3, 4), beta 2 (lanes 5, 6), gamma  (lanes 7, 8), and supernatants (lanes 9, 10) from both resting (lanes 1, 3, 5, 7) or activated (lanes 2, 4, 6, 8) PMNs were analyzed for leukolysin by Western blot as described in Fig. 5. The arrows indicate the major leukolysin species; the arrowheads indicate those for degraded products.

Cytokine- and Chemokine-regulated Release of Leukolysin from Neutrophils-- The observed release of soluble leukolysin from PMA-stimulated neutrophils would argue that there must be regulators capable of mediating a similar function under physiological conditions. A wide range of cytokines and chemokines has been implicated in neutrophil functions by regulating activities such as degranulation and transendothelial and transepithelial migrations (1, 36). To further explore the physiological significance of leukolysin expression and release, neutrophils were treated with inflammatory cytokines and chemokines such as IL-1alpha and beta ; IL-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -15, -17, -18; and tumor necrosis factor alpha . Surprisingly, most of these factors appeared to have stimulated the release of leukolysin from neutrophils, albeit with different efficiency (data not shown). In general, two categories of stimulation emerged as represented by cytokines such as IL-1alpha and beta , and chemokine IL-8. As shown in Fig. 7, IL-1alpha and beta  triggered the release of MMP-9 (panel A, lanes 6, 7) and that of leukolysin with similar efficiency (panel B, lane 6, 7). IL-8, the main chemokine for neutrophil chemotaxis in vivo, however, triggered the most robust release of leukolysin and MMP-9 (Fig. 7, A and B, lanes 8). Interestingly, a new leukolysin species migrating ~5 kDa faster than the 56-kDa form was detected for the first time in the IL-8-treated supernatants (Fig. 7B, lane 8, asterisk), suggesting that it may be a processed product as observed for MMP-11 (32).


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Fig. 7.   Cytokine- and chemokine-stimulated secretion of leukolysin from PMNs. A and B, PMNs (4 × 106 cells in 100 µl) were incubated either alone (lanes 1, 5) or with IL-1alpha (100 ng/ml; lanes 2, 7), IL-1beta (100 ng/ml; lanes 3, 7) and IL-8 (100 ng/ml; lanes 4, 8) at 37 °C for 15 min. The cells were then lysed in 1% Triton X-100 in PBS (100 µl). The supernatants (lanes 5-8) and lysates (lanes 1-4) were analyzed for MMP-9 (panel A) or leukolysin (panel B) by Western blotting using anti-MMP-9 or anti-leukolysin antibodies. The arrows indicate the full-length molecules for MMP-9 and leukolysin. The asterisk depicts the novel processed species for leukolysin. C, PMNs (4 × 106 cells in 100 µl) were incubated without (lanes 1, 2, 5, 6) or with (lanes 3, 4, 7, 8) IL-8 (100 ng/ml), in the absence (lanes 1, 3, 5, 7) or presence (lanes 2, 4, 6, 8) of proteinase inhibitors (see "Materials and Methods") at 37 °C for 15 min. The cell lysates and supernatants were analyzed as in A and B.

The 56-kDa leukolysin species detected in the supernatants of PMN cells treated with both IL-1alpha /beta and IL8 is almost indistinguishable from the one in their lysates (Fig. 7B), suggesting that the secreted or shed species is a result of cleavage at the extreme end of its C terminus (i.e. with minimal loss of molecular mass). The apparent conversion of the 56-kDa species into a 51-kDa one in IL-8-treated supernatants (Fig. 7B, lane 8) suggests that further proteolytic cleavage might have occurred after shedding. To identify the potential proteinases responsible for the observed shedding and processing, we stimulated PMNs in the presence of proteinase inhibitors against the serine-, cysteine-, aspartyl-, and metalloproteinases as described under "Materials and Methods." As shown in Fig. 7C, these proteinase inhibitors failed to inhibit the shedding as well as processing, suggesting that these two processes may be mediated by novel mechanisms independent of proteolysis or novel proteinases resistant to the inhibitors applied. Thus, cytokines and chemokines may differ not only quantitatively but also qualitatively in mobilizing leukolysin from neutrophils, presumably under different physiological conditions.

