Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Circulating neutrophils, when sufficiently activated by an inflammatory stimulus, migrate out of the bloodstream at the site of inflammation by adhering to and then traversing the endothelial cell layer of the blood vessel wall. After penetrating the endothelium, the migrating neutrophil encounters the subendothelial basement membrane (BM), a specialized nonfibrillar matrix composed predominantly of collagen IV and laminin (1). One of the functions of BMs is to impede the passage of cells by acting as passive selective molecular sieves between tissue compartments. Specific mechanisms must exist that permit inflammatory cells to emigrate across these barriers (2). Although histologic studies have demonstrated that subendothelial BM is indeed a structural barrier that causes neutrophils to pause during the migratory process (reviewed in 3), the specific mechanism(s) mediating neutrophil penetration of subendothelial BM have not been characterized to date.

Matrix metalloproteinases (MMPs), a family of Zn- endopeptidases, are implicated in cell movement during development, angiogenesis, tissue remodeling, and tumorgenesis (4). A subgroup of the MMP family, the gelatinases or type IV collagenases, display a particular specificity for BM collagens (5). Two gelatinases, A (MMP2) and B (MMP9), have been identified to date. In common with the other members of the MMP family, the gelatinases are produced and secreted in latent proenzyme form and activated extracellularly (reviewed in 6). The active enzymes are inhibited by endogenous tissue inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Unlike the other MMP family members, the latent, inactive forms of both gelatinases form stable complexes with TIMP inhibitors, progelatinase B associating preferentially with TIMP-1 and progelatinase A with TIMP-2. The biologic significance of this association is the subject of much speculation, with several investigators suggesting that it may be a mechanism to ensure containment of extracellular proteolysis during cell migration (6).

The involvement of gelatinases, particularly gelatinase B, in extravasation of inflammatory cells has been postulated. Neutrophils store significant quantities of preformed gelatinase B in their secretory vesicles and tertiary granules (7). Monocytes also produce gelatinase B (8), and constitutive production of gelatinase B by T lymphocytes has recently been reported (9). In neutrophils, stored gelatinase B is released rapidly when cells are exposed to chemotactic stimuli that induce migration (10). In in vitro studies, neutrophil adhesion and migration are accompanied by concomitant release of significant quantities of gelatinase B (11, 12); and in vivo, migration of neutrophils is accompanied by release of gelatinase B-containing secretory granules (13). The association of gelatinase B release with neutrophil migration, combined with the histologic observations that migrating neutrophils pause in the subendothelial space between endothelial cells and BM, has led to widespread speculation that gelatinase B is involved in proteolysis of BM during neutrophil extravasation (14).

Neutrophil elastase and cathepsin G are two serine proteases contained within the azurophil granules of neutrophils that play important roles in neutrophil-mediated proteolytic events. They degrade a variety of extracellular matrix (ECM) macromolecules, including the major BM components, laminin (15), and native type IV procollagen (16), and have also been implicated in mediating proteolytic digestion of BM to facilitate neutrophil transmigration.

Direct evidence that MMPs, or indeed any proteinases, are involved in neutrophil endothelial transmigration is, however, scant. Several researchers have examined protease involvement during neutrophil migration through Matrigel (Becton-Dickinson, Bedford, MA), an acellular BM preparation from a neoplasmic lesion in mice (Engelbrecht-Holm-Swarm murine tumor). TIMP-1 was found to inhibit n-formylmethionyl leucylphenylalanine (FMLP)- stimulated neutrophil migration through Matrigel by 50% (17); whereas a hydroxamic acid inhibitor of MMPs, GM-6001, was reported to decrease interleukin (IL)-2-stimulated T-cell migration through Matrigel by > 90% (9). Using a similar Matrigel-based system, eosinophil migration in response to platelet-activating factor and IL-5 was decreased by inhibitors of serine proteinases and gelatinase B (18). By contrast, in the two reports to date that have examined the effect of protease inhibitors on neutrophil migration across endothelial cells as opposed to Matrigel, migration was found to be independent of proteolytic activity (19, 20). Although Allport and colleagues (20) demonstrated that the hydroxamic acid-based MMP inhibitor Ro 31-9790 did not inhibit neutrophil migration across tumor necrosis factor (TNF)--activated human umbilical vein endothelial cell (HUVEC) monolayers, the presence of subendothelial basal lamina was not confirmed. The single study examining the involvement of proteases during neutrophil migration across endothelial cells (HUVECs) and underlying basal lamina (19) found that, in contrast to results with Matrigel, protease inhibitors did not inhibit neutrophil transmigration of basal lamina. Thus, proteolytic digestion of BM by migrating neutrophils remains controversial.

