3 Department of Medicine, Division of Infectious Diseases, University of Maryland School of Medicine Baltimore, MD 21201; 4 Immunology and Disease Resistance Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705; 5 Program in Oncology Greenebaum Cancer Center, University of Maryland School of Medicine Baltimore, MD 21201; and 6 Department of Veterans Affairs Medical Center, Mucosal Biology Research Center, 10 North Greene Street, University of Maryland School of Medicine Baltimore, MD 21201
Received on January 5, 2003; revised on February 18, 2004; accepted on February 19, 2004
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Abstract |
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Key words: adhesion molecules / endothelial cells / neuraminidase / neutrophils / sialidase
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Introduction |
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Neuraminidases (NANases) are enzymes that cleave sialyl residues from cell surface glycoconjugates and have been reported in a variety of microorganisms, such as viruses, bacteria, and protozoa (Schauer, 1982) as well as in mammalian cells, where they are referred to as sialidases (Ferrari et al., 1994
; Miyagi et al., 1992
). Each NANase/sialidase possesses a conserved structural motif that has been proposed as the operative catalytic domain for the cleavage of specific sialic acid residues (Roggentin et al., 1993
). Cleavage of dissimilar sialic acid glycosidic linkages requires distinct forms of NANase. In addition, each sialidase may differ in its tissue and subcellular distribution as well as in its biochemical properties (e.g., molecular weight, thermal stability). Mammalian sialidases have been cloned from the cytosol of rat skeletal muscle (Miyagi et al., 1993
), the Chinese hamster ovary cell (Ferrari et al., 1994
), and from the major histocompatibility complex of human Epstein-Barr virustransformed lymphoblastoid cells (Milner et al., 1997
). However, their role in the physiologic function of these cells has not been well defined.
Endogenous sialidase activity has been demonstrated in PMNs (Chari and Nath, 1984; Cross and Wright, 1991
; Verheijen et al., 1983
). We have previously observed that in human PMNs, sialidase exists in a preformed pool within a rapidly mobilizable compartment (Cross and Wright, 1991
). On activation by various stimuli, the granule- associated sialidase is translocated to the plasma membrane. This translocated sialidase then removes sialic acid from glycoconjugates on its own and adjacent PMN surfaces (Cross and Wright, 1991
). One functional consequence of this desialylation is an enhanced PMN capacity for aggregation and adherence to substrata. Based on the similarity among amino acid sequences of NANases from a diverse array of bacteria and viruses, a sialidase enzyme superfamily has been proposed (Roggentin et al., 1993
). We have extended this superfamily to include mammalian PMN sialidase. Polyclonal antibody obtained from rabbits immunized with clostridial NANase bound to sialidase on the surface of IL-8-activated human and murine PMNs both in vitro and in vivo (Cross et al., 2003
). This anti-NANase IgG inhibited pulmonary leukostasis in mice systemically infused with cobra venom factor and extravasation of PMNs into the alveoli of mice intranasally administrated IL-8.
Because anti-NANase antibody inhibited PMN adhesion to the pulmonary vascular endothelial surface (i.e., pulmonary leukostasis) as well as subsequent transendothelial migration (TEM) of PMNs into the bronchoalveolar compartment, we asked whether endogenous sialidase activity might regulate these PMNendothelial cell (EC) interactions. We reasoned that the mobilized PMN sialidase might play an important role in adhesion through surface desialylation not only of PMNs but also of pulmonary vascular ECs. To more directly test this hypothesis in vitro, in the absence of the many variables seen in vivo, we studied the effect of desialylation with exogenous, bacteria-derived NANase in the absence of EC activation and the contribution of endogenous sialidase activity to PMN adhesion to and migration across the pulmonary vascular EC barrier. In these studies, we present evidence that PMN-associated sialidase activity renders the pulmonary vascular endothelial surface hyperadherent for circulating unstimulated PMNs, promoting their paracellular movement across the endothelial barrier. This novel hyperadherent state did not require de novo protein synthesis or expression of the EC adhesion molecules, endothelial leukocyte-adherence molecule type 1 (ELAM-1 or E-selectin) or intercellular adhesion molecule (ICAM)-1 or CD54, or involvement of the PMN ß2 integrin, CD11b/CD18.
