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* Vascular Research Division, Departments of Pathology, Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts;
Washington University School of Medicine, St. Louis, Missouri;
Department of Dermatology, Kyoto University, Japan; and
Institute of Cell Biology, University of Münster, Germany
Correspondence: Francis W. Luscinskas, Ph.D., Vascular Research Division, Brigham & Women’s Hospital, 221 Longwood Ave., LMRC 414a, Boston, MA 02115. E-mail: fluscinskas{at}rics.bwh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • protease • leukocyte • endothelium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we and others [2 , 4 , 11 ] demonstrated that neutrophil proteases are capable of rapidly degrading components of the vascular endothelial (VE)-cadherin complex during neutrophil-endothelial contact. Degradation of VE-cadherin could be prevented by pretreatment of the neutrophils with a combination of recombinant human elastase inhibitor and phenylmethylsulfonyl fluoride, an inhibitor of serine proteases [3 ]. During certain inflammatory conditions, such as acute respiratory distress syndrome, or in chronic conditions, such as chronic obstructive pulmonary disorder [12 , 13 ], there is progressive destruction of the endothelium and basement membrane that is linked closely to elastase activity [9 , 13 14 15 ]. Although extensive degradation of endothelial and matrix proteins is detrimental, focal loss of these barrier proteins under tightly regulated conditions may facilitate leukocyte extravasation during an inflammatory response. For example, neutrophil-derived serine proteases can increase matrix metalloproteinase-1 (MMP-1)-dependent tumor-cell invasion by activation of MMP-1 [16 ], however the precise role for neutrophil proteases in neutrophil migration across the endothelium remains undetermined.
Neutrophils contain multiple proteases that fall into three classes: serine proteases, including elastase, cathepsin G, and proteinase 3; MMPs, including gelatinase B (MMP-9) and collagenase (MMP-8); and urokinase-type plasminogen activator, all of which can be released from neutrophil granules upon activation by chemoattractants or other stimuli. The goal of these studies was to determine whether the absence of MMP-9 or elastase would result in an impaired ability to interact with or transmigrate endothelium under flow conditions and secondly, if either of these proteases was capable of degrading endothelial adherens junction proteins specifically (directly or via activation of other proteases). We made use of neutrophils isolated from mice deficient in MMP-9 or neutrophil elastase and investigated their interactions with murine cardiac endothelium in an in vitro flow chamber. Previous studies [17 18 19 ] have indicated that MMP-9 or neutrophil elastase (NE)-deficient mice have no impairment in emigration, however these studies were performed in vivo at long time points (>4 h) by quantification of leukocyte cell number and cell type in bronchoalveolar lavage fluid or the peritoneal cavity. Because the present study is performed in vitro under flow conditions, it is possible for us to directly observe the phenotype of the leukocyte-endothelial interactions and the exact time for each transmigration event.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-; produced in Escherichia coli) was purchased from Genzyme Corporation (Cambridge, MA) and contained <10 pg/ml endotoxin, as determined by the manufacturer. The maximal dose was determined to be 120 ng/ml by surface immunofluorescence assay of murine endothelium [20
]. Bovine serum albumin (BSA; fraction V) was obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest grade available and were purchased from Baker Chemical (Phillipsburg, NJ).
Antibodies
Purified rat anti-PECAM-1 monoclonal antibody [mAb; CD31, clone MEC13.3, rat immunoglobulin G (IgG)2a] and purified rat anti-mouse ICAM-2 mAb (CD102, clone 3C4, rat IgG2a), used in the isolation of murine EC, were obtained from Pharmingen (San Diego, CA). The following anti-mouse antibodies have been published previously and were used as purified IgG: Anti-ICAM-1 {clone YN1.1, American Type Culture Collection (ATCC), Manassas, VA; ref. [21
]}, anti-vascular cell-adhesion molecule 1 (VCAM-1; clone MK-2, ATCC; ref. [21
]); anti-E-selectin (clone RME-1), and anti-P-selectin (clone RMP-1) were a generous gift of Dr. Andrew Issekutz (Dalhousie University, Halifax, CA; ref. [22
]). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG and FITC-conjugated goat anti-mouse IgG were obtained from Caltag Laboratories (San Francisco, CA). The following antibodies were used for neutrophil fluorescence-activated cell-sorter (FACS) analysis and were all available commercially from Pharmingen: anti-CD11a-FITC [lymphocyte function-associated antigen-1 (LFA-1), clone 2D7, rat IgG2a], anti-CD11b-phycoerythrin (Mac-1, clone M1/70, rat IgG2b), anti-Ly-6-allophycocyanin (APC; Gr-1, clone RB6-8C5, rat IgG2b), anti-Ly-76-biotin (clone TER119, rat IgG2b), anti-CD62L (Ly-22, clone Mel-14, rat IgG2a), and anti-CD16/CD62 [Fc receptor for IgG (Fc
R) III/II Fc block, clone 2.4Gr, rat IgG2b]. For immunoprecipitation and Western blotting, the following antibodies were used: anti-PECAM-1 (MEC13.3; Pharmingen), anti-ß-catenin (mouse IgG1; Research Diagnostics, Piscataway, NJ), and anti-mouse VE-cadherin (clone VECD1 supernatant; described previously in ref [23
]).
