Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

Serhan Sakarya1,3, Salahaldin Rifat3, Jie Zhou3, Douglas D. Bannerman4, Nicholas M. Stamatos3, Alan S. Cross3,5 and Simeon E. Goldblum2,3,6

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor–activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.

Key words: adhesion molecules / endothelial cells / neuraminidase / neutrophils / sialidase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sialic acid consists of a family of amino sugars expressed on all living cells as glycoconjugates, which impart a negative electric charge to the cell surface. Within the bone marrow, myeloid precursors are characterized in part by a high neuraminidase-susceptible sialic acid content, negative net surface charge, a high degree of cellular rigidity, hypoadhesiveness, as well as impaired pseudopod extension and phagocytic activity (Lichtman and Weed, 1972Go; Lund-Johansen and Terstappen, 1993Go). As these myeloid cells mature and leave the bone marrow, sialic acid is progressively lost from their surface (Lichtman and Weed, 1972Go). This desialylation is associated with increases in cellular deformability, motility, adhesiveness and phagocytic potential (Lichtman and Weed, 1970Go, 1972Go). Once in the peripheral circulation, activation of neutrophils (PMNs) is associated with an additional loss of cell surface sialic acid with a further decrease in negative surface charge (Cross and Wright, 1991Go; Gallin, 1980Go).

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, 1982Go) as well as in mammalian cells, where they are referred to as sialidases (Ferrari et al., 1994Go; Miyagi et al., 1992Go). 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., 1993Go). 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., 1993Go), the Chinese hamster ovary cell (Ferrari et al., 1994Go), and from the major histocompatibility complex of human Epstein-Barr virus–transformed lymphoblastoid cells (Milner et al., 1997Go). 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, 1984Go; Cross and Wright, 1991Go; Verheijen et al., 1983Go). We have previously observed that in human PMNs, sialidase exists in a preformed pool within a rapidly mobilizable compartment (Cross and Wright, 1991Go). 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, 1991Go). 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., 1993Go). 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., 2003Go). 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 PMN–endothelial 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Effect of NANase on PMN adhesion to pulmonary vascular EC monolayers
Adhesion of NANase-treated PMNs to unstimulated bovine pulmonary artery EC monolayers was determined (Figure 1A). NANase treatment of PMNs (>=10 mU/ml, 0.5 h) increased their adherence to untreated bovine EC monolayers with further dose-dependent increments at 30 and 100 mU/ml. Similarly, NANase treatment of bovine ECs (>=10 mU/ml, 4 h) increased their adhesiveness for untreated PMNs (Figure 1B). However, maximal adhesion occurred at a NANase concentration of 10 mU/ml without further increments at higher concentrations. Exposure of human pulmonary artery ECs to a fixed concentration of NANase (30 mU/ml) increased adhesiveness for resting PMNs after exposure times as brief as 10 min (Figure 1C). PMN adhesion after NANase treatment of both PMNs and ECs was greater than that seen after treatment of either PMNs or ECs alone (Figure 1D). Thus removal of sialyl residues from either PMNs or ECs enhanced PMN–EC heterotypic interaction.



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Fig. 1. Effect of NANase on PMN adhesion to pulmonary vascular EC monolayers. Each bar represents mean (±SE) percent adhesion of PMNs to bovine (A, B, D) or human (C) EC monolayers after treatment of PMNs for 0.5 h with increasing concentrations of NANase (open bars) or media alone (closed bar) (A), after treatment of bovine ECs for 4 h with increasing concentrations of NANase (open bars) or media alone (closed bar) (B), after treatment of human ECs with a fixed concentration of NANase (30 mU/ml) (opens bars) or media alone (closed bars) for increasing exposure times (C), or after treatment of bovine ECs or PMNs alone (open bars), or both EC and PMN (cross-hatched bars) with NANase (10 mU/ml) or media alone (closed bar) (D). The n for each group is indicated in the bars. *, significantly increased compared to the media control at p < 0.001. **, significantly increased adhesion compared to NANase treatment of either PMNs or ECs alone at p < 0.001.