Recombinant Leukolysin Is Processed at the RRRR Site into a Fully Active Enzyme with ECM-degrading Activities-- Although PMNs release leukolysin efficiently in response to chemokines, we failed to purify enough enzyme for enzymatic analysis and N-terminal sequencing. We estimated, based on signals on Western blots (Fig. 3), that 105 PMNs express ~20 ng of leukolysin protein, an equivalent of ~4 × 105 molecules/neutrophil. 1 liter of whole blood should be sufficient to provide enough PMNs for 200 µg of leukolysin, which is sufficient for microscale purification. However, leukolysin in the exudates, known to contain destructive agents such oxidants and proteinases (1), has a short half-life (a few hours). In fact, we were able to detect leukolysin species only by boiling the samples under reducing condition immediately after IL-8 or similar treatments. Prolonged storage, a step necessary for purification by chromatography, rendered leukolysin undetectable.2 Until we develop a rapid method of purification, perhaps by immunoaffinity chromatography, we have to rely on recombinant techniques to produce leukolysin similar to those described for several MMPs reported in recent years (32, 37). We have previously expressed the pro- and catalytic domains of leukolysin in MDCK cells (16). To further define the structure and function of the secreted enzyme, a microscale purification was attempted with ~150 ml of the conditioned media using an M2 affinity column as described previously (25). As shown in Fig. 8A, the FLAG-tagged leukolysin (lane 2) was absorbed to the column and eluted as a single species (lanes 4, 7, and 9) as characterized by SDS-PAGE, Western blotting, and zymography. Approximately 30 µg of leukolysin was recovered from 150 ml of supernatants. Its N terminus was determined by direct N-terminal sequencing to be YALSG, which matches exactly to the predicted processing site at RRRR107 (Fig. 8A). Consistent with the idea that it is the active form, the purified enzyme degrades denatured type I collagen efficiently without further activation in solution (Fig. 8B, lane 2). The observed activity can be blocked by MMP inhibitors both synthetic (BB-94) and natural such as TIMP1 and TIMP2 (Fig. 8B, lanes 3-5). To examine its likely substrates, the purified leukolysin was incubated with laminin (LN), fibronectin (FN), chondroitin sulfide proteoglycan (CSPG), and dermatin sulfide proteoglycans (DSPG) as well as type I, II, and III collagens as described (23). As shown in Fig. 8C, leukolysin was able to cleave FN, CSPG, and DSPG but not LN or type I, II, and III collagens (data not shown). We have recently developed a more robust expression system for leukolysin in E. coli and confirmed the cleavage of the above substrates (data not shown). Additional substrates such as alpha 1-PI, pro-MMP-9, and pro-MMP-2 can also be cleaved by leukolysin, albeit with different efficiency (data not shown). Therefore, the substrate spectrum for leukolysin could be broader than that of its closest relative, MT4-MMP, as recently described (37).


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Fig. 8.   Purification and characterization of active leukolysins. A, purification and sequencing of leukolysin catalytic domain. Conditioned media (lanes 2, 5) from MDCK cells stably expressing the N-terminal half (MMP251-280F) were cleared of any debris, loaded onto an M2 column (lanes 3 and 6 as the pass-through materials), washed (lane 8), and eluted with 2 molar excess of FLAG peptide (lanes 4, 7, and 9). These collected materials were analyzed by Western blotting (lanes 2-4), zymography (lanes 5-7), and R-250 staining (lanes 8 and 9). The eluted materials were also blotted onto PVDF membranes and sequenced N-terminally for five cycles, and the results are shown on the right (arrows). B, proteolytic activity of leukolysin. Aliquots (5 µg) of gelatin were either incubated alone (lane 1) or with the purified leukolysin (50 ng) in the absence (lane 2) or presence of synthetic inhibitor (BB-94, 5 µM, lane 3), TIMP-1 (100 ng, lane 4) or TIMP-2 (100 ng, lane 5) for 2 h at 37 °C. The reaction mixtures were analyzed by SDS-PAGE as described (22). C, leukolysin degrades ECM molecules. Purified ECM molecules such as laminin (LN, 5 µg, lanes 1-3), fibronectin (FN, 5 µg, lanes 4-6), chondroitin sulfate proteoglycan (CSPG, 8 µg, lanes 7-9) and dermatin sulfate proteoglycan (DSPG, 8 µg, lanes 10-12) were either incubated alone (lanes 1, 4, 7, 9) or with purified leukolysin (50 ng) in the absence (lanes 2, 5, 8, 11) or presence of BB-94 (5 µM, lanes 3, 6, 9, 12) for 24 h at 37 °C. The reactions were analyzed by SDS-PAGE as described (22). The arrowhead indicates the cleaved products.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PMNs are known for their stored arsenal of proteolytic enzymes in host defense and tissue injury (1). It has been assumed for a long time that PMNs only express two secretory MMPs, MMP-8 and MMP-9 (2, 38). In this report, we present evidence that leukolysin/MT6-MMP/MMP-25 is a PMN-specific enzyme, which is localized to the membranes of intracellular granules as well as the cell surface, and that leukolysin can be released into the extracellular milieu from neutrophils stimulated with cytokines and chemokines (see illustrations in Fig. 9). These features differ dramatically from either MMP-8 or MMP-9 stored in specific or gelatinase granules, respectively (4, 28), and leukolysin may confer to neutrophils another versatile weapon in mediating host defense. Due to its robust proteolytic activities (16, 17), leukolysin could serve as a potential target for the development of therapeutic agents against tissue damage in acute inflammation and malignant cancers.