Mice
129S6/SvEv wild-type (WT) mice were purchased from Taconic Farms (Germantown, NY). MMP-9- (gelatinase B; refs [17
, 19
]) and NE-deficient [18
] mice were generated as described previously and were backcrossed to 129S6/SvEv pure background. All mice were maintained in our approved pathogen and viral-free housing facilities at the Longwood Medical Research Center (Boston, MA). For EC isolation, mice were used at 8–12 weeks. For isolation of neutrophils from bone marrow, mice were used from 8 to 24 weeks. Animals were sacrificed by carbon dioxide asphyxiation as approved by the panel on Euthanasia at the American Veterinary Association.
Isolation of murine EC from cardiac tissue (MCEC)
Methods for isolation and purification of EC were modified from published protocols [21
, 24
, 25
]. Briefly, the hearts of three mice were harvested, minced finely with scissors, and then digested in 25 ml collagenase [0.2% (w/v); Worthington Biochemicals, Lakewood, NJ] at 37°C for 45 min. The crude cell preparation was pelleted and resuspended in DPBS+, and the cell concentration was adjusted to 3 x 107 cells/ml in DPBS+. Sheep anti-rat IgG Dynal beads (Dynal Corp., Great Neck, NY) were coated with anti-PECAM-1 or anti-ICAM-2 mAb, according to the manufacturer’s instructions. The cell suspension was incubated with PECAM-1-coated beads (35 µl/ml cells) at room temperature (RT) for 10 min with end-over-end rotation. Using a magnetic separator, the bead-bound cells were recovered, washed with DMEM-20%, suspended in 12 ml complete culture medium [DMEM containing 20% fetal calf serum (FCS), supplemented with 100 µg/ml porcine heparin, 100 µg/ml EC growth supplement (ECGS; Biomedical Technologies, Stoughton, MA), and nonessential amino acids, sodium pyruvate, L-glutamine, and antibiotics at standard concentrations], and then plated in a gelatin-coated 75 cm2 tissue-culture flask. Once the cells reached 70–80% confluence, they were detached with trypsin-ethylenediaminetetraacetate (EDTA) to generate a single-cell suspension, pelleted, and then resuspended in 1–3 ml DPBS+ and incubated with ICAM-2-coated beads (35 µl/ml cells) at RT for 15 min with rotation. The bead-bound cells were washed and plated in complete culture medium at a 1:2 split and passaged further at a 1:2 ratio. Confluent monolayers of MCEC were used at passages 1–3.
Isolation of mature murine neutrophils from bone marrow
Isolation of mature neutrophils from murine bone marrow was achieved using modifications of published methods [26
, 27
]. Briefly, the femur and tibia were removed and stripped of all muscle and sinew, and the bones were placed in RPMI containing 5% FCS (RPMI-5%) on ice. The bone marrow was flushed from each bone with RPMI-5%, and the plugs were disrupted by tituration to generate a unicellular suspension. Cells were pelleted, and mature anucleate erythrocytes were removed by hypotonic lysis. The entire bone marrow preparation was resuspended at 5 x 107 cells/ml in Hanks’ balanced saline solution (HBSS). Mature neutrophils were separated from the remaining cells by centrifugation over discontinuous Percoll gradients at 500 g for 30 min at 4°C, consisting of 55% (v/v), 65% (v/v), and 75% (v/v) Percoll in PBS. Mature neutrophils were recovered at the interface of the 65% and 75% fractions and were >90% pure and >95% viable in the neutrophil-rich fraction as determined by Wright-Giemsa [28
] and trypan blue exclusion, respectively. Neutrophils were determined to be mature by their size, morphology, and surface expression of specific markers (see Fig. 2
, and refs. [28
, 29
]).