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Results |
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PMN-associated sialidase activity directly desialylates the EC surface
Activated PMNs induce increments in adhesion and TEM of resting PMNs that can be blocked by the sialidase inhibitor 2-deoxy NANA (Figures 6 and 8). To allow direct observation of whether activated PMNs desialylate the EC surface, EC monolayers preincubated with resting or activated PMNs were studied by fluorescein isothiocyanate (FITC)-PNA lectin fluorescence microscopy (Figure 9). The A.hypogaea (PNA) lectin detects subterminal galactose residues unmasked by desialylation (Novogrodsky et al., 1975). Monolayers treated with clostridial NANase revealed numerous discrete areas of increased PNA lectin binding compared to the media control (Figure 9AB). EC monolayers preincubated with activated PMNs revealed an increase in both the number and size of discrete ellipsoid areas of increased PNA binding compared to monolayers preincubated with resting PMNs (Figure 9CD). This increase in areas of desialylation on the EC surface was blocked by prior sialidase inhibition (Figure 9E) but not by KDO (Figure 9F). Therefore activated PMNs in close proximity to the endothelium leads to spatially restricted areas of desialylation over the EC surface. It is possible that these discrete areas of desialylation offer hyperadhesive sites for PMN adhesion and eventual extravasation.
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Discussion |
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We recently have demonstrated the physiological relevance of PMN-associated sialidase activity in vivo (Cross et al., 2003). To extend our findings to an in vitro system that precludes multiple confounding variables that come into play in vivo, we introduced established inhibitors of NANase into our adhesion/TEM assays. Inhibition of endogenous sialidase activity by either immunoblockade with anti-NANase antibody or competitive pseudosubstrate inhibition with 2-deoxy NANA (Table II), decreased adhesion of fMLP-activated PMNs to resting ECs (Figure 5A) but did not decrease adhesion of resting PMNs to TNF-activated EC (Figure 5B). These findings suggested that the endogenous sialidase activity was PMN-associated. In fact, mobilization of this PMN-associated sialidase activity directly desialylated the EC surface (Figure 9CD), and enhanced resting PMN adhesion to (Figure 6) and migration across (Figure 8C) the endothelium. These data indicate that PMN-associated sialidase activity that is mobilized to and remains associated with the plasma membrane (Cross et al., 2003
; Cross and Wright, 1991
), may remove sialic acid residues not only from the PMN surface but from the EC surface as well. Prior protein synthesis inhibition and blocking antibodies against E-selectin, CD54, and CD18 each blocked PMN adhesion to TNF-activated EC, whereas these same interventions failed to diminish the increased adhesiveness seen after PMN-mediated desialylation of the EC surface. That such desialylation increases adhesive properties of ECs and is not blocked by several adhesion moleculeblocking antibodies might implicate a novel regulatory mechanism(s) for PMN-EC adhesion.
In selected experiments, NANase extracted from C. perfringens by ammonium sulfate fractionation was introduced to desialylate either PMNs (Figures 1A, 1C, and 2) and/or ECs (Figures 1BC, 2, 3, and 8AB). Clostridial NANase hydrolyzes a range of glycosidic linkages of terminal sialic residues including -(2
3),
-(2
6), and
-(2
8) linkages (Cassidy et al., 1965
; Roggentin et al., 1995
). Because NANase preparations may contain proteases, LPS, or other contaminants, multiple interventions were introduced to help establish that its effect on PMNEC adhesion was mediated solely through NANase activity. First, activity was inhibited by either 2-deoxy NANA or anti-NANase IgG but not by preimmune IgG (Table I), demonstrating the specificity of both the antiNANase antibody and the assay. That the NANase activity was destroyed by boiling but undiminished by PMB preadsorption, together with the lack of sialidase activity in a pure LPS preparation, excluded endotoxin contamination as a contributing factor. Finally, preincubation of the clostridial NANase preparation with a cocktail of three potent protease inhibitors (aprotinin, leupeptin, and PMSF), did not decrease its NANase activity. In the PMNEC adhesion assay, the NANase-induced increments in PMN adhesion were evident after exposure times as brief as 10 min (Figure 1C) and were inhibited by either prior boiling or coadministration with 2-deoxy NANA but not by KDO, protease inhibitors, or ENP (Figure 2). The clostridial NANase preparation also failed to increase EC expression of either the adhesion molecules, E-selectin and ICAM-1 (Figure 3A) or the cytokines IL-8 and IL-6 (Figure 3B), whereas LPS did. These combined data indicate that the effects of the NANase preparation on either PMN or EC function cannot be ascribed solely to either LPS or protease contamination but can be attributed to true NANase activity.