To address this controversy, the objective of this study was to assess the effects of MMP and serine protease inhibitors on neutrophil migration across endothelial cells and associated basal lamina. In an in vitro system characterized at the ultrastructural level, we demonstrate that inhibitors of MMPs and serine proteases fail to block neutrophil transmigration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

FMLP, human placental type IV collagen, 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (ABTS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (MEOSAAPVPNA) were purchased from Sigma-Aldrich Chemical Company Ltd. (Dorset, UK). Anti-CD18 and isotype-matched immunoglobulin (Ig)G₁ monoclonal antibodies, phycoerythrin (PE)-conjugated anti-L-selectin (Leu 8, IgG2a) and PE-labeled isotype-matched IgG2a were from Becton Dickinson (Oxford, UK). Human recombinant TIMP-1 was kindly provided by Prof. Gillian Murphy, Nottingham University (Nottingham, UK). The dipeptide analogue hydroxamic acid MMP inhibitor GM-6001 (OHNHCOCH CH[i-Bu]CO-L-Trp-NHMe) (21) was a generous gift from Dr. Richard Galardy, Guilford, CT. BB-3101 was provided by British Biotech Pharmaceuticals Ltd. (Oxford, UK). Ro 31-9790 was provided by Roche Products Ltd. (Hertfordshire, UK). Human recombinant secretory leukoprotease inhibitor (SLPI) was a gift from Dr. Jan Stolk, University Hospital, Leiden, The Netherlands. 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc SC) was purchased from Boehringer Mannheim GmbH (Mannheim, Germany).

Cell Culture

Human pulmonary artery endothelial cells (HPAECs) were grown in 100% humidity and 5% CO₂ at 37°C in endothelial growth medium (EGM) supplemented with 5% fetal calf serum, epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), bovine brain extract containing heparin (10 µg/ml), gentamicin (50 ng/ml), and amphotericin B (50 ng/ml) (Clonetics Corp., San Diego, CA). When approximately 80% confluent, cells were harvested, resuspended in fresh EGM, and seeded at a density of 1.5 × 10⁵ cells in 200 µl onto Transwell polycarbonate membrane filters (6.5 mm diameter, 3.0 µm pore size; Corning Costar Corp., Cambridge, MA) that had been previously coated with human type IV collagen as described by the manufacturers. The Transwell filter inserts were suspended in 24-well culture plates so that the filter separated the upper and lower compartments. Culture medium, 600 µl, was placed in the lower compartment and the cells were cultured for 4 d. All experiments were carried out on HPAECs between passages 6 and 9 and, after completion of experiments, the cells were confirmed to be free of Mycoplasma infection. Chromosome analysis also confirmed that cells remained diploid.

Electron Microscopy

Following gentle rinsing with prewarmed phosphate-buffered saline (PBS), endothelial cells grown on Transwell filters were fixed at room temperature in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h, rinsed with PBS, and then postfixed for 1 h in 1% osmium tetroxide. After dehydrating in increasing concentrations of ethanol up to 100%, filters to be examined by scanning electron microscopy (SEM) were critical-point dried, mounted on 10-mm aluminum stubs, and coated with gold in a Polaron E 5100 sputter coater. They were then scanned in a JEOL 35C scanning electron microscope with an accelerating voltage of 15 kV. For transmission electron microscopy (TEM), filters were fixed, postfixed, and dehydrated in ethanol as described previously. They were then immersed in an ethanol/Epon (1:1 vol/vol) mixture for 1 h before being transferred to pure Epon and embedded at 37°C for 2 h. The final polymerization was carried out at 60°C for 24 h. Sections were prepared, mounted on copper grids, and stained with uranyl acetate and lead citrate before being examined in a JEOL 1200 transmission electron microscope.

Assessment of Permeability of HPAEC System

Permeability of the HPAEC bilayer system was assessed by measuring the transendothelial transfer of Trypan blue- labeled albumin by adaptation of previously described methods (22, 23). HPAECs were grown on Transwell filters for 4 d, washed, and preincubated with either Hanks' balanced salt solution (HBSS) or FMLP as detailed in the transmigration assay procedure below. Trypan blue- labeled albumin (4%; 100 µl) was added to the upper compartment and the system incubated for 3 h at 37°C. Trypan blue-labeled albumin transfer across the HPAEC bilayer was quantified by measuring the absorbance at 570 nm of aliquots sampled from the lower Transwell compartment.

Isolation of Neutrophils

Human venous blood was collected by venipuncture into sterile vacutainers containing 0.105 M sodium citrate as an anticoagulant and allowed to cool to room temperature for 10 to 15 min. Neutrophils were isolated using density gradient centrifugation on Polymorphprep (Nycomed Pharma As, Oslo, Norway) at 450 g for 35 min at 20°C. Contaminating erythrocytes were removed by hypotonic lysis, and the isolated neutrophils were resuspended in HBSS without Ca²⁺ or Mg²⁺ for counting and viability assessment and then resuspended in complete HBSS at a concentration of 1 × 10⁷ neutrophils/ml immediately before addition to the transmigration assay. Neutrophils isolated in this way were  97% pure and  95% viable.

