The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsikitis, V. L.
Right arrow Articles by Reichner, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsikitis, V. L.
Right arrow Articles by Reichner, J. S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2004, 173: 1284-1291.
Copyright © 2004 by The American Association of Immunologists

The Lectin-Like Domain of Complement Receptor 3 Protects Endothelial Barrier Function from Activated Neutrophils1

Vassiliki L. Tsikitis*, Nicole A. Morin{dagger}, Elizabeth O. Harrington{dagger}, Jorge E. Albina* and Jonathan S. Reichner2,*

* Department of Surgery, Rhode Island Hospital and Brown University Medical School, Providence, RI 02903; and {dagger} Pulmonary Vascular Biology Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Brown University Medical School, Providence, RI 02912


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of {beta}2 integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the {beta}2 integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist {beta}-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates {beta}-glucan protection while activation of the LLD by VIM12 cross-linking mimics the {beta}-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. {beta}-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Integrins are heterodimeric molecules that mediate cell-cell and cell-extracellular matrix interactions (1). Leukocyte integrins of the {beta}2 family (CD11/CD18) are necessary for the migration of leukocytes into sites of inflammation or infection as demonstrated by the recurrent, life-threatening infections suffered by patients lacking expression of the CD18 subunit (leukocyte adhesion deficiency) (2). Conversely, excessive neutrophil adhesion to the vascular endothelium mediated by the {beta}2 integrins has been implicated in diseases such as myocardial infarction, ischemia, and reperfusion injury (3, 4, 5).

Complement receptor 3 (CD11b/CD18) (CR3),3 one of four members of the {beta}2-integrin family, is a multifunctional adhesion molecule in which a common {beta}2 (CD18) subunit is noncovalently bound to the {alpha}M subunit (CD11b). A number of studies have shown that CR3 plays a role in neutrophil adhesion and transmigration through an endothelial cell layer, and in migration through the extracellular matrix (6). CR3 also mediates leukocyte phagocytosis of opsonized and unopsonized pathogens, homotypic aggregation, and adhesion-dependent respiratory burst (7, 8). The extensive repertoire of CR3 ligands renders this integrin able to mediate a multitude of key functions in host defense. A region in the extracellular domain of CR3 referred to as the inserted or I-domain has been shown to bind >30 ligands including iC3b, fibrinogen, and ICAMs 1, 2, and 3 (9, 10). The I-domain can also bind a number of extracellular matrix proteins including fibronectin, laminin, collagen, vitronectin, thrombospondin, and Cyr61 (11, 12, 13, 14). This ability of the I-domain of CR3 to bind diverse ligands has recently been attributed to a consensus binding site within CD11b (Lys245-Arg261) (15).

In addition to the I-domain, CR3 contains a unique lectin-like domain (LLD), which permits binding of microbial polysaccharides such as {beta}1,3-linked glucose polymers (viz {beta}-glucan). This aspect of CR3 mediates the recognition of several pathogens such as Candida albicans, Leishmania sp., Bordetella pertussis, and Pneumocystis carinii (16, 17, 18). Furthermore, the LLD participates in the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b, CD14, and CD87 enabling CR3 to mediate cytoskeleton-dependent functions including phagocytosis, degranulation, and adhesion (19, 20, 21). Taken together, these findings underscore the functional significance of the LLD in host defense against infection and injury.