 
Specificity of NANase activity
NANase activity was measured as the release of sialic acid from an artificial N-acetylneuraminyl-lactose substrate (Table I). Anti-NANase IgG, the NANase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (2-deoxy NANA) or boiling (1.5 h) each decreased NANase activity compared to the untreated NANase, whereas the preimmune rabbit IgG did not. To exclude proteases (Park and Labbe, 1991Go) and bacterial lipopolysaccharide (LPS) contamination in our NANase preparation, we preincubated NANase with protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride [PMSF]) or polymyxin B (PMB); NANase activity was not decreased. As anticipated, protease inhibitors, LPS, PMB (400 µg/ml), LPS + PMB, and 2-deoxy NANA each lacked intrinsic NANase activity.


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Table I. Characterization of clostridial NANase

 
Because clostridial NANase activity was decreased by prior boiling or preincubation with 2-deoxy NANA (Table I), we assayed PMN adhesion to ECs following these same treatments of both PMNs and ECs. Both EC monolayers and PMNs were incubated with NANase, boiled NANase, NANase together with 2-deoxy NANA, or media alone. Prior boiling of NANase or its coadministration with 2-deoxy NANA diminished NANase-enhanced adhesion of PMNs to ECs, whereas the 2-keto-3-deoxyoctulosonic acid (KDO) control did not (Figure 2). Similarly, protease inhibitors as well as recombinant endotoxin neutralizing protein (rENP) did not protect against the NANase-enhanced adhesion, and neither ENP nor the protease inhibitors in themselves altered PMN adhesion to ECs compared to the simultaneous media controls.



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Fig. 2. Effect of NANase inhibitors, protease inhibitors and ENP on NANase-enhanced PMN adhesion to NANase-treated pulmonary vascular EC monolayers. Each bar represents mean (±SE) percent adhesion of PMNs to bovine EC monolayers after treatment of both PMNs and ECs with media alone, NANase (10 mU/ml), boiled NANase (10 mU/ml), NANase (10 mU/ml) preincubated with 2-deoxy NANA (200 µg/ml), KDO (200 µg/ml), protease inhibitors (aprotinin 10 µl/ml, leupeptin 10 µl/ml, PMSF 0.1 M) or ENP (100 µg/ml), or ENP or protease inhibitors alone. The n for each group is indicated in the bars. *, significantly decreased compared to NANase treatment at p < 0.001.

 
NANase effect on pulmonary vascular EC surface expression of adhesion molecules and cytokine release
EC surface expression of ICAM-1, also known as CD54 and E-selectin, also known as CD62E (Figure 3A) as well as release of IL-8 and IL-6 (Figure 3B) were determined following exposure of ECs to NANase, tumor necrosis factor alpha (TNF), LPS, or media alone. TNF and LPS increased EC surface expression of E-selectin and ICAM-1, whereas NANase treatment did not. No increase in either CD54 or E-selectin expression was seen in response to any of the stimuli in the presence of the species- and isotype-matched irrelevant antibody control. Similarly, TNF and LPS increased EC release of IL-8 and IL-6, whereas NANase did not. Although NANase treatment of EC was associated with a small increment in IL-8, this elevation did not reach statistical significance (p > 0.05). Therefore, NANase increased pulmonary vascular EC adhesiveness without a detectible increase in adhesion molecule expression.



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Fig. 3. Effect of NANase on pulmonary vascular EC activation. (A) Each bar represents mean (±SE) fluorescence units after incubation of EC monolayers with fluorescein-labeled anti-E-selectin or anti-ICAM-1 monoclonal antibodies or a murine monoclonal antibody (IgG1) control, and (B) IL-8 and IL-6 release from EC after incubation for 4 h with NANase (10 mU/ml), TNF (10 U/ml), LPS (10 ng/ml), or media alone. The n for each group is indicated in the bars. *, significantly increased compared to the simultaneous media control at p < 0.05.