Transmigration Assay

After 4 d in culture, HPAECs on the Transwell filter inserts were transferred to a fresh 24-well tissue culture plate. For each migration assay, one filter was retained and processed for SEM to confirm the presence of an intact continuous cell layer covering the filter. Culture medium was carefully removed from other filter inserts and, after gentle rinsing of the cells with HBSS (prewarmed to 37°C), medium was replaced with 100 µl HBSS. A total of 600 µl of either FMLP (0.1 to 100 nM) in HBSS or HBSS alone was added to the lower compartments of the Transwell system and the cells were preincubated for 45 min at 37°C. The migration assay was then initiated by the addition of 1 × 10⁶ neutrophils in 100 µl HBSS to all upper compartments. An additional 200 µl of either FMLP or HBSS was added to the bottom compartment to equalize the liquid levels in the top and bottom compartments of the Transwells. The plates were incubated at 37°C in 100% humidity and 5% CO₂. For TEM studies, duplicate filters were removed at time points from 5 to 30 min, washed, fixed, and processed as described previously. For transmigration assays, Transwells were incubated for 3 h, after which time the plate was placed on ice. Nonmigrated and migrated neutrophil populations were separated by lifting the filter inserts (upper compartment) out of the well (lower compartment) in which they had been suspended. Any migrated neutrophils that were loosely adhered to the filter undersurface were rinsed into the population of migrated neutrophils in the lower compartment of the Transwell. Nonadherent neutrophils that had not migrated remained in the volume contained in the filter insert (i.e., in the upper compartment of the Transwell system) and were collected by gentle washing with HBSS. Both neutrophil populations and their respective supernatants were collected by centrifugation at 300 × g for 10 min. The neutrophils were then lysed by suspension in HBSS containing 0.25% (wt/vol) Brij-35. The HPAEC monolayer with associated adherent neutrophils was removed by carefully cutting the filter membrane out of the insert, and was lysed by addition of HBSS containing 0.25% Brij-35. For each experiment, a range of neutrophil concentrations were prepared and incubated in either HBSS or FMLP for 3 h at 37°C, after which time the neutrophils were collected by centrifuging at 300 × g for 10 min before being resuspended in HBSS containing 0.25% Brij-35 and lysed concurrent with the migration assay samples. All neutrophil lysates were assayed for myeloperoxidase (MPO) activity. A standard curve of number of neutrophils versus MPO activity was constructed and the numbers of nonadhered, adherent, and migrated neutrophils were quantified by extrapolation of the MPO activity present in upper, monolayer, and lower compartments, respectively. The involvement of neutrophil ₂ adhesion molecules in the neutrophil migration across HPAECs was analyzed by preincubating neutrophils for 15 min at 37°C with either anti-CD18 or an isotype-matched IgG₁ control monoclonal antibody (1 µg of purified immunoglobulin per 5 × 10⁵ neutrophils) before adding these preincubating neutrophils to the HPAEC monolayers. When the effects of MMP inhibitors on neutrophil transendothelial migration were being analyzed, either TIMP-1 (0.1 µM), GM-6001 (10 µM), BB-3103 (30 µM), or Ro 31-9790 (30 µM) was present in both the upper and lower compartments throughout the preincubation period and during the migration experiment. In assays with BB-3103 and Ro 31-9790, dimethyl sulfoxide (DMSO) was maintained at a final concentration of 1% (vol/vol).

MPO Assay

An adaptation of the method of Madara and associates (24) was used to quantify MPO activity in neutrophil lysates. Briefly, the pH of neutrophil lysates was adjusted to 4.2 by the addition of 100 mM citrate buffer; pH 4.2. 50-µl aliquots of each pH-adjusted sample were then transferred to a 96-well microtiter plate and incubated for 20 min at 37°C with 100 µl 2 mM ABTS in 100 mM citrate buffer, pH 4.2, containing 0.06% H₂O₂. The reaction was terminated by the addition of sodium dodecyl sulfate to a final concentration of 0.5% and the absorbance measured at 405 nm using a Microplate EL309 Autoreader (Bio-tek Instruments, Inc., Winooski, VT). A linear relationship between number of neutrophils and MPO activity was obtained in the range of 0.05 to 1.0 × 10⁶ neutrophils/ml.

Assessment of Cytotoxicity

Cytotoxicity of the inhibitors at the concentrations used in the transmigration assay to HPAECs was analyzed using an MTT assay as described by Mosmann (25). Confluent monolayers of HPAEC in 96-well plates were exposed to each inhibitor in HBSS for 4 h at 37°C. The inhibitor was then removed and the cells were incubated for a further 4 h with 0.5 mM MTT in HBSS. MTT is a tetrazolium salt that is reduced to a purple formazan product by viable and metabolically active cells (25). After 4 h the MTT solution was carefully removed. The purple formazan crystals were lysed out of the cells and dissolved by addition of 50 µl of 10% Triton X-100 in 0.1 M HCl to each well and shaking at room temperature for 20 min. The absorbance at 570 nm was measured using a microplate reader.