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Fig. 9.   A model for the distribution and secretion of leukolysin in maturing neutrophils. Various granules and vesicles were indicated in the boxes in the lower left corner. According to the targeting-by-timing mechanism, progenitor cells undergoing granulopoiesis begin to transcribe leukolysin message, synthesize its protein products, and package them with other proteins into the gelatinase granules and secretory vesicles. The content of secretory vesicles is exported to cell surface constantly, thus, allowing the displaying of leukolysin on the cell surface (Fig. 5). The stored leukolysin is discharged from granules when stimulated by cytokines or chemokines such as IL-8 (Fig. 7). The inset in the lower right corner depicts the shedding of leukolysin enzymatically by GPI-PLC treatment or IL-8 treatment by an unknown mechanism. PLC, GPI-PLC.

Leukolysin as the First MT-MMP for PMNs-- Initially, leukolysin was named after the cell populations in which it is specifically expressed, i.e. human leukocytes (16). To begin to uncover its biological functions, we further defined the cells within the leukocyte lineage that express leukolysin. Surprisingly, only PMNs, not lymphocytes and monocytes, express leukolysin (Fig. 1). This degree of specificity is quite unprecedented given the known complexity in the patterns of expression for MMPs by both normal and diseased cells (2, 18-20, 39, 40). Although it is clearly premature to proclaim that leukolysin is only expressed by PMNs, the fact that other leukocytes such as monocytes do not express it suggests that it may possess properties that contribute to the functions of PMNs specifically. Interestingly, Velasco and colleagues (17) reported that leukolysin is up-regulated in brain tumor samples. Because MMPs are known to be dysregulated during carcinogenesis, it is reasonable to reconcile these two observations by suggesting that brain tumor cells or their surrounding stromal cells acquired the ability to express leukolysin during malignant transformation. However, it is not clear which cell type in the brain tumor expresses leukolysin. Further studies are required to identify these cells and compare their properties with PMNs to determine if they share similar behavior properties such as motility. With the availability of mono-specific antisera against leukolysin (Figs. 2 and 3), we envisage that these issues may be resolved by performing immunohistochemistry on brain tumor samples. Should it be proven that brain tumors are positive for leukolysin, it may be argued that leukolysin may confer an invasive and motile phenotype to the tumor cells as implied for PMNs. Nonetheless, biochemical as well as cell biological analysis of leukolysin products in PMNs as demonstrated in this report would gain valuable insight into the biological function of leukolysin, which may contribute to our understanding of its role in brain tumor biology.