 
Effect of inhibition of endogenous sialidase activity on PMN adhesion to bovine and/or human pulmonary vascular EC
Treatment of PMNs or ECs with bacterial NANase increased the adherence of PMNs to EC monolayers (Figure 1); however, unless there is an endogenous mechanism for this desialylating activity, this observation would have little physiologic significance. We (Cross et al., 2003Go; Cross and Wright, 1991Go) and others (Chari and Nath, 1984Go; Verheijen et al., 1983Go), have demonstrated sialidase activity in PMNs, but this enzyme has been neither purified nor cloned. On PMN activation, sialidase activity is translocated to the PMN plasma membrane (Cross et al., 2003Go; Cross and Wright, 1991Go). PMN sialidase activity was measured as the release of sialic acid from an artificial N-acetylneuraminyl-lactose substrate (Table II). Anti-NANase IgG (40 µg/ml) and 2-deoxy NANA (200 µg/ml) each decreased PMN lysate sialidase activity, whereas pretreatment with preimmune IgG did not. Thus human PMN sialidase activity, like its bacterial homolog, was inhibited by either immunologic or pharmacologic blockade.


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Table II. Inhibition of PMN lysate sialidase activity

 
To determine whether endogenous sialidase(s) contributes to PMN–EC adhesion in the activated state, adhesion of n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs to TNF-activated bovine EC monolayers was studied in the presence of anti-NANase IgG, preimmune rabbit IgG, 2-deoxy NANA, KDO, anti-NANase IgG + 2-deoxy NANA, preimmune rabbit IgG + KDO, or media alone (Figure 4A). Adhesion of fMLP-activated PMN to TNF-activated EC was decreased by anti-NANase IgG, 2-deoxy NANA, and the two in combination, but not by preimmune rabbit IgG, KDO, or KDO in the presence of preimmune rabbit IgG. There was no further decrease in adhesion in the presence of both anti-NANase IgG and 2-deoxy NANA compared to either intervention alone. When similar studies were performed with human ECs, introduction of anti-NANase IgG or 2-deoxy NANA was associated with comparable inhibition (Figure 4B). Thus endogenous sialidase activity appeared to play a role in PMN-EC adhesion.



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Fig. 4. Effect of inhibition of endogenous sialidase(s) activity on activated PMN adhesion to activated pulmonary vascular EC monolayers. Each bar represents mean (± SE) percent adhesion of fMLP (10–7 M)/cytochalasin B (5 µg/ml)–activated PMNs to TNF (25 U/ml)-activated EC monolayers. (A) Adhesion of PMNs to bovine EC in the presence of media alone, 2-deoxy NANA (200 µg/ml), anti-NANase IgG (50 µg/ml), anti-NANase IgG + 2-deoxy NANA, preimmune IgG (50 µg/ml), KDO (200 µg/ml), or preimmune IgG + KDO. (B) Adhesion of PMNs to human EC in the presence of media alone, 2-deoxy NANA (200 µg/ml), anti-NANase IgG (50 µg/ml), or preimmune IgG (50 µg/ml). The n for each group is indicated in each bar. *, significantly decreased compared to adhesion of fMLP/cytochalasin B-activated PMN to TNF-activated ECs at p < 0.01.

 
To determine whether the endogenous sialidase activity was associated with PMNs, ECs, or both, adhesion of either fMLP-stimulated PMNs to resting bovine EC monolayers (Figure 5A) or unstimulated PMNs to TNF-activated bovine EC (Figure 5B) was studied in the presence of anti-NANase IgG, preimmune rabbit IgG, 2-deoxy NANA, or media alone. Adhesion of fMLP-activated PMNs to resting bovine ECs was decreased ~50% by either anti-NANase IgG or 2-deoxy NANA (Figure 5A). These same inhibitors had no significant effect on adhesion of unstimulated PMNs to TNF-activated bovine EC (Figure 5B). These combined data suggest that either the endogenous sialidase activity responsible for PMN–EC adhesion is PMN- associated and/or that the inhibitors used in these studies are not active for any putative EC sialidase(s).