Gelatin Zymography

Gelatin zymography was carried out on diluted supernatants collected from the migration-assay upper and lower compartments as described by Overall and coworkers (26). In brief, samples were applied to 7.5% acrylamide gels containing 0.01% gelatin. Following electrophoresis, gels were rinsed twice for 30 min in 2.5% Triton X-100 before being incubated overnight at 37°C in assay buffer containing 10 mM Tris-HCl, 0.04 M NaCl, 1 mM CaCl₂, and 0.01% Brij, pH 7.2. Gels were then stained in Coomassie Brilliant Blue R-250 before being transferred to water. Zones of enzymatic activity were visualized as clear bands against a blue background. A preparation containing gelatinases A and B from gelatinase B-transfected human kidney cells (obtained from Dr. S. McDonnell, Dublin City University, Dublin, Ireland) was used as a positive control. Densitometry was carried out on negative images of the zymograms using the GDS-8000 Complete Imaging System (Phoretix International, Newcastle-Upon-Tyne, UK). The densities of bands in individual samples were standardized using the gelatinase A/gelatinase B positive standard on the corresponding zymogram. Gelatinolytic activity was expressed in arbitrary densitometric units (adu).

Analysis of Antiproteolytic Activity of Inhibitors

The ability of the MMP inhibitors to inhibit gelatinase activity in 1 × 10⁵ neutrophils and in the supernatants of FMLP-stimulated migration assays was determined using an adaptation of the gelatin zymography technique described previously. In brief, samples were loaded onto gels and, after electrophoresis, were then incubated overnight at 37°C in assay buffer with and without each MMP inhibitor at the concentration used in the migration assays, i.e., 0.1 µM TIMP-1, 10 µM GM-6001, 30 µM BB-3103, and 30 µM Ro 31-9790. Gels were then stained and destained and gelatinolytic enzyme activity was quantified as described previously. Inhibition of gelatinase activity was expressed as a percentage of the gelatinolytic activity quantified in gels without inhibitor.

Inhibition of L-selectin shedding was determined by preincubating neutrophils for 30 min at 37°C with either TIMP-1 (0.1 µM), GM-6001 (10 µM), BB-3103 (30 µM), or Ro 31-9790 (30 µM) before activating with FMLP (10 nM) for a further 30 min at 37°C. Neutrophil surface expression of L-selectin was determined by fluorescent labeling with PE-conjugated anti-L-selectin and then quantifying using a fluorescence-activated cell sorter (FACS) as described previously (27).

The inhibitory capacity of SLPI and Pefabloc SC was assessed by their ability to inhibit neutrophil elastase activity in a homogenate of 5 × 10⁶ neutrophils. Neutrophil elastase activity was measured using the chromogenic peptide substrate MEOSAAPVPNA at a concentration of 4.2 mM (28). Using an extinction coefficient of 8,800 l¹ mol¹ cm¹, molar concentrations of peptide hydrolyzed were determined and enzyme units, defined as the release of 1 M p-nitroanilide min¹ ml¹, were calculated.

Statistical Analysis

Results are summarized as means ± SEM. Treatment-versus-control effects were analyzed by paired, two-tailed Student's t test and were considered significantly different at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of Endothelial Cell System

SEM demonstrated the formation of a continuous layer of endothelial cells covering the entire filter surface at 4 d after seeding on type IV collagen-coated filters (Figure 1A). TEM of sections made perpendicular to the filter demonstrated the presence of a continuous subendothelial basal lamina that typically had a lamina densa of 12 nm width and a total structure thickness of 40 nm (Figure 1D). TEM also revealed the presence of two monolayers of HPAECs covering both surfaces of the Transwell filter (Figure 1B), with cells on the underside (the nonseeded side) oriented with their luminal surfaces facing the bottom well of the Transwell system. Both HPAEC monolayers displayed occluding (tight) intercellular junctions (Figure 1C).

View larger version (118K): [in this window] [in a new window]   Figure 1.   HPAECs after 4 d growth on Transwell microporous filters under conditions described in MATERIALS AND METHODS. A continuous sheet of cells covering the filter surface was observed by SEM (A). Transmission electron micrographs revealed the presence of endothelial cell monolayers on both sides of the filter (B). The monolayers formed by HPAECs displayed (C ) intact intercellular junctions (arrow) and (D) complete subendothelial basal lamina (arrow). Scale bars: 35 µm (A), 5 µm (B), 0.5 µm (C ), and 0.2 µm (D).

Collagen-coated filters without cells were found to be 54% permeable to Trypan blue-labeled albumin. The presence of confluent monolayers of HPAEC decreased this permeability to 20%. Incubation with FMLP for 3 h had no effect on the permeability of the HPAEC system.

Neutrophil Transmigration

Neutrophil migration through the HPAEC bilayer was considerably stimulated by FMLP at concentrations of 10 and 100 nM, with greatest migration observed with 10 nM. Less than 10% of neutrophils migrated in response to 0.1 nM FMLP (Figure 2A). Addition of a monoclonal antibody to CD18, the  component of the neutrophil adhesion molecule, Mac-1, significantly reduced FMLP-stimulated migration (Figure 2B), whereas an isotype-matched control Ig had no significant effect. In the absence of FMLP, migration was minimal.

View larger version (54K): [in this window] [in a new window]   View larger version (60K): [in this window] [in a new window]   Figure 2.   Neutrophil migration across HPAEC bilayers in response to different nM concentrations of FMLP (A) and in the presence of antibody to CD18 and isotype-matched IgG1 (B). Neutrophils were allowed to migrate for 3 h across HPAECs that had been pretreated with FMLP for 45 min. The percentage of neutrophils migrated was determined by comparison of MPO in neutrophils recovered from the lower compartment of the Transwell system with the total MPO content of neutrophils added to the system. In B, FMLP concentration was 10 nM. Figure data are from three independent experiments with different neutrophil samples.