Desialylation of PMNs through a brief exposure (0.5 h) to exogenous NANase induced dose-dependent increments in PMN adhesion to resting ECs at concentrations 10 mU/ml (Figure 1A). Desialylation of EC monolayers with both brief (
10 min) (Figure 1C) and more prolonged (46 h) (Figure 1B) exposures to the same NANase preparation also increased resting PMN adhesion and TEM (Figure 8A) at concentrations
10 mU/ml, at which point the effect on PMN adhesion plateaued. It is possible that after the more prolonged duration of desialylation with 10 mU/ml, almost all NANase-susceptible sialic acid linkages were already cleaved, preventing further increases in adhesion with dose escalation. Of interest, desialylation of EC monolayers increased their adhesiveness for PMNs independent of EC expression of either adhesion molecules and/or cytokines (Figure 2). In our studies, where NANase exposure times as brief as 10 min were sufficient to increase adhesion (Figure 1C) and intervals of
1 h, were sufficient for increments in TEM (Figure 8B), protein synthesisdependent adhesion molecule expression was unlikely (Cines et al., 1998
). These data are compatible with the previous demonstration that PMN sialidase activity increases PMN adhesion to nonbiological substrata that lack adhesion molecules (Cross and Wright, 1991
).
The minimal NANase concentrations that increased PMNEC adhesion (Figure 1AB-) and TEM (Figure 8A) were 10 mU/ml and 1.0 mU/ml, respectively. How this level of NANase activity compares with endogenous sialidase activity is unclear. We quantified NANase activity in human PMN lysates (Table II) and from this data estimated that 1.0 mU sialidase activity would reflect 3.7 x 107 human PMNs. The number of circulating PMNs that would reflect these levels of sialidase activity is difficult to estimate, especially if one considers the rolling and either loosely tethered or tightly adherent PMNs that make up the marginated pool as well as the known pH optima and substrate specificities for each sialidase. Under pathological conditions where tissue leukostasis precedes PMN recruitment to extravascular tissues and body compartments (Cross et al., 2003
), such levels of PMNs and sialidase activity might be anticipated (e.g., meningitis, pneumonia, abscess). Finally, desialylation of both PMNs and ECs resulted in greater PMNEC adhesion than did desialylation of either PMNs or EC alone (Figure 1D). This suggests that if a sialidase associated with either cell type could desialylate glycoconjugates on the surface of both myeloid and endothelial cells, its effect on adhesion would be further enhanced.
We have previously demonstrated that on activation, PMN sialidase activity is mobilized to the cell surface, where it is strategically positioned to remove sialic acid residues (Cross et al., 2003; Cross and Wright, 1991
). Because desialylation of EC monolayers with exogenous NANase increases their adhesiveness for PMNs (Figure 1B and C), whereas endogenous sialidase activity appears to be PMN-associated (Figure 5 and Table II), we asked whether PMN-associated sialidase(s) might regulate the sialylation state and adhesive properties of the EC surface. Preincubation of EC monolayers with increasing concentrations of activated PMNs for increasing times, enhanced adhesion of resting PMNs to EC monolayers in a PMN concentration and exposure timedependent manner (Figure 6BC). This increased adhesion did not require de novo protein synthesis and was not diminished by blocking antibodies raised against the adhesion molecules, E-selectin, ICAM-1, and CD18 (Figure 7BC). That this PMN-EC interaction is blocked by selective sialidase inhibition (Figure 6A) but not protein synthesis inhibition (Figure 7B) and is independent of adhesion molecule engagement (Figure 7BC) suggests a novel PMN sialidasemediated mechanism(s) for PMNEC adhesion. Although PMN activation can generate multiple products that can elicit an EC response, including proteases (Carden et al., 1998
; Varani et al., 1989
) and oxygen intermediates (Varani et al., 1989
), the PMN- associated activity described here was inhibitable by a specific sialidase inhibitor, 2-deoxy NANA, but not by KDO (Figures 4AB, 5A, 6A, 8C).