Cellular Localizations and Likely Function of Leukolysin-- Because MMP-8 and MMP-9 are stored in the specific and gelatinase granules of resting PMNs, respectively (4, 8, 28), we reasoned that leukolysin would be localized with either or both of them to complement their proteolytic activities. Surprisingly, this assumption is only partly true given the small percentage of leukolysins found in the specific granules, a substantial amount (~40%) in the gelatinase granules, but almost 50% of leukolysins in the secretory vesicles and plasma membranes (Figs. 4, 5, 9). In fact, almost one of five leukolysins appears to be displayed on the cell surface of resting PMNs (Fig. 5). This conclusion was reinforced by the fact that GPI-PLC released leukolysin from PMN cells (Fig. 5). As a GPI-anchored protein, leukolysin joins a class of cell surface molecules expressed by resting PMNs that have been implicated in a wide range of biological functions, including signal transductions (41). The prospect of leukolysin being a signaling molecule via a GPI-anchor would raise the possibility that it may act as a sensory apparatus for cells such as PMNs migrating through rapidly changing environments of plasma, tissues, and inflammatory sites. On the other hand, the cell surface-associated leukolysin may also cleave other cell surface molecules such as selectins, growth factors, or their receptors either during migration or at inflammatory sites, providing a mechanism of down-regulation after stimulations (1, 40).

The Role of Leukolysin in Inflammation and Tissue Damage-- The swift discharge of stored leukolysin by chemokine- and cytokine-treated PMNs (Fig. 7 and 9) supports the hypothesis that leukolysin is one of the proteolytic enzymes important for host defense and tissue damages (1, 2, 35). Although both cytokines and chemokines are effective in regulating leukolysin secretion, IL-8 is the most effective in triggering such a discharge (Fig. 8), indicating that leukolysin may be important for neutrophil chemotaxis or transmigration through blood vessels or tissues. Despite the fact that the particular physiological targets for leukolysin remain unknown, we suggest that it may assist neutrophil migration through both the basement membrane and interstitium by degrading select constituents. Intriguingly, we have shown that leukolysin is an efficient gelatinase (16); thus, it may have a similar substrate specificity as MMP-2 or -9. Given the report that MMP-9 acts upstream of neutrophil elastase by proteolytically inactivating neutrophil elastase inhibitor alpha 1-PI in experimental bullous pemphigoid (15), secreted leukolysin may be able to accomplish a similar task. Indeed, we have determined that leukolysin cleaves alpha 1-PI efficiently using bacterially derived leukolysin (data not shown). On the other hand, leukolysin may interact with other proteinases in the secreted materials from PMNs, such as neutrophil elastase, protease 3 as well as the metalloproteinases, MMP-8 or MMP-9, for zymogen activation and matrix destruction (1). In fact, the membrane-type MMPs such as MT1-, 3-, and 5-MMPs are known activators for secreted MMPs such as MMP-13 and MMP-2 (for reviews, see Refs. 18, 38). Indeed, Velasco and colleagues (17) reported that leukolysin is capable of activating pro-MMP-2 in co-transfection experiments. Thus, a proteolytic cascade could be formed in the released mixtures of activated PMNs dedicated to the destruction of ECM components at inflammatory sites (1). Further experiments both in vitro and in vivo are required to define its role in the normal physiology of PMNs and pathological states such as acute inflammation or carcinogenesis.

    ACKNOWLEDGEMENTS

We thank Mary Dietz of R&D Systems for supplying the purified leukocytes and Drs. Stephen J. Weiss (University of Michigan), Mike Wilson (University of Minnesota) for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA76308 and by a small grant from the Minnesota Chapter of Arthritis Foundation (to D. P.) and the Danish Medical Research Council (to N. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church St., S.E. Minneapolis, MN 55455. Tel.: 612-626-1468; Fax: 612-625-8408; E-mail: peixx003@tc.umn.edu.

Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M007997200

2 T. Kang, X. Wang, and D. Pei, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PMN, polymorphonuclear leukocytes; MMP, matrix metalloproteinase; MT, membrane-type; IL, interleukin; GPI-PLC, glycosylphosphatidylinositol phospholipase C; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; HLA, human leukocyte antigen; alpha 1-PI, alpha 1-proteinase inhibitor; MDCK, Madin-Darby canine kidney cells; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol myristate acetate; KRP, Krebs-Ringer phosphate; PVDF, polyvinylidene difluoride; ECM, extracellular membrane; DSPG, dermatin sulfide proteoglycans; CSPG, chondroitin sulfide proteoglycan; LN, laminin; FN, fibronectin.

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