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(120 ng/ml). The coverslips were then placed in the flow chamber, and murine neutrophils (0.5x106 cells/ml) were drawn across the monolayers for 10 min at 0.52 ml/min (estimated wall shear stress of 1.0 dynes/cm2). Leukocyte-endothelial interactions were recorded live by video-linked, phase-contrast microscopy and adhesion/rolling, and transmigration was determined offline from 8–10 high-power (60x obj.) fields after 10 min of perfusion. Transmigrated neutrophils were determined as those that were phase dark and appeared in a separate plane of focus from neutrophils adherent to the apical surface.
Immunoprecipitation and immunoblotting
Immunoprecipitation of endothelial proteins from the VE-cadherin complex was performed as detailed previously [2
]. Briefly, 4-h TNF--activated endothelial monolayers were incubated with neutrophils or media alone for 10 min at 37°C. Nonadherent cells were removed, and the monolayers were lysed in ice-cold Tris-buffered saline, pH 7.4, containing 1% TX-100, 1% Nonidet-P-40, 2 mM CaCl2, and a cocktail of protease inhibitors. Specific antibodies directed against VE-cadherin, ß-catenin, or PECAM-1 were coupled to protein-G-sepharose and were used to immunoprecipitate the proteins of interest from the cell lysate (10 µg purified IgG or 50 µl hybridoma supernatant per 30 µl protein-G-sepharose). Proteins were then solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and resolved on a 7.5% polyacrylamide gel under reducing conditions. Subsequently, proteins were transferred to a nitrocellulose membrane and detected by immunoblotting with specific antibodies coupled to horseradish peroxidase, and then visualized with enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Flow cytometric analysis of cell-surface protein expression
Cell-surface protein expression was analyzed on murine EC and neutrophils using a modification of published protocols [29
]. Briefly, neutrophils (1x106 cells) were FcR-blocked with anti-CD16/CD62 mAb and incubated with 1/50 dilution of TER119-biotin for 30 min on ice. After washing, they were resuspended in a 1/50 dilution of directly conjugated antibodies to LFA-1, Mac-1, or L-selectin in combination with 1/50 dilution of streptavidin-CYC and Gr-1-APC. Subsequently, neutrophils were washed and fixed in 200 µl 2% formalin in PBS. A total of 20,000 cells were analyzed in each case on a Becton Dickinson (San Jose, CA) FACScan.
For EC, confluent monolayers of MCEC in 100 mm diameter dishes were incubated with media alone or media containing 120 ng/ml TNF- for 5 h. EC were then washed extensively with warm HBSS to remove serum and then suspended by brief trypsinization in 2 ml warm trypsin EDTA for no more than 2 min. Proteolysis was arrested by addition of 10 ml ice-cold RPMI-20% fetal bovine serum. EC were then pelleted and resuspended in ice-cold RPMI-5%. Analysis of cell-surface expression of ICAM-1, ICAM-2, VCAM-1, P-selectin, and PECAM-1 was performed using two-step immunofluorescence staining. The cells were stained first with purified mAb (10 µg/ml, 30 min on ice), washed, and detected with FITC anti-rat IgG or FITC anti-mouse IgG (1/50 dilution 30 min on ice). The cells were then washed and fixed as described above. A minimum of 10,000 cells per sample were analyzed.
Statistics
Adhesion and transmigration data were collected by analysis of variance, and Student’s two-sample t-test was used to calculate statistical significance (Excel, Microsoft Corp., Redmond, WA). P values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-activated endothelium. From the data presented in Figure 1
, it can be observed that more than 99% of MCEC express the EC markers PECAM-1 (Fig. 1a)
and ICAM-2 (Fig. 1b)
and that the level of PECAM-1 and ICAM-2 expression is not altered by murine TNF- treatment. It was not surprising that the endothelium expressed PECAM-1 and ICAM-2 because the cells were selected via those antigens, however they also exhibited a high constitutive expression of ICAM-1 [Fig. 1c
; mean channel fluorescence (mcf)=90] that was induced further by exposure of the endothelium to 120 ng/ml murine TNF- (Fig. 1c)
. A similar high constitutive expression pattern of VCAM-1 and low constitutive VE-cadherin expression were observed in these cells (unpublished results). Additionally, unactivated endothelium did not express P-selectin (Fig. 1d
, 8% positive), but cell-surface expression could be induced following activation with TNF- (Fig. 1d
, 74% positive). These data indicated that murine EC isolated by this method were more than 95% pure and were valid cells to use in this model.