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Fig. 5. Effect of inhibition of endogenous sialidase(s) on adhesion of either activated PMNs to resting EC monolayers or resting PMNs to activated EC monolayers. Each bar represents mean (±SE) percent adhesion of fMLP (10–7 M)/cytochalasin B (5 µg/ml)–activated PMNs to resting EC (A), or adhesion of unactivated PMNs to TNF (25 U/ml, 4 h)–activated EC (B) in the presence of media alone, 2-deoxy NANA (200 µg/ml), anti-NANase IgG (50 µg/ml) or preimmune IgG (50 µg/ml). The n for each group is indicated in each bar. *, significantly decreased compared to adhesion of fMLP/cytochalasin B–activated PMN to resting EC monolayers at p < 0.01.

 
Effect of PMN-associated sialidase activity on human pulmonary vascular EC adhesiveness for unstimulated PMNs
Because EC desialylation with exogenous NANase clearly increases EC adhesiveness for PMNs (Figure 1B and C) but endogenous sialidase activity was associated with PMNs (Figure 5A) and not ECs (Figure 5B), we asked whether PMN-associated sialidase activity could regulate the adhesive state of ECs. Preincubation of resting EC monolayers with activated PMNs increased EC adhesiveness for unstimulated PMNs > twofold relative to EC adhesiveness after preincubation with resting PMNs (Figure 6A). This PMN-dependent increase in EC adhesiveness was completely blocked by the sialidase inhibitor 2-deoxy NANA, but not by its negative control, KDO. In this experimental system, preincubation with activated PMNs only at concentrations of >=106 cells/ml enhanced EC adhesiveness compared to preincubation with equivalent concentrations of unstimulated PMNs and 3 x 106 cells were no more active than were 106 cells (Figure 6B). When EC monolayers were preincubated with a fixed concentration of activated PMNs (106 cells/ml) for increasing times, only preincubation times of >=1 h increased EC adhesiveness, and a preincubation time of 2 h was associated with a greater increment in adhesion than was a 1-h preincubation time (Figure 6C). These combined data indicate that PMN activation with surface presentation of sialidase activity can render the endothelial surface hyperadherent for unstimulated PMNs in a PMN concentration– and exposure time–dependent manner.



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Fig. 6. Effect of mobilized PMN-associated sialidase activity on human pulmonary vascular EC adhesiveness for unstimulated PMNs. Human EC monolayers were preincubated with either unstimulated PMNs or PMNs activated with fMLP 10–6 M/cytochalasin B 5 µg/ml; washed; incubated with unstimulated, calcein AM-labeled PMNs; and again washed; then the attached PMNs were fluorometrically assayed. Each bar represents mean (±SE) adhesion of unstimulated, labeled PMNs to human EC monolayers. The n for each group is indicated in the bars. (A) PMN adhesion to EC monolayers preincubated for 1 h with 106 unstimulated PMNs or 106 PMNs activated in the absence or presence of equimolar concentrations of either the sialidase inhibitor 2-deoxy NANA or its negative control, KDO. *, significantly increased compared to the unstimulated PMN/unstimulated EC controls at p < 0.01. **, significantly decreased compared to EC preincubated with activated PMNs at p < 0.01. (B) PMN adhesion to EC monolayers preincubated with increasing concentrations of either unstimulated or activated PMNs. *, significantly increased compared to EC monolayers preincubated with equivalent concentrations of unstimulated PMNs at p < 0.01. (C) PMN adhesion to EC monolayers preincubated for increasing times with equivalent concentrations (106 PMNs/ml) of unstimulated or activated PMNs. *, significantly increased compared to EC monolayers preincubated for equivalent times with equivalent concentrations (106 PMNs/ml) of unstimulated PMNs at p < 0.01.