Dual ligation of the I-domain and the LLD by their corresponding ligands has been shown to affect CR3-dependent functions differently than ligation of either site alone. Co-occupancy of the I-domain with the extracellular matrix protein fibronectin, and of the LLD with {beta}-glucan, resulted in an increase in the chemotactic capacity of neutrophils toward fMLP (22). Furthermore, tumor cells, otherwise resistant to NK cells, became susceptible to killing when opsonized with the CR3 I-domain ligand iC3b and treated with the LLD ligand {beta}-glucan (23). The current investigation sought to determine the effect of LLD activation on neutrophil:endothelial cell interactions. The rationale for this study is that the I-domain of CR3 will be ligated by endothelial ICAM and thereby mediate the interaction between the neutrophil and the endothelial cell (9, 10). The hypothesis to be tested is that coligation of the I-domain with ICAM and the LLD with {beta}-glucan would alter this CR3-dependent intercellular interaction. Findings to be presented show that neutrophils, activated with fMLP, impaired the barrier function of intact endothelial monolayers, which was preserved by {beta}-glucan or by Ab cross-linking of the CR3 LLD. Ligation of the CR3 LLD significantly reduced polymorphonuclear neutrophil (PMN) transmigration. Additional findings show that {beta}-glucan did not affect the adhesion of neutrophils to endothelial cells. These findings suggest that activation of the CR3 LLD prevents endothelial barrier dysfunction in the presence of activated neutrophils and may thereby attenuate tissue damage during autoimmune or hyperinflammatory processes. This work identifies the CR3 LLD as a potential target site for the development of oligosaccharide-based therapeutics with anti-inflammatory applications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Highly purified, pharmaceutical grade, soluble {beta}-glucan (BETAFECTIN, 150,000 ± 20,000 m.w.) was obtained from Biopolymer Engineering (Eagan, MN). Rat-tail collagen type I and fibronectin were purchased from BD Biosciences (Bedford, MA). Rat TNF-{alpha} was obtained from BioSource International (Camarillo, CA). Dextran, thrombin, fMLP, Evans blue dye-conjugated albumin, and endotoxin-free BSA were purchased from Sigma-Aldrich (St. Louis, MO). PBS and HBSS were obtained from Invitrogen Life Technologies (Grand Island, NY). DMEM was purchased from Invitrogen Life Technologies and FBS was obtained from HyClone (Logan, UT). CFSE was purchased from Molecular Probes (Eugene, OR). All reagents used contained <0.1 pg/ml endotoxin, as determined by Limulus amoebocyte lysate screening (BioWhittaker, Walkersville, MD). Some reagents were treated with Detoxi-Gel (Pierce, Rockford, IL) to remove residual endotoxin. The CD11b-specific Ab VIM12 was purchased from Caltag Laboratories (Burlingame, CA). The F(ab')2 of goat anti-mouse IgG and human HLA class I Ab were purchased from Sigma-Aldrich. The CR3 I-domain specific Ab LM2.1 was obtained from Bender MedSystems (San Bruno, CA) and the CD18-specific Ab TS1/18 was obtained from Pierce (Woburn, MA). CBRM1/23 was a gift from Dr. T. Springer (Center for Blood Research and Harvard University Medical School, Boston, MA).

Isolation and activation of human neutrophils

Human neutrophils were isolated from heparinized venous blood of healthy volunteers by gradient centrifugation on Ficoll-Hypaque (Sigma-Aldrich), followed by erythrocyte sedimentation with 3% dextran (500,000 m.w.). The leukocyte-rich supernatant underwent hypotonic lysis of residual erythrocytes. Cells were resuspended in ice-cold DMEM and counted. PMN purity and viability were consistently >90% as determined by trypan blue dye exclusion. Where indicated, neutrophils were treated with fMLP (10–6 M) for 20 min on ice and then {beta}-glucan (10 µg/ml) was added as a second treatment agent for 20 min at room temperature.

Ab treatment of human neutrophils

Neutrophils (3 x 106 cells/ml), were incubated with no Ab, LM2.1 (20 µg/ml), TS1/18 (10 µg/ml), VIM12 (5 µg/106 cells), CBRM1/23 ascitic fluid (10 µl/ml), and anti-HLA class I IgG (5 µg/106 cells) for 30 min on ice and then, where indicated, with {beta}-glucan for 20 min at room temperature. Anti-HLA class I IgG was used to control nonspecific effects of Ab binding to cell surface Ags. For cross-linking of the LLD, purified neutrophils (3 x 106 cells/ml) were treated with VIM12 as described above, washed, and then incubated with goat anti-mouse F(ab')2 (1/20 dilution) for 20 min on ice. All neutrophil groups were warmed to room temperature for 20 min before addition onto the endothelial monolayers.