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Neutrophils deficient in elastase or MMP-9 have no impairment in transendothelial migration under in vitro flow conditions
Neutrophils from WT and MMP-9- and NE-deficient mice were resuspended at 0.5 x 106 cells/ml in warm 1:1 DPBS:DPBS+ containing 0.1% (w/v) BSA and drawn across 4-h TNF--activated MCEC monolayers for 10 min at 0.52 ml/min. For WT and deficient mice, large numbers of neutrophils interacted with the endothelial surface [924±170 (WT), 993±75 (MMP-9-/-), and 922±255 cells/mm2 (NE-/-); n
3; Table 1
], and greater than 30% interacting cells ultimately transmigrated the endothelial monolayers in all cases. The total interaction and transmigration events for each strain were determined from multiple fields at 8–10 min of perfusion (Fig. 3
). Figure 3
is representative of three similar experiments, showing no significant difference between WT and MMP-9 (left graph) or WT and NE-deficient mice (right graph) in total interactions or migration events. Neutrophils from WT and deficient mice typically transmigrated the endothelial monolayer in 30–60 s, in similar fashion to that observed previously for human neutrophils traversing HUVEC [21
]. Similar behavior of WT and NE- and MMP-9-deficient neutrophils was observed. Overall, there was no significant difference among any group for numbers of adherent or transmigrated cells or for the percentage of migration, indicating that neither MMP-9 nor neutrophil elastase is required for murine neutrophil transendothelial migration under these conditions.
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. WT or MMP-9- or NE-deficient neutrophils (1x106 cells or M199 media alone, 1 ml) were added to each well and incubated at 37°C for 10 min. After this time, the nonadherent cells were removed, and the monolayers were lysed in a cocktail of protease inhibitors. Lysates were stored at -80°C until analysis. VE-cadherin, ß-catenin, and PECAM-1 were immunoprecipitated from these lysates (equivalent of one C6 used for each lane), as described in Materials and Methods, and a typical result is shown in Figure 4
. In the absence of neutrophils (lanes 1 and 4), full-length VE-cadherin (140 kDa) and ß-catenin (105 kDa) are present, with low levels of truncated proteins. Preincubation of murine EC with WT neutrophils (lanes 2 and 5) resulted in a significant reduction in the native forms of VE-cadherin (twofold decrease) and ß-catenin (tenfold decrease, as determined by densitometric scanning using NIH Image software) and a significant increase in the corresponding degradation products. For VE-cadherin, the 100 kDa fragment showed a fourfold increase in the presence of WT neutrophils, and ß-catenin (75 kDa fragment) showed a 15-fold increase in both cases. In the presence of NE-deficient (lane 6) or MMP-9-deficient neutrophils (lane 3), VE-cadherin and ß-catenin were degraded to a similar extent as seen with the WT neutrophils, and there was a similar concomitant increase in the degradation products at 100 and 75 kDa, respectively. As published previously for human neutrophils [2
, 4
], PECAM-1 remained unchanged under all conditions (Fig. 4)
, indicating some specificity in the degradation process. Taken together, these results indicate that in this system, murine neutrophil elastase or MMP-9 is not responsible for the degradation of murine VE-cadherin complex, and because MMP-9 or NE is not required for neutrophil transendothelial migration, they further support the concept that degradation of VE-cadherin complex is a nonphysiological event unrelated to migration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To overcome problems inherent in use of inhibitors, such as specificity and cellular toxicity, we used a deficient mouse model of transmigration under conditions of in vitro flow. Functionally mature neutrophils were isolated from the bone marrow of WT mice or mice deficient in NE or MMP-9, and their interactions with confluent monolayers of TNF--activated murine EC from WT strain-matched mice were observed under flow conditions. Using this model, no differences were observed in the behavior of neutrophils from either of the deficient mice strains when compared with WT with respect to attachment, rolling, adhesion, or transmigration. Although these data contrast strongly with those published previously for human neutrophils using elastase inhibitors and a static assay [5
6
7
], there are further data to support these observations.
Previously, we had demonstrated that human neutrophil elastase inhibitor could prevent human neutrophil-dependent degradation of HUVEC VE-cadherin [3 ], and this is supported by Carden et al. [32 ], who demonstrated that activated human neutrophils can degrade VE-cadherin but that these events could be inhibited by a specific elastase inhibitor. In addition, our laboratory (unpublished results) and others [32 ] have demonstrated directly that human neutrophil elastase can degrade VE-cadherin in vitro. In contrast, in our murine system, mice deficient in neutrophil elastase or MMP-9 retained the ability to degrade VE-cadherin, implying that these enzymes alone are not responsible for VE-cadherin degradation in this model. Importantly, although WT murine neutrophils exhibited similar levels of elastase activity as compared with human neutrophils, direct activation of NE-deficient neutrophils did not elicit any elastase-type activity (unpublished results), supporting the hypothesis that other enzymes are capable of degrading VE-cadherin. These data do not preclude the idea that in WT mice, the principle enzyme responsible for VE-cadherin degradation is elastase, however, because there may be redundancy in the deficient mice. It is interesting that Cepinskas et al. [6 ] could not inhibit PAF-dependent neutrophil transendothelial migration with a specific inhibitor of neutrophil elastase at high PAF concentrations, implying that at least one other mechanism exists to mediate these events, even in the human system.