 
Effect of prior protein synthesis inhibition or adhesion molecule immunoblockade on cytokine-induced or PMN sialidase–mediated adhesion
To further discriminate between inducible EC adhesiveness that requires de novo synthesis and surface expression of adhesion molecules and adhesiveness associated with PMN sialidase activity, interventions with the protein synthesis inhibitor cycloheximide, as well as blocking antibodies raised against E-selectin, CD54, and CD18, were introduced. Prior protein synthesis inhibition completely protected against the TNF-induced increment in EC adhesiveness (Figure 7A), whereas hyperadhesiveness after preincubation with activated PMNs was not (Figure 7B). Similarly, anti-E-selectin and anti-CD54 antibodies each blocked the TNF-induced EC hyperadhesiveness, whereas the control Ig did not (Figure 7A). In contrast, preincubation with either anti-E-selectin or anti-CD54 blocking antibodies failed to prevent the increased adhesiveness mediated through PMN sialidase activity (Figure 7B). Finally, anti-CD18 antibodies decreased adhesion of activated PMNs to TNF-treated EC but not adhesion of resting PMNs to ECs preincubated with activated PMNs (Figure 7C). These data indicate that EC adhesiveness mediated through PMN-mediated desialylation, unlike cytokine-inducible adhesion, requires neither de novo protein synthesis nor expression of certain adhesion molecules, including E-selectin, CD54, and CD18.



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Fig. 7. Effect of prior protein synthesis inhibition or adhesion molecule immunoblockade on cytokine-induced or PMN sialidase–mediated adhesion. (A) Cytokine-induced adhesion. Each bar represents mean (± SE) percent adhesion of fMLP (10–7 M)/cytochalasin B (5 µg/ml)–activated, labeled PMNs to resting (open bar) or TNF (25 U/ml, 4 h)-treated EC (cross-hatched bars) in the presence of media alone, cycloheximide (50 µg/ml), anti-CD62 antibody (20 µg/ml), anti-CD54 antibody (20 µg/ml), or a species- and isotype-matched irrelevant antibody control. *, significantly increased compared to adhesion of fMLP/cytochalasin B–activated PMNs to resting EC. **, significantly decreased compared to adhesion of fMLP/cytochalasin B–treated PMNs to TNF-treated EC. (B) PMN sialidase–mediated adhesion. Each bar represents mean (±SE) percent adhesion of unstimulated labeled PMNs to human EC monolayers preincubated for 1 h with resting (open bar) or fMLP/cytochalasin B–activated unlabeled PMNs (106 PMNs/ml) (cross-hatched bars) in the presence of media alone, cycloheximide (50 µg/ml), anti-CD62 antibody (20 µg/ml), anti-CD54 antibody (20 µg/ml), or a species- and isotype-matched irrelevant antibody control. *, significantly increased compared to adhesion of resting PMNs to EC preincubated with fMLP/cytochalasin-activated PMNs. (C) Effect of CD18 immunoblockade on both cytokine-induced and PMN sialidase–mediated adhesion. Each bar represents mean (±) % adhesion of either fMLP/cytochalasin B–activated labeled PMNs to TNF-treated human ECs (cross-hatched bars) or unstimulated labeled PMNs to human ECs preincubated with fMLP/cytochalasin B–activated unlabeled PMNs (closed bars) in the presence of either anti-CD18 antibody (20 µg/ml) or a species-and isotype-matched irrelevant antibody control. *, significantly decreased compared to PMN adhesion in the presence of the antibody control. The n for each group is indicated in each bar.

 
Effect of NANase on TEM of PMNs
Because exogenous NANase increases PMN adhesion to EC (Figure 1B) and adhesion is an early step in TEM of PMNs (Carlos and Harlan, 1994Go; Cines et al., 1998Go), we asked whether NANase treatment of EC monolayers might increase TEM of PMNs. Treatment of EC with NANase >= 1 mU/ml increased TEM of resting PMNs in response to IL-8, plateauing at NANase >=3 mU/ml (Figure 8A). Treatment of EC monolayers with a fixed concentration of NANase (10 mU/ml) for >=1 h increased PMN TEM (up to 40%) compared to simultaneous media controls (<10%) with the maximal effect (>fourfold) at 4 h (Figure 8B). At 5 h and 6 h, TEM decreased compared to 4 h but remained greater than their simultaneous media controls.