TEM demonstrated that neutrophils added to the seeded side of FMLP-treated HPAECs were seen to extend a pseudopod toward the endothelium, followed by intimate adherence between the apical plasmalemma of the endothelium and the surface of the neutrophil (Figure 3A). The migrating neutrophils penetrated the endothelial monolayer through the intercellular junctions, which immediately re-formed, leaving the endothelium intact and covering the neutrophils in an attenuated position (Figure 3B). At this point, the migrating neutrophils were positioned between the cell monolayer and the underlying basal lamina. After penetrating the subendothelial basal lamina, neutrophils entered and passed unimpeded through the 3-µm pores of the polycarbonate filter (Figure 3C) and arrived at the basal lamina and endothelium on the underside of the filter (Figure 4A). Groups of neutrophils were often seen gathering at this point, where they were closely apposed to the basal lamina (Figure 4B). Despite creating an apparant barrier to migration, the basal lamina was successfully traversed and neutrophils penetrated the second cell monolayer through the intercellular junctions (Figure 4C).

View larger version (108K): [in this window] [in a new window]   Figure 3.   Transmission electron micrographs of neutrophil interactions with the HPAEC monolayer on the seeded side of the filter. Before neutrophil penetration of the interendothelial junctions, intimate adherence between added neutrophils and the endothelium was observed (A). After penetration, the endothelial junctions re-formed to cover the migrating neutrophil (B). The neutrophil was positioned between the endothelium and the basal lamina (arrow), which it penetrated to move through the 3-µm filter pore (C ). Scale bars: 0.8 µm (A), 1.1 µm (B), 0.25 µm (C ).

View larger version (125K): [in this window] [in a new window]   Figure 4.   Transmission electron micrographs of neutrophils that have migrated from the seeded side of the filter to contact the HPAEC monolayer on the filter underside. Migrated neutrophils were found between the filter undersurface and the monolayer of HPAEC (A), where they were tightly apposed to the basal lamina (arrow) (B). The basal lamina appeared to form a real barrier to neutrophil migration, as groups of neutrophils were often found "lining up" at this point (C ) before successfully penetrating the endothelial monolayer through the intercellular junctions (D). Scale bars: 2 µm (A), 0.7 µm (B), 4 µm (C ), 1.6 µm (D).

Gelatinase Release by Migrating Neutrophils

Zymography revealed the presence of several gelatinase bands in supernatants from both upper and lower compartments of the Transwell system after FMLP-stimulated migration. These bands corresponded to latent gelatinase B (97 kD), gelatinase B associated with lipocalin (130 kD), and higher molecular-weight multiple forms of the enzyme (7). No bands with molecular weights lower than that of latent gelatinase B, corresponding to active gelatinase or gelatinase A (72 kD), were observed in any sample. Negligible levels of gelatinase were present in lower compartments from samples not stimulated with FMLP (0.42 ± 0.26 adu of gelatinase activity), indicating that in the absence of migrated neutrophils, little gelatinase was released into the lower compartments. Although measurable amounts of gelatinase B were released by neutrophils into the upper compartments of wells containing only HBSS (1.52 ± 0.21 adu), the total amount of gelatinase released (i.e., into both upper and lower compartments) was significantly higher when FMLP was present (3.7 ± 0.69 adu in the presence of FMLP, compared with 1.95 ± 0.38 adu in HBSS alone; P < 0.05). Values are gelatinase B levels assayed in the supernatants of six independent transmigration experiments.

Effects of MMP Inhibitors on Neutrophil Transmigration

The presence of the physiologic inhibitor of gelatinase B TIMP-1 (0.1 µM), or the hydroxamic acid-based synthetic inhibitors GM-6001 (10 µM), BB-3103 (30 µM), or Ro 31-9790 (30 µM) in the system before and during assessment of neutrophil migration had no significant effect on FMLP- stimulated neutrophil migration through the endothelial cells and associated basal lamina (Figure 5). At the concentrations used in the transmigration assay, the inhibitors had no cytotoxic effect on HPAECs, as demonstrated by MTT assay (results not shown).

View larger version (63K): [in this window] [in a new window]   Figure 5.   Effects of MMP inhibitors on neutrophil migration across HPAEC bilayers in response to 10 nM FMLP. HPAECs were pretreated with TIMP-1 (0.1 µM), GM-6001 (10 µM), BB-3103 (30 µM), or Ro 31-9790 (30 µM), either alone or in combination with FMLP, for 45 min before assessment of neutrophil migration. The effect of 1% DMSO (vehicle for BB-3103 and Ro 31-9790) on FMLP-stimulated migration was also assessed. The percentage of neutrophils migrated was determined by comparison of MPO in neutrophils recovered from the lower compartment of the Transwell system with the total MPO content of neutrophils added to the system. Figure data are from at least three independent experiments with different neutrophil samples.