Because prior desialylation of the EC surface increases PMN adhesion (Figures 1BC, 6AC) and adhesion to the EC barrier is a prerequisite step for PMN diapedesis (Cines et al., 1998), we asked whether preincubation of EC monolayers with activated PMNs might also facilitate TEM of unstimulated PMNs (Figure 8C). TEM was increased compared to migration across EC monolayers preincubated with resting PMNs, and this increase was inhibitable by the sialidase inhibitor, 2-deoxy NANA. When FITC-PNA fluorescence microscopy was applied to similarly treated EC monolayers, ECs incubated with activated PMNs revealed discrete areas of increased PNA binding (Figure 9D) not seen on monolayers incubated with resting PMNs (Figure 9C); this increase in signal was diminished by the sialidase inhibitor 2-deoxy NANA (Figure 9E) but not the KDO control (Figure 9F). These findings demonstrate that PMN activation mobilizes sialidase activity that prepares the EC surface through direct desialylation that is both temporally (Figure 6C) and spatially (Figure 9D) restricted resulting in regionally increased adhesion of unstimulated PMNs. It is possible that mobilization of PMN sialidase provides a mechanism by which activated PMNs can amplify the inflammatory response through the recruitment of unstimulated resting PMNs. The requirement for a sustained PMNEC interaction (
1 h) (Figure 6C) involving a sufficient number of PMNs (
106 cells) (Figure 6B) indicate that this amplification mechanism is reserved for a committed inflammatory response and not transient perturbations.
The PMN-associated sialidase activity may regulate PMNEC interactions through one or more mechanisms. We previously have demonstrated, using flow cytometry, that activation of PMNs is associated with increased surface expression of sialidase as detected by anti-NANase IgG and decreased surface sialic acid as detected by both decreased L. flavus lectin binding and increased PNA lectin binding (Cross et al., 2003). These surface changes were temporally coincident with increased PMNPMN aggregation (Cross and Wright, 1991
). These data suggest that through autocrine and/or paracrine desialylation, PMN homotypic adhesion was increased. Whether this hyperadhesiveness results from selective desialylation of specific surface glycoconjugates is unknown. PMNs and ECs both express membrane-spanning glycoproteins with ectodomains that participate in homophilic adhesion with identical molecules on neighboring cells (e.g., platelet-endothelial cell adhesion molecule-1 [PECAM-1]; Sun et al., 1996
). CD43, also known as leukosialin, is a highly sialylated molecule on the surface of PMNs that participates in PMN adhesion and spreading (Nathan et al., 1993
). Of note, PMN sialidase activity has been colocalized to the same subcellular compartment as ß2 integrin or CD11b/CD18 (Cross and Wright, 1991
), the ligand for CD54. Whether one or more of these surface structures are substrates for PMN-associated sialidase is unknown.
It also is conceivable that PMN-associated sialidase can regulate PMN adhesion through global desialylation with decreases in both net negative surface charge and cellcell repulsion (Gallin, 1980; Lichtman and Weed, 1970
, 1972
). Such desialylation is known to alter cellular deformability, motility, and adhesiveness. That the desialylation associated with activation enhances PMN adhesion to uncoated nylon and plastic substrata (Cross and Wright, 1991
) is compatible with an interaction that does not require binding between a specific ligand and counterligand.