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Fig. 8. Effect of desialylation of the pulmonary vascular endothelium on TEM of resting PMNs. (AB). Dose- and time-dependent effect of NANase treatment of EC monolayers on TEM of PMNs. (A) Each bar represents mean (± SE) percent migration of untreated PMNs across bovine EC monolayers in response to IL-8 (100 ng/ml) treated for 6 h with increasing concentrations of NANase (open bars) or media alone (closed bar), and (B) treated for increasing times with either NANase (10 mU/ml) (open bars) or media alone (closed bars). *, significantly increased compared to the simultaneous media control at p < 0.05. (C) Effect of mobilized PMN-associated sialidase activity on TEM of unstimulated PMNs. Human EC monolayers cultured on filter supports were preincubated for 1 h with either unstimulated PMNs or PMNs activated with fMLP (10–6 M)/cytochalasin B (5 µg/ml) (0.5 x 106 cells/chamber) in the absence or presence of equimolar concentrations of either 2-deoxy NANA or KDO, washed, and inserted into wells containing 10–6 M fMLP. Calcein AM-labeled PMNs were introduced into the upper compartment, after which the lower compartment was sampled and fluorometrically assayed. Each bar represents mean (±SE) TEM of PMNs. *, significantly increased compared to EC monolayers preincubated with unstimulated PMNs at p < 0.05; **, significantly decreased compared to EC monolayers preincubated with activated PMNs at p < 0.05. The n for each group is indicated in each bar.

 
Effect of PMN-associated sialidase activity on PMN migration across human EC monolayers
Because PMN-associated sialidase activity increases PMN adhesion to EC (Figure 6), we asked whether mobilization of PMN sialidase activity at the EC surface might also increase PMN movement across the EC barrier. Preincubation of resting EC monolayers with activated PMNs increased TEM of unstimulated PMNs compared to TEM after preincubation of monolayers with unstimulated PMNs (Figure 8C). This increment in PMN migration across EC monolayers preincubated with activated PMNs was completely blocked by the sialidase inhibitor 2-deoxy NANA but unaffected by the negative control, KDO. These data indicate that prior exposure of the EC surface to PMN-associated sialidase activity not only increases its adhesiveness for unstimulated PMNs but facilitates their TEM.

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., 1975Go). Monolayers treated with clostridial NANase revealed numerous discrete areas of increased PNA lectin binding compared to the media control (Figure 9A–B). 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 9C–D). 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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Fig. 9. Activated PMNs directly desialylate the pulmonary vascular EC surface. Postconfluent human pulmonary artery EC monolayers were preincubated for 6 h with media alone (A), clostridial NANase 100 mU/ml (B), or resting (C) or fMLP/cytochalasin B–activated (D) PMNs in the absence or presence of equimolar concentrations (300 µg/ml) of either the sialidase inhibitor, 2-deoxy NANA (E), or the KDO control (F). EC monolayers were washed, incubated for 0.5 h with 50 µg/ml FITC-conjugated PNA lectin, again washed, fixed (glutaraldehyde 0.5%, 10 min RT), and examined by fluorescence microscopy. Arrows in B and D indicate areas of increased PNA binding. Magnification, 200x.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
These studies demonstrate that perturbation of surface sialic acid profoundly influences PMN adhesion to and migration across the endothelium. Exogenous NANase treatment of either PMNs or ECs increased PMN adhesion to EC monolayers in a dose- and time-dependent manner (Figure 1A–C), and treatment of both PMNs and ECs increased PMN adhesion to ECs more than the treatment of either cell type alone (Figure 1D). These same treatments also increased TEM of PMNs in a dose- and time- dependent manner (Figure 8A–B). Importantly, although NANase treatment of ECs enhanced their adhesiveness for resting PMNs, it did so in the absence of EC activation as measured by increased adhesion molecule and cytokine expression (Figure 3).