Endothelial cell culture

HUVEC (Cambrex, Walkersville MD), were grown in endothelial cell growth medium containing 2% FBS, 10 ng/ml human recombinant epidermal growth factor, 50 mg/ml gentamicin, 50 ng/ml amphotericin B, 12 mg/ml bovine brain extract, and 1 mg/ml hydrocortisone as provided by the manufacturer. Cells were used between passages 5 and 9. Rat pulmonary artery endothelial cells (RPAEC) and rat lung microvascular endothelial cells were obtained from Dr. P. Lee (Yale University School of Medicine, New Haven, CT) and grown in DMEM with 20% FBS supplemented with antibiotics. Cells were used between passages 17 and 24.

Measurement of electrical resistance

Transendothelial electrical resistance, an index of endothelial cell barrier function, was measured in real time using an electric cell-substrate impedance sensor system (Applied Biophysics, Troy, NY) (24). For resistance measurements, endothelial cells were plated on sterile eight-chambered gold-plated electrode arrays precoated with collagen type I (30 µg/ml) and grown to confluence. The electrode arrays were then mounted on the electric cell-substrate impedance sensor system within an incubator (37°C, 5% CO2) and connected to its recorder device. Confluence of each endothelial monolayer was established before use from the resistive portion of the impedance tracing. Only monolayers, showing a resistance of 1–1.3 kilo-ohms were used in experiments (25). Naive neutrophils (6 x 105 cells per well in 200 µl of DMEM) or neutrophils pretreated with fMLP (10–6 M) for 20 min on ice, were added to each well of the array. Thrombin (0.8 U/ml), which causes instant retraction of the endothelial monolayer, was used as a positive control. Monolayer resistance was recorded over 3 h in 1-min intervals after PMN addition. Values are reported as percentage change of resistance normalized to initial baseline resistance (observed resistance/initial resistance x 100).

Permeability assays

Permeability assays were performed using a two-compartment chamber system obtained from Costar (Acton, MA). This system contained 0.4-µm pore polycarbonate transwell supports. HUVEC were plated to confluence in the rehydrated transwell supports at a density of 105 cells per well and permitted to adhere overnight in complete medium. The following day, the medium was changed in both chambers. Thrombin and HRP (50 µM HRP) were then placed in the upper chamber. The appearance of HRP in the lower chamber was determined by retrieving 20-µl aliquots of medium from the lower compartment every 20 min, over 2 h. The HRP concentration was determined using a spectrophotometric assay. The assay was conducted by incubating the sample in a solution containing 0.4 mg/ml-1 O-phenylenediamine HCl and 0.012% H2O2 in 0.5M phosphate-citrate buffer, pH 5.0, for 30 min at 25°C. The reaction was stopped with HCl, a final concentration of 0.55 M, and the absorbance was taken at 492 nm. The data are presented as the number of moles of HRP that have diffused to the lower chamber over time.

Neutrophil adhesion

Neutrophils (5 x 106 cells/ml) were fluoresceinated using CFSE at 1.0 µg/ml by incubating at room temperature for 30 min in the dark. Cells were washed and resuspended to 3 x 106 cells/ml in DMEM and kept on ice until use. Viability of fluorescent PMNs was monitored by trypan blue dye exclusion and cells were >85% viable at time of use. Polystyrene 96-well tissue culture plates (BD Biosciences, Franklin Lakes, NJ) were coated with collagen type I (30 µg/ml) for 1 h at 37°C and then blocked with BSA (10 mg/ml, endotoxin-free) for 1 h. After rinsing twice with sterile PBS, RPAEC (105 cells/well) were plated and grown to confluence which was confirmed microscopically. Endothelial cells were stimulated, or not, with rat TNF-{alpha} (50 ng/ml) for 6 h and then washed three times with sterile PBS. When HUVECs were used, plates were precoated with fibronectin (30 µg/ml) and then blocked with BSA as described above. HUVECs were stimulated with human TNF-{alpha} (20 ng/ml) for 6 h before the addition of neutrophils. Fluorescent neutrophils (3 x 105 cells) were added to each well and incubated for 45 min at 37°C in 5% CO2. Nonadherent cells were removed by washing twice with serum-free DMEM and fluorescence was quantified in a FL 500 Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT) with excitation/emission wavelength settings of 485/530 nm and a sensitivity setting of 40–44. All determinations were conducted in six-well replicates and repeated three times.