In the current report, we show that PMN-associated sialidase activity not only regulates PMN surface adhesiveness but EC adhesiveness as well (Figure 6A). Again, whether selective desialylation of constitutively expressed surface glycoproteins (e.g., leukosialin or CD43, PECAM-1) that participate in PMNEC interactions (Nathan et al., 1993; Sun et al., 1996
) and/or less specific modifications augment adhesion is unclear. The ability of PMNs to desialylate the EC surface may also influence EC adhesiveness for other circulating cells, including erythrocytes, monocytes, lymphocytes, platelets, and even tumor cells. It is known that circulating bone marrowderived EC stem cells participate in vascular repair (Takahashi et al., 1999
). A hyperadherent EC surface might help recruit these cells to areas of EC injury. We have shown that sialidase activity presented to the EC surface through PMN activation enhances TEM (Figure 8C). Although the most likely explanation for this increase in TEM is the increase in adhesion, a proximal step in diapedesis, it is also possible that desialylation of one or more EC receptors is coupled to intracellular signaling events that open the paracellular pathway. We have preliminary data that desialylation of EC monolayers increases tyrosine phosphorylation of EC proteins and increases transendothelial albumin flux (unpublished observation). There also is evidence that PMNs play a role in angiogenesis in the context of wound healing (Gaudry et al., 1997
; Kibbey et al., 1994
). Perhaps PMN-mediated EC desialylation modulates EC adhesion and motility, thereby promoting vascular sprouting and new vessel formation.
Because all mammalian cells are sialylated and PMNs can migrate through all tissues, PMN-associated sialidase activity may desialylate cells other than PMNs and ECs. Prior desialylation of epithelial cells are known to unmask glycosylated receptors for attaching bacteria (Galen et al., 1992). For example, prior influenza infection increases risk of bacterial superinfection and prior neuraminidase inhibition projects against this increased risk (McCullers and Bartmess, 2003
). It is conceivable that PMN recruitment to inflamed airways allows for increased colonization and promotes ongoing bronchitis. Finally, it is possible that PMN sialidase activity may selectively desialylate sialylated receptors engaged by informational molecules that are involved in key biological processes. Because multiple sialidases in concert with sialylotransferases, each with distinct substrate specificities, reside within specific subcellular compartments of cells in diverse tissues, the ability to regulate the state of sialylation of cell surface glycoproteins surely provides an additional layer of dynamic regulation over proteinprotein and cellcell interactions, signaling, and cell behavior.
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Materials and methods |
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EC culture
Bovine pulmonary artery ECs (American Tissue Culture Collection, Rockville MD) were cultured at 37°C under 5% CO2 in Dulbecco's modified Eagles medium (Sigma) enriched with 20% heat-inactivated (56°C, 30 min) fetal bovine serum (Hyclone Laboratories, Logan, UT), L- glutamine, nonessential amino acids, and vitamins in the presence of penicillin and streptomycin as described (Goldblum and Sun, 1990). Human pulmonary artery EC (Clonetics, San Diego, CA) were cultured as described (Campbell et al., 1997
) with minor modifications using medium containing human recombinant epidermal growth factor, insulin-like growth factor-1, vascular endothelial growth factor, human recombinant basic fibroblast growth factor, hydrocortisone, 5% fetal bovine serum, gentamicin, amphotericin B, and ascorbic acid (Clonetics). Only ECs from passages 26 were studied.
PMN preparation and fluorescent labeling
Whole peripheral blood from healthy human volunteers was collected under a protocol approved by the University of Maryland, Baltimore, Institutional Review Board, into acid citrate dextran (Sigma) solution, and PMNs were isolated by dextran erythrocyte sedimentation and density gradient centrifugation through ficoll-hypaque (Sigma) as previously described (Cross and Wright, 1991). PMNs were resuspended in HBSS without divalent cations at 107 PMNs/ml and were incubated with 5 µM calcein AM (Molecular Probes, Eugene, OR) for 40 min with gentle agitation in the dark (Akeson and Woods, 1993
). Calcein AM fluoresces on cleavage by intracellular esterases (Weston and Parish, 1990
). PMNs were washed three times with HBSS after which their purity was >95% and viability >98% by trypan blue dye exclusion.