We recently have demonstrated the physiological relevance of PMN-associated sialidase activity in vivo (Cross et al., 2003Go). 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 9C–D), 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., 2003Go; Cross and Wright, 1991Go), 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 molecule–blocking 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 1B–C, 2, 3, and 8A–B). Clostridial NANase hydrolyzes a range of glycosidic linkages of terminal sialic residues including {alpha}-(2->3), {alpha}-(2->6), and {alpha}-(2->8) linkages (Cassidy et al., 1965Go; Roggentin et al., 1995Go). Because NANase preparations may contain proteases, LPS, or other contaminants, multiple interventions were introduced to help establish that its effect on PMN–EC 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 PMN–EC 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 (4–6 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 synthesis–dependent adhesion molecule expression was unlikely (Cines et al., 1998Go). 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, 1991Go).

The minimal NANase concentrations that increased PMN–EC adhesion (Figure 1A–B-) 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., 2003Go), 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 PMN–EC 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., 2003Go; Cross and Wright, 1991Go). 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 time–dependent manner (Figure 6B–C). 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 7B–C). 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 7B–C) suggests a novel PMN sialidase–mediated mechanism(s) for PMN–EC adhesion. Although PMN activation can generate multiple products that can elicit an EC response, including proteases (Carden et al., 1998Go; Varani et al., 1989Go) and oxygen intermediates (Varani et al., 1989Go), the PMN- associated activity described here was inhibitable by a specific sialidase inhibitor, 2-deoxy NANA, but not by KDO (Figures 4A–B, 5A, 6A, 8C).

Because prior desialylation of the EC surface increases PMN adhesion (Figures 1B–C, 6A–C) and adhesion to the EC barrier is a prerequisite step for PMN diapedesis (Cines et al., 1998Go), 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 PMN–EC 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 PMN–EC 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., 2003Go). These surface changes were temporally coincident with increased PMN–PMN aggregation (Cross and Wright, 1991Go). 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., 1996Go). 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., 1993Go). Of note, PMN sialidase activity has been colocalized to the same subcellular compartment as ß2 integrin or CD11b/CD18 (Cross and Wright, 1991Go), 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 cell–cell repulsion (Gallin, 1980Go; Lichtman and Weed, 1970Go, 1972Go). 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, 1991Go) 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 PMN–EC interactions (Nathan et al., 1993Go; Sun et al., 1996Go) 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 marrow–derived EC stem cells participate in vascular repair (Takahashi et al., 1999Go). 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., 1997Go; Kibbey et al., 1994Go). 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., 1992Go). For example, prior influenza infection increases risk of bacterial superinfection and prior neuraminidase inhibition projects against this increased risk (McCullers and Bartmess, 2003Go). 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 protein–protein and cell–cell interactions, signaling, and cell behavior.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Reagents
Recombinant human TNF (Endogen, Cambridge, MA) with a specific activity of 25 U/ng and 0.094 endotoxin units/ng was reconstituted in 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 10 µg/ml, aliquoted, and stored at –70°C. IL-8 (Calbiochem, San Diego, CA) was reconstituted in sterile PBS, aliquoted, and stored at –20°C. fMLP (Sigma, St. Louis, MO) was reconstituted in PBS at 10–3 M, aliquoted, and stored at –20°C. NANase extracted from Clostridum perfringens (Sigma), the NANase inhibitor 2-deoxy NANA (Calbiochem Biochemical, La Jolla, CA), and N-acetylneuraminic acid (Sigma) were each reconstituted in PBS. N-acetylneuraminyl-lactose (Sigma) was reconstituted in PBS immediately prior to use. Specific inhibitors of LPS bioactivity, PMB (Sigma), and recombinant ENP (Associates of Cape Cod, Woods Hole, MA) were each reconstituted in PBS, aliquoted, and stored at –20°C as previously described (Bannerman et al., 1998Go). Purified native LPS derived from Escherichia coli serotype 0111:B4 (Sigma) was dissolved in PBS at 5 mg/ml. The protease inhibitor PMSF (Sigma) was reconstituted in isopropyl alcohol, whereas aprotinin and leupeptin (Sigma) were reconstituted in Hanks balanced salt solution (HBSS) (Life Technologies, Gaithersburg, MD). The murine monoclonal anti-human CD54 antibodies and purified mouse IgG1 (Pharmingen, San Jose, CA), anti-human E-selectin antibodies (Serotec, Raleigh, NC), and the anti-CD18 antibodies (Immunotech, Marseilles, France) were for adhesion blocking studies. To prepare anti-NANase IgG, New Zealand white rabbits were immunized with highly purified C. perfringens NANase (Sigma) as described (Cross et al., 2003Go). At 1 week after the last boost, serum was obtained and passed through a protein G affinity column (Pharmacia, Piscataway, NJ), eluted with glycine-HCL buffer (0.15 M, 2.5 pH) and immediately neutralized with 0.15 M Tris to a pH of 6.8–7.2. The combined fractions were passed through a sterile filter (0.2 µm) and stored at –20°C. Rabbit IgG (Sigma), preimmune rabbit IgG, and KDO (Sigma) were used as negative controls for anti-NANase IgG and 2-deoxy NANA, respectively.