To confirm that the MMP inhibitor preparations used were active and that the concentrations used were capable of inhibiting gelatinase activity released in the transmigration assay, we performed experiments to assess their inhibitory effect on neutrophil gelatinase. Using gelatin zymography, TIMP-1 (0.1 µM), GM-6001 (10 µM), BB-3103 (30 µM), and Ro 31-9790 (30 µM) were tested for their ability to inhibit total gelatinolytic activity in both the upper and lower supernatants of FMLP-stimulated migration assays. All of the hydroxamic acid inhibitors completely inhibited total gelatinolytic activity in supernatants from assays where migration was stimulated by FMLP. In addition to blocking the amount of gelatinase released from migrating neutrophils, the hydroxamic acid-based MMP inhibitors were also able to block the total amount of gelatinase contained in 1 × 10⁵ neutrophils (Table 1). Therefore, although neutrophils activated by FMLP release less than 30% of their stored gelatinase (10) and, in situations where activation of latent gelatinase is observed, less than 20% of the released enzyme is activated (17, 29), even if a migrating neutrophil in the present system released all of its gelatinase in active form, GM-6001, BB-3103, and Ro 31-9790 were present in sufficient excess to ensure complete inhibition of released activity.

In addition to inhibiting gelatinase activity, the MMP inhibitors BB-3103 and Ro 31-9790 were capable of preventing FMLP-stimulated shedding of L-selectin from the surface of neutrophils (Figure 6), a function of the membrane-bound MMP, L-selectin sheddase (30). However, despite being able to inhibit both released gelatinase activity and membrane-bound MMP activity, these inhibitors did not block neutrophil transendothelial migration (Figure 5).

View larger version (44K): [in this window] [in a new window]   Figure 6.   Effects of MMP inhibitors on the shedding of surface L-selectin from neutrophils in response to 10 nM FMLP. The 100% expression level (line) represents the amount of L-selectin on the surfaces of unstimulated neutrophils. Surface L-selectin expression was determined by fluorescent labeling with PE-conjugated anti-L-selectin followed by quantification using FACS as described in MATERIALS AND METHODS. Results are from three independent experiments with different neutrophil samples.

Effects of Serine Protease Inhibitors on Neutrophil Transmigration

The serine protease inhibitors SLPI (1 µg/ml) and Pefabloc SC (1 mM), were capable of inhibiting neutrophil elastolytic activity in a homogenate of 5 × 10⁶ neutrophils (i.e., five times the number of neutrophils in the transmigration assay) by 100 and 84%, respectively. Despite this, neither serine protease inhibitor (either alone or in combination with the MMP inhibitor GM-6001) blocked neutrophil migration across endothelium and basal lamina (Figure 7). The concentrations of serine protease inhibitors used in transmigration assays did not affect monolayer permeability and were not cytotoxic to HPAEC, as determined by MTT assay.

View larger version (60K): [in this window] [in a new window]   Figure 7.   Effects of serine protease inhibitors on neutrophil migration across HPAEC bilayers in response to 10 nM FMLP. HPAECs were pretreated with SLPI (1 µg/ml), Pefabloc SC (1 mM), and/ or GM-6001 (10 µM), either alone or in combination with FMLP, for 45 min before assessment of neutrophil migration. The percentage of neutrophils migrated was determined by comparison of MPO in neutrophils recovered from the lower compartment of the Transwell system with the total MPO content of neutrophils added to the system. Figure data are from three independent experiments with different neutrophil samples.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we describe how HPAECs grown on 3-µm porous filters form a complete monolayer of cells on both sides of the filter. The presence of a complete monolayer of endothelium on the filter undersurface was a very surprising finding. Filters with 3-µm pores are standardly used in transendothelial leukocyte migration studies, and the filter coating (i.e., human placental type IV collagen, 50 µg/ml) and endothelial cell culture conditions (i.e., seeding density and length of time growing on filters) used in this study were identical to those used by others with HUVECs (for example, see references 31 and 32). However, the presence of a second endothelial monolayer on the filter undersurface has never been reported. In this study, HPAECs were able to migrate through the 3-µm pores of the filter to form a complete monolayer of endothelium with underlying basal lamina on the filter underside. Follow-up studies on HUVECs and human lung microvascular endothelial cells (HLMVECs) demonstrated that both of these cell types also form a monolayer on both sides of 3-µm Transwell filters, indicating that this is not a property specific to HPAECs (33). Because neutrophil migration to the lung occurs out of the capillaries, HLMVECs would be the ideal type of endothelial cells to use in an in vitro model of pulmonary inflammation. However, when HLMVECs were grown on collagen IV-coated porous filters, unlike HPAECs, they did not form a continuous monolayer of cells on the filter surface but often grew in patches with large areas of the filter remaining uncovered. The HLMVEC intercellular junctions were not always intact and the subendothelial basal lamina, if present at all, was discontinuous. In contrast, HPAEC consistently formed complete monolayers with intact intracellular junctions and continuous subendothelial basal lamina. Because the aim of this study was to examine whether MMP and/or serine protease activities are required for neutrophils to traverse endothelium and associated basal lamina, the presence of a complete monolayer of endothelium with intact, intracellular junctions and continuous subendothelial basal lamina was crucial. For this reason, HPAECs were used instead of HLMVECs as a source of human pulmonary endothelial cells in this study.