PMN adhesion
ECs were seeded (2 x 105 bovine ECs/well, 1 x 105 human ECs/well) into the wells of 24-well culture plates (Costar, Cambridge, MA), in 1 ml media and cultured to confluence (72 h, 37°C, 5% CO2). The EC monolayers were preincubated with TNF (25 U/ml, 4 h), NANase (0.3100 mU/ml, 10 min4 h), or media alone and gently washed with PBS. For 0.5 h prior to and throughout the TNF exposure, EC were treated with cycloheximide (50 µg/ml) or media alone. This treatment inhibits 95% of EC protein synthesis as measured by 35S-methionine incorporation into trichloroacetic acid precipitable protein (Goldblum and Sun, 1990
). Calcein AM-labeled PMNs were pretreated with NANase (0.3100 mU/ml), fMLP (107 M), or media alone, washed, and incubated for 30 min with EC monolayers (5 x 105 PMNs/well). In selected experiments, labeled PMNs were incubated with TNF-treated EC monolayers in the presence of antibodies raised against E-selectin (20 µg/ml) (Serotec), CD54 (20 µg/ml) (Pharmingen), or CD18 (20 µg/ml) (Immunotech) or an equivalent concentration of a species- and isotype-matched irrelevant antibody control (Pharmingen). The ECs were preincubated with anti-E-selectin and anti-CD54 antibodies, whereas PMNs were preincubated with anti-CD18 antibody. After gentle washing to remove nonadherent PMNs, the attached PMNs were flourometrically assayed (excitation 485 nm, emission 530 nm) in a Cytofluor II Multi-well Fluorescence plate reader (PerSeptive Biosystems, Framingham, MA).
For each experiment, serial dilutions of labeled PMNs (1 x 105103 cells/ml) were used to generate a standard curve from which PMN numbers could be interpolated from fluorescence units and PMNEC adhesion was expressed as % adhesion. In other experiments, postconfluent human EC monolayers were preincubated for increasing times with increasing concentrations of either resting or activated unlabeled PMNs. For 0.5 h prior to and throughout the preincubation of ECs with fMLP-activated PMNs, the ECs were treated with cycloheximide (50 µ/ml) or media alone. The monolayers were washed four times. These four washes removed >98% PMNs (56,536 ± 9624 to 771 ± 61 fluoresence units, n = 5) as measured in separate experiments using calcein AM-labeled cells. The EC monolayers were then incubated for 0.5 h with unstimulated calcein AM-labeled PMNs (5 x 105 PMNs/ml) in the presence of antiCD62, antiCD54, species- and isotype-matched antibodies, or media alone and again washed; the attached PMNs were fluorometrically assayed. The unlabeled PMNs were activated (fMLP 106 M/cytochalasin B 5 µg/ml x 15 min) in the presence and absence of equimolar concentrations (250 µg/ml) of either 2-deoxy NANA, a sialidase inhibitor, or KDO, a molecule of similar molecular weight and charge in a separate container, not in the presence of the EC monolayers.
TEM of PMNs
Polycarbonate filters (13 mm diameter, 3 mm pore size; Nucleopore, Pleasanton, CA) were treated with 0.5% acetic acid (50°C, 20 min), washed in distilled H2O, and immersed in boiling pig skin gelatin (Fisher Scientific, Pittsburgh, PA) solution (5 mg/L distilled H2O) for 60 min as previously described (Goldblum and Sun, 1990). The filters were dried, glued to polystyrene chemotactic chambers (ADAPS, Dedham, MA), and gas sterilized with ethylene oxide. These chambers, which serve as the upper compartment for the assay chambers, were inserted into the wells of 24-well plates, each well containing 1.5 ml media and serving as the lower compartment of the assay chamber. The upper compartment of each assay chamber was seeded with 1.5 x 105 HPAEC/chamber in 0.5 ml media and cultured to confluence (72 h, 37°C, 5% CO2). To establish functional integrity for each monolayer, transendothelial 14C-BSA flux was determined as previously described (Goldblum and Sun, 1990
). Only EC monolayers retaining
97% of the 14C-BSA tracer were studied. The EC monolayers cultured on filter supports were treated with increasing concentrations of NANase for increasing times or preincubated for 1 h with either unstimulated or activated unlabeled PMNs (106 cells/ml), washed, and inserted into wells containing IL-8 (100 ng/ml), fMLP (106M), or media alone. Calcein-AM-labeled PMNs (5 x 105 cells/well) were introduced into the upper compartments of assay chambers, incubated for 2 h at 37°C, after which time each lower compartment was sampled and fluorometrically assayed. After migration through EC monolayers, >99% of fluorescence remained PMN-associated (data not shown). Again, a standard curve was established for each experiment from which PMN numbers could be interpolated from fluorescence units and PMN TEM was expressed as % migration.