Neutrophil transmigration assay

Assays were performed as described in Taooka et al. (26). HUVECs or RPAECs were plated onto type I collagen polycarbonate inserts (transwell inserts, 6.5-mm diameter, 3-µm pore for 24-well plates; VWR, Boston, MA). HUVECs (105 cells) and RPAECs (2 x 105 cells) were grown in 200 µl of cell-specific complete medium as described above. Additional complete medium was added to the lower chamber of each well and endothelial cells were grown to confluence over 72 h. Evans blue dye-conjugated albumin was used as a marker for macromolecular permeability of the endothelial cell monolayers before the start of the experiment as described in Gautam et al. (27). Dye exclusion was determined by spectrophotometric reading at OD550. Monolayers allowing <10% leakage of the dye relative to maximal leakage (thrombin x 100%) were accepted for further study. PMN (106 cells in 200 µl of DMEM) activated with fMLP (10–9 M) and then treated, or not, with {beta}-glucan for 40 min at room temperature, were added to the upper chamber of the transwells. The lower chamber was replaced with 500 µl of serum-free DMEM or serum-free DMEM with 10–6 M fMLP. After 3 h at 37°C in 5% CO2 in air, medium from the lower chambers was collected and rinsed several times and the migrated cells were counted in a hemacytometer. All determinations were conducted in quadruplicate and repeated three times. Results are presented as percentage of cells migrated.

Data presentation and statistical analysis

Data are presented are mean ± SEM unless otherwise stated, with n being the number of independent experiments. Statistical assessments were made using the Student t test or ANOVA with Newman-Keuls posthoc analysis as appropriate and statistical significance set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{beta}-glucan protects the barrier function of endothelial monolayers in the presence of activated neutrophils

Electrical resistance across confluent RPAEC monolayers was measured in the absence or presence of PMNs over a 3-h period. The change of the resistance shown correlates with macromolecular leakage and it is used here as an index of endothelial monolayer barrier function (Fig. 1). PMNs activated with fMLP caused a greater drop in transendothelial resistance (33 ± 4%) than unstimulated PMNs, suggesting an increase in endothelial monolayer permeability. In contrast, when fMLP-activated PMNs were pretreated with {beta}-glucan, the integrity of the endothelial monolayer was preserved with a drop in the endothelial monolayer resistance of 8.2 ± 3%. The extent of endothelial monolayer permeability caused by fMLP-activated neutrophils exceeds that of thrombin, a benchmark for instantaneous disruption of endothelial cell barrier function. {beta}-glucan did not affect thrombin-induced endothelial barrier dysfunction (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. {beta}-glucan diminishes endothelial barrier dysfunction induced by activated PMNs. a, Representative tracing of electrical resistance across RPAEC monolayers measured in the absence (medium) or presence of untreated PMNs, PMNs treated with fMLP (10–6 M) alone, PMNs treated with fMLP (10–6 M) and {beta}-glucan (10 µg/ml), or thrombin (0.8 U/ml) as detailed in Materials and Methods. Arrow indicates the time of addition of media, cells, or thrombin. b, Values are mean ± SEM of the percent change of normalized resistance at 60 min of PMN addition from six independent experiments. Supernatants from PMNs activated with fMLP– (Sup/fMLP) or with fMLP and {beta}-glucan (Sup/fMLP + {beta}-glucan) caused no change on the normalized resistance recorded. The presence of either {beta}-glucan alone (10 µg/ml) or fMLP (10–6 M) alone in the media caused no change in normalized resistance. *, p < 0.01 vs fMLP-treated PMNs.