These other mechanisms may not require neutrophil enzyme activity at all. The hypothesis that VE-cadherin and other adherens junction-associated proteins or proteins localized to the lateral junctions (such as PECAM-1) are the barrier to transmigration has been tested by many investigators. Although it is apparent that these lateral junction proteins play a prominent role in neutrophil transendothelial migration, they cannot account for all of these migratory events. For example, in the PECAM-1-null mouse, there appears to be no impediment to leukocyte emigration [33 ]. Indeed the fact that PAF-dependent migration cannot be inhibited at high concentrations of PAF suggests that a second mechanism of transmigration exists. It has been shown [34 ] that lateral-junction protein-independent migration occurs away from the lateral junctions via transcellular routes in response to chemoattractants rather than via more accepted paracellular routes. This would require an active participation by the endothelium that diminishes a role for neutrophil-derived proteases.
So, what role do elastase or other neutrophil-derived proteases have to play in neutrophil transendothelial migration? It would be irresponsible to dismiss these serine proteases as irrelevant to migration and required only for clearance of bacteria or other foreign bodies. Certainly there are data demonstrating tight control of elastase localization during transmigration in vitro, indicating that elastase is mobilized to the migrating front of neutrophil pseudopodia [7
], but perhaps the role of elastase has been misinterpreted in this setting. Recent data [35
] have demonstrated high expression of neutrophil elastase, MMP-9, and activation of MMP-2 in the subepithelial stroma of the colitic gut localized to infiltrating leukocytes. Translocation of these enzymes to the extracellular membrane appears to occur during transendothelial migration as circulating leukocytes leave the vasculature rather than as a prerequisite to transmigration as suggested by Cepinskas and coworkers [7
]. However, the target(s) of these proteases appears to be the subepithelial extracellular matrix, and most extensive degradation is seen distal to the endothelium within the subepithelial compartment and the least damage, observed at the site of emigration, suggesting that elastase or MMP-9 remains relatively inactive until the infiltrating leukocytes reach their target. Therefore, it is possible that in vivo, endogenous inhibitors exist in the circulation to prevent erroneous degradation of endothelial junction components by activated neutrophils. In the absence of these inhibitors, this control mechanism does not exist, and the elastase becomes active and is permitted to degrade components of the endothelial monolayer or more likely, the subendothelial matrix. Alternatively, the pathway of neutrophil activation might dictate the activity of elastase or MMP-9. For example, transforming growth factor-ß activates many serine proteases but down-regulates MMPs, whereas TNF- up-regulates most MMPs [36
]. In the present study, endothelial monolayers were activated with TNF-, presumably up-regulating endothelial MMPs. In contrast, previous studies have used PAF to activate neutrophils directly and as a chemoattractant, and undoubtedly, the local environment is different from that induced by TNF- activation, which could help explain differences between the studies.
The strongest support for our notion that neutrophil proteases are not a necessary component of transendothelial migration comes from a recent publication by Shaw et al. [37 ]. In their study, transmigration of human neutrophils was evaluated live-time under flow conditions using HUVEC infected with fluorescent constructs of VE-cadherin (VE-cadGFP). The authors demonstrate a rapid but focal dispersion of VE-cadGFP contacts to the local cell membrane during transmigration and more importantly, a rapid reformation of those contacts with what appears to be the same VE-cadGFP immediately after the neutrophil has passed into the basement membrane. These data and our previous study [3 ] strongly suggest that leukocyte transmigration, at least in this in vitro system, does not require neutrophil-derived proteases.
The present study has demonstrated that neutrophil elastase or gelatinase B alone is not required for neutrophil transendothelial migration in vitro and that other enzymes are present in the murine neutrophil that are capable of degrading VE-cadherin in murine cardiac endothelium. However, the role of these proteases in physiological transmigration is questionable. Because neutrophils from elastase and MMP-9-deficient mice were capable of transendothelial migration, no definitive role for these enzymes in transmigration has been ascertained, suggesting that the mechanisms mediating neutrophil transendothelial migration in vivo are numerous and complex, involving not only the leukocyte but also the underlying endothelium. The roles of cytokines, adhesion molecules, state of activation, and differing vascular beds have not been addressed or understood adequately, and there is an extensive scope for further study in this regard.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received March 5, 2001; revised November 18, 2001; accepted December 28, 2001.
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REFERENCES |
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