NANase effect on EC expression of adhesion molecules and cytokine release
Surface expression of ICAM-1 and E-selectin and release of IL-8 and IL-6 were used as markers of EC activation (Carlos and Harlan, 1994; Cines et al., 1998
; Schleiffenbaum and Fehr, 1996
). Confluent EC monolayers cultured in 24-well plates were preincubated with NANase, TNF, LPS, or media alone for 4 h and gently washed with ice-cold PBS. Monolayers were incubated with FITC-conjugated monoclonal anti-human CD54 (ICAM-1) (Sigma), monoclonal anti-human E-selectin (Sigma), or mouse isotype IgG1 (Sigma) for 0.5 h in the dark at 28°C. After gentle washing with cold PBS with 0.1% azide three times, EC monolayers were flourometrically assayed (excitation 485 nm, emission 530 nm) in a multiwell fluorescence plate reader. Concentrations of IL-8 and IL-6 in culture supernatants were measured by standard two-antibody enzyme-linked immunosorbent assay using recombinant standards and paired antibodies from Biosource (Camarillo, CA) and Endogen (Woburn, MA), respectively, and biotin-strepavidin- peroxidase detection as previously described (Jiang et al., 1999
). The detection limit of each assay was 6 pg/ml.
Assay of NANase/sialidase activity
C. perfringens NANase and PMN sialidase activities were measured by the thiobarbituric acid assay as previously described (Cross and Wright, 1991). Samples to be assayed for NANase/sialidase activity were incubated (10 min, 37°C) with the artificial substrate, N-acetylneuraminyl-lactose in 100 mM sodium acetate, 2 mM CaCl2, pH 5.0, and the liberated sialic acid was measured by the thiobarbituric acid assay. For each assay, increasing concentrations of NANA standard solution were used to generate a standard curve from which mU of NANA liberated from N-acetylneuraminyl-lactose was determined. For NANase/sialidase activity inhibition, samples were preincubated with anti-NANase IgG, 2-deoxy NANA, or preimmune IgG for 0.5 h at 37°C before addition to N-acetylneuraminyl-lactose and assayed with thiobarbituric acid assay.
PNA lectin fluorescence microscopy
To demonstrate that PMN-associated sialidase activity directly desialylates the EC surface, a peanut lectin that specifically recognizes subterminal ß-galactose after sialic acid removal and reacts strongly with desialylated glycoproteins was used (Novogrodsky et al., 1975). Human ECs cultured to postconfluence in Lab-Tek chamber slides (Nalge Nunc, Naperville, IL) were preincubated for 6 h with clostridial NANase 100 mU/ml or media alone, or resting or fMLP-activated unlabeled PMNs (1.6 x 107 PMNs/chamber) in the presence or absence of equimolar concentrations of either 2-deoxy NANA or KDO (300 µg/ml). The EC monolayers were washed and incubated for 0.5 h with 50 µg/ml FITC-conjugated, affinity-purified PNA, a lectin isolated from A. hypogaea (EY Lab, San Mateo, CA). Monolayers were again washed, fixed (glutaraldelhyde 0.5%, 10 min, RT) and visualized and photographed through a Zeiss Axioscop 20 microscope (Carl Zeiss, Thornwood, NY) equipped for epifluorescence.
Statistical methods
Analysis of variance was used to compare the mean responses among experimental and control groups. A p-value of <0.05 was considered significant. Linear regression analyses of standard curves were used to calculate cell number.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Medicine, Division of Microbiology and Infectious Diseases, Adnan Menderes University School of Medicine, Aydin, Turkey
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Abbreviations |
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References |
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