 
Coincubation of PMNs with fMLP (10–6 M) and dextran (10 µg/ml), a polyglucose polymer of similar size {beta}-glucan, did not prevent endothelial barrier dysfunction suggesting that the effect of {beta}-glucan is not due to a nonspecific response to polysaccharide (data not shown). Moreover, supernatants from naive or fMLP-activated neutrophils that were pretreated with or without {beta}-glucan did not alter endothelial monolayer resistance indicating the requirement of neutrophils for the observed increase in endothelial monolayer permeability (Fig. 1b). Direct addition of fMLP and {beta}-glucan into the medium of endothelial cells was also without effect on recorded resistance.

To further confirm that the presence of neutrophils is necessary for endothelial cell retraction, neutrophils were fixed with 2% paraformaldehyde and added onto the endothelial monolayer in the presence or absence of {beta}-glucan. Retraction was observed in both conditions suggesting that {beta}-glucan only regulates the interaction between viable neutrophils and endothelial monolayers (data not shown).

{beta}-glucan prevents barrier dysfunction by activated neutrophils on different TNF-{alpha}-activated endothelial monolayers

Experiments were performed to determine whether the {beta}-glucan protection of endothelial barrier function is maintained when both PMN and endothelial cells are activated and when different endothelial cell lines are used. Confluent RPAEC or HUVEC monolayers were treated with TNF-{alpha} before the addition of fMLP-activated PMNs. To validate that the monolayers were activated by TNF-{alpha}, PMN:endothelial cell adhesion assays were performed, as described in Materials and Methods. There was a 2.7-fold increase in the adhesion of fMLP-activated neutrophils to TNF-{alpha}-treated endothelial cells as compared with untreated monolayers (4700 ± 199 vs 1730 ± 148 fluorescent units, respectively). Similar results were obtained with either RPAEC or HUVEC endothelial monolayers. FMLP-activated neutrophils caused the same decrease in relative resistance (40 ± 5.2%) of monolayers of activated HUVEC (Fig. 2a) as well as RPAEC and RLMVC (not shown). This drop in normalized resistance was reduced to 20 ± 4.8% by 10 µg/ml {beta}-glucan and to 9 ± 2.8% by 50 µg/ml. Thus, the barrier protective effect of {beta}-glucan was independent of the activation state of the endothelial monolayers and was seen with three different endothelial cell lines.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 2. {beta}-glucan preserves barrier function of TNF-{alpha}-treated endothelium in the presence of activated PMNs. a, HUVEC monolayers were incubated with TNF-{alpha} (20 ng/ml) for 6 h at 37°C. fMLP-activated PMN were added to the TNF-{alpha} activated HUVEC in the presence of 10 or 50 µg/ml {beta}-glucan. Transendothelial resistance was measured at 60 min and presented as mean ± SEM from three independent experiments. *, p < 0.01 vs fMLP treatment alone for both {beta}-glucan concentrations. b, {beta}-glucan preserves paracellular permeability of HUVEC monolayers in the presence of fMLP-activated PMNs. Transendothelial flux of HRP is shown at 1 h. Data are mean ± SD from representative of three identical experiments. *, p < 0.01 vs fMLP-activated PMNs.

 
To determine whether the effect of {beta}-glucan is demonstrable on a model of endothelial paracellular permeability, PMNs were activated with fMLP (10–6 M) and placed together with HRP on HUVEC monolayers grown on permeable inserts (Fig. 2b). Activated PMNs caused a significant increase in the permeability of the endothelial monolayer to an extent comparable to thrombin. Inclusion of {beta}-glucan had a protective effect on monolayer permeability as shown by a decrease in the flux of HRP, consistent with the protection measured by transcellular electrical resistance shown above.