The presence of two monolayers of endothelial cells prompted us to assess thoroughly the characteristics of neutrophil migration through the HPAEC system. The HPAEC system was found to support neutrophil transmigration in a manner similar to that described for other in vitro systems in that (1) more than 60% of added neutrophils migrated across the monolayers in response to the chemoattractant FMLP, (2) minimal migration was observed in the absence of chemoattractant, and (3) stimulated migration was significantly inhibited by antibody to the ₂ integrin neutrophil adhesion molecule subunit CD18 (34, 35). Further characterization of neutrophil transmigration by TEM demonstrated that the manner by which neutrophils traversed the HPAEC system closely resembled that which is observed during extravasation in vivo, i.e., (1) neutrophil adherence to the endothelial surface, (2) penetration through the endothelial intracellular junctions in close apposition to the endothelial plasma membrane, and (3) a "hold-up" at the BM after the endothelial junction had reapposed, followed by (4) migration through the endothelial BM (36). At all times, intimate neutrophil-endothelial contact was maintained. Thus, although composed of two monolayers, the characteristics of neutrophil migration through the HPAEC system mimicked that observed in vivo.

It has long been hypothesized that neutrophil transendothelial migration is dependent on degradation of the subendothelial BM (3, 40). In light of its substrate specificity for BM collagens and its release during neutrophil migration, the MMP gelatinase B is a favored candidate for facilitating such BM degradation. Indeed, despite the absence of direct evidence to this effect, the weight of indirect evidence is such that some authors assume its involvement (29). In the present study we found that neutrophil migration across HPAECs and associated basal lamina in response to FMLP was accompanied by a significant increase in gelatinase B release but was not inhibited by TIMP-1 or by a range of potent, synthetic MMP inhibitors. Inhibition experiments demonstrated that, at the concentrations used, all inhibitors were capable of inhibiting activated gelatinase B extracted from 1 × 10⁵ neutrophils. Thus, the lack of inhibition observed is unlikely to be due to the presence of insufficient quantities of MMP inhibitors.

In addition to confirming the MMP inhibitors' capacity to block released MMP activity (i.e., gelatinase), their ability to inhibit cell-surface metalloproteinase activity was also determined by testing their ability to prevent shedding of surface L-selectin from activated neutrophils, a function of cell surface-associated metalloproteinase activity (30). Similar to previous observations with phorbol dibutyrate-stimulated L-selectin shedding (30), TIMP-1 did not inhibit shedding of L-selectin from neutrophils exposed to FMLP. The hydroxamic acid-based inhibitors varied in their L-selectin shedding inhibitory capacity: GM-6001 and BB-3103 caused 30 and 82% inhibition of L-selectin shedding, respectively; whereas, in accordance with observations by Allport and coworkers (20), the inhibitor Ro 31-9790 completely blocked FMLP-stimulated shedding of L-selectin. Regardless of their ability to inhibit cell-surface MMP sheddase activity, none of the inhibitors blocked neutrophil migration across HPAECs in response to FMLP.

Although the lack of effect of MMP inhibitors in this study was in accordance with the one previous report examining MMP involvement during neutrophil migration across endothelial-derived basal lamina (19), results from these cell-based studies are in contrast to results obtained in an acellular system of neutrophil migration across Matrigel (17). It could be suggested that the discrepancies between these observations and those of studies carried out in cellular systems may reflect a lack of penetration of MMP inhibitors through endothelial cells to the BM in cellular models. In the present study, however, inhibitors were added to the upper and lower compartments of HPAEC cultures for 45 min before the initiation of migration assays. Because the system was 20% permeable to Trypan blue-labeled albumin (66 kD), it is unlikely that significant diffusion of TIMP-1 (28 kD) across the HPAEC monolayers did not occur during this preincubation period. It is even more improbable that the synthetic inhibitors, with molecular weights less than 0.4 kD (i.e., similar size to FMLP), did not diffuse throughout the culture system. Thus, the reported differences between cellular and acellular systems are unlikely to be due to the failure of MMP inhibitors to access the "target" BM in this cellular system.

Although MMP activity did not appear to be required for neutrophil transmigration across HPAEC bilayers, significant quantities of latent and complexed forms of gelatinase B were secreted during migration. Stimulated endothelial cells can produce gelatinase B (41). However, the zymographic banding pattern observed in the migration assay supernatants was highly characteristic of gelatinase B secreted from neutrophils (42). In addition, significant levels of enzyme were present only in compartments that contained neutrophils, suggesting that this cell is the major source of the gelatinase B observed. FMLP stimulates rapid release of large quantities of latent gelatinase B from neutrophils (7), and significantly higher levels of gelatinase B were detected during FMLP-stimulated migration. However, activation of secreted gelatinase B was not observed. Delclaux and colleagues (17) report activation of approximately 15% of the gelatinase B secreted into supernatants by neutrophils migrating across Matrigel BM constructs. As components of Matrigel, a matrix derived from murine sarcoma, have been shown to stimulate neutrophils directly to secrete elastase and gelatinase (43), it is unclear from these Matrigel studies whether gelatinase B release and activation is a feature of neutrophil migration through this specialized BM preparation or a characteristic associated with neutrophil migration per se. In this context, endothelial cells with subendothelial basal lamina laid down in situ provide a system that more closely resembles the barrier neutrophils traverse during inflammation in vivo.