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, 1990Go). Human pulmonary artery EC (Clonetics, San Diego, CA) were cultured as described (Campbell et al., 1997Go) 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 2–6 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, 1991Go). 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, 1993Go). Calcein AM fluoresces on cleavage by intracellular esterases (Weston and Parish, 1990Go). 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 (0.3–100 mU/ml, 10 min–4 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, 1990Go). Calcein AM-labeled PMNs were pretreated with NANase (0.3–100 mU/ml), fMLP (10–7 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 105–103 cells/ml) were used to generate a standard curve from which PMN numbers could be interpolated from fluorescence units and PMN–EC 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 10–6 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, 1990Go). 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, 1990Go). 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 (10–6M), 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, 1994Go; Cines et al., 1998Go; Schleiffenbaum and Fehr, 1996Go). 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 2–8°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., 1999Go). 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, 1991Go). 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., 1975Go). 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.


    Acknowledgements
 
We acknowledge the technical support of Katie Buffum and Kathy Strauss and Ms. Shirley Taylor for manuscript preparation. This work was supported in part by the Office of Research and Development, Department of Veterans Affairs (S.E.G.), and grants RO1HL63217 (S.E.G.) and RO1AI42818 (A.S.C.) from the National Institutes of Health. D. D. Bannerman was a recipient of a department of Defense Augmentation Award for Science and Engineering Research Training.


    Footnotes
 
2 To whom correspondence should be addressed; e-mail: simeon.goldblum{at}med.va.gov

1 Present address: Department of Medicine, Division of Microbiology and Infectious Diseases, Adnan Menderes University School of Medicine, Aydin, Turkey Back


    Abbreviations
 
BSA, bovine serum albumin; EC, endothelial cell; FITC, fluorescein isothiocyanate; fMLP, n-formyl-methionyl-leucyl-phenylalanine; HBSS, Hanks balanced salt solution; ICAM-1, intercellular adhesion molecule-1; KDO, 2-keto-3-deoxyoctulosonic acid; LPS, bacterial lipopolysaccharide; NANA, N-acetylneuraminic acid; NANase, neuraminidase; PBS, phosphate buffered saline; PECAM-1, platelet-endothelial cell adhesion molecule; PMB, polymyxin B; PMN, neutrophil; PMSF, phenylmethylsulfonyl fluoride; PNA, lectin isolated from Arachis hypogaea; rENP, recombinant endotoxin neutralizing protein; TEM, transendothelial migration; TNF, tumor necrosis factor alpha


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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