Neutrophils have been documented to form "sealed" extracellular compartments in the zones of contact with BM. Secretion of proteases into these compartments by chemoattractant-stimulated neutrophils has been postulated as a means of both localizing ECM degradation and protecting protease activity from the high molecular- weight protease inhibitors present extracellularly in vivo (44). Proteins greater than 40 kD in size are excluded from these protected environments. It could be argued that TIMP-1, as a 28-kD protein, may have limited ability to penetrate such microenvironments and that this may account for TIMP-1 having no inhibitory effect on neutrophil migration in this cell system. However, this would not explain the lack of inhibition of the small molecular-weight hydroxamic acid inhibitors. Because inhibitors of 12 kD have been shown to penetrate such protected compartments (45), it is highly unlikely that these MMP inhibitors (molecular weights < 0.4 kD) are excluded from cell- matrix microenvironments.

Results of the current study, together with those described by Huber and Weiss (19) and, more recently, by Allport and associates (20), strongly suggest that neutrophils can migrate through endothelial cells and subendothelial BM without the involvement of active MMPs. Migration stimuli used in these studies include FMLP, IL-8, TNF- (20), and zymosan-activated plasma (19). Whether MMP activity might be required to facilitate neutrophil transendothelial migration toward other chemoattractants remains to be elucidated.

Proteases other than MMPs, particularly the serine proteases, neutrophil elastase, and cathepsin G, are capable of degrading basal lamina components (16, 46, 47) and may be involved in digestion of BM during neutrophil transendothelial migration (3, 40). Loike and colleagues (44) demonstrated that chemoattractant-stimulated neutrophils secrete elastase into a site that is inaccessible to high molecular-weight antiproteases but is accessible to low molecular-weight antiproteases such as SLPI. In the present study, SLPI did not prevent FMLP-stimulated neutrophil transmigration of endothelium and basal lamina, suggesting that serine proteases released from neutrophils do not mediate their migration.

In addition to causing release of proteases, exposure of neutrophils to FMLP increases cell-surface expression of serine proteases (48). Degradation of the BM by such proteases would occur only at the point of contact of the cell surface with the matrix. Owen and coworkers (48) reported that this cell surface-bound elastase and cathepsin G are resistant to inhibition by naturally occurring proteinase inhibitors, a fact that may account for the lack of effect on migration observed with SLPI. To investigate the possibility that this cell-surface elastolytic activity may be responsible for facilitating transmigration, we tested a new, potent, nontoxic, and irreversible serine proteinase inhibitor, Pefabloc SC, which is capable of inhibiting membrane-associated serine protease activity (49) and is small enough (< 0.3 kD) to penetrate occlusion zones. Similar to SLPI, despite being present in concentrations that could inhibit neutrophil elastase activity from five times the number of neutrophils present in the migration system, Pefabloc SC did not block neutrophil migration. Lack of inhibition of neutrophil transmigration by SLPI and Pefabloc SC alone or in combination suggests that serine-protease activity, whether released or membrane-bound, does not have a direct role in digesting basal lamina during neutrophil extravasation. This finding supports the report by Huber and Weiss (19) that serine protease inhibitors did not prevent the loss of BM integrity associated with neutrophil migration across HUVECs grown on a collagen matrix.

Serine proteases have been suggesed to function indirectly to facilitate basal lamina digestion during neutrophil extravasation by activating pro-MMPs (17). Observations that pro-gelatinase B released from FMLP-stimulated neutrophils can be activated by neutrophil serine proteinases in vitro (50) and that proteolytic activity involving neutrophil elastase associated with neutrophil membranes can activate 92-kD progelatinase (51) provide support for this suggestion. However, a combination of the serine proteinase inhibitors SLPI and Pefabloc SC with GM-6001, the smallest of the MMP inhibitors tested, did not prevent neutrophil transmigration. This demonstrates that serine proteinases do not indirectly mediate basal lamina disruption by FMLP-stimulated neutrophils via the activation of pro-gelatinases.

In conclusion, neither naturally occurring nor synthetic inhibitors of MMP or serine proteases inhibited FMLP-stimulated neutrophil migration across endothelial cells and associated basal lamina, suggesting that neutrophils traverse subendothelial basal lamina by a mechanism(s) other than proteolytic digestion. Recent reports of normal neutrophil transendothelial migration in both gelatinase B (52) and elastase (53) knockout mice support this conclusion. Indeed, Walker and colleagues (54), using TEM and serial thin sections, examined neutrophil migration in the lungs of rabbits during streptococcal pneumonia and concluded that neutrophils traversed subendothelial basal lamina by displacing fibroblasts from pre-existing "slits" in the capillary basal lamina before using these discontinuities as avenues through which to migrate into the extracellular compartment of the alveolar wall. This observation suggests that neutrophils use mechanical rather than proteolytic means to traverse basal lamina.