{beta}-glucan does not alter PMN:endothelial cell adhesion and decreases neutrophil transmigration

To investigate whether the role of {beta}-glucan on barrier function is based on altering adhesive interaction between neutrophil and endothelial cells, adhesion assays were performed as described in the Materials and Methods. Results in Fig. 3 show the relative adhesion of fMLP-activated neutrophils vs fMLP + {beta}-glucan cotreated neutrophils: 4890 ± 750 vs 4786 ± 586 fluorescent units on resting endothelium, respectively. Experiments also compared neutrophil adhesion to TNF-{alpha}-activated endothelium and show similar adhesion of fMLP-activated neutrophils vs fMLP + {beta}-glucan cotreated neutrophils: 5810 ± 516 vs 4774 ± 593 fluorescent units, respectively (p > 0.05, t test). In this series of experiments, endothelial cells were activated with TNF-{alpha} 6 h prior to the adhesion assay as described in Materials and Methods. Adhesion assays were also performed where endothelial cells were pretreated with the same concentration of TNF-{alpha} for 18 h with similar results: (adhesion of fMLP-activated neutrophils vs fMLP + {beta}-glucan cotreated neutrophils: 9280 ± 406 vs 9704 ± 1000 fluorescent units, respectively). Fig. 3 also includes results obtained from nonactivated, naive neutrophils added to resting and activated endothelium and while the extent of adhesion is less than seen with activated neutrophils, the addition of {beta}-glucan was without affect. These results suggest that {beta}-glucan did not significantly alter adhesion of resting or activated neutrophils to resting or activated endothelial cells.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. {beta}-glucan does not alter PMN adhesion to endothelial cells. RPAEC monolayers were activated, or not, with TNF-{alpha} as described in Materials and Methods. Fluorescent naive (unstimulated) or fMLP-activated (10–6 M) PMNs were added to endothelial cells in the presence or absence of {beta}-glucan (10 µg/ml). Adhesion was determined after incubation for 45 min. Data are presented as mean ± SEM from six independent experiments.

 
Neutrophil adhesion and transmigration can occur with or without increased vascular permeability (28), thus we next sought to determine whether the effect of {beta}-glucan was germane to neutrophil extravasation and migration across endothelial monolayers. RPAECs were grown and activated with TNF-{alpha} as described in Materials and Methods and migration of activated neutrophils was measured. The presence of {beta}-glucan resulted in neutrophil transmigration of 31 ± 1.2% as compared with 60 ± 8% for neutrophils in the absence of {beta}-glucan (Fig. 4). This decrease in neutrophil transmigration induced by {beta}-glucan directly correlates with its inhibitory effect on neutrophil-dependent endothelial barrier dysfunction. Identical results were obtained using resting RPAEC as well as with resting and activated HUVEC (data not shown).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. Pretreatment of activated PMNs with {beta}-glucan decreased diapedesis. Monolayers of RPAECs were activated with TNF-{alpha} on collagen precoated semipermeable membranes as detailed in Materials and Methods. The percentage of neutrophils pretreated with fMLP (10–9 M) that migrated through an intact monolayer toward an fMLP gradient (10–6 M) in the presence or absence of {beta}-glucan (50 µg/ml) was determined after 1 h at 37°C. Data are expressed as means ± SEM from three independent experiments; *, p < 0.05 vs no {beta}-glucan.

 
Ab blocking of the CR3 LLD obviates the protective role of {beta}-glucan of endothelial barrier function

To test the hypothesis that the prevention of endothelial barrier dysfunction is mediated by ligation of the neutrophil CR3 LLD, Ab-blocking experiments were performed as described in Materials and Methods. Results in Fig. 5a using RPAEC monolayers show that neutrophils treated with VIM12 or CBRM1/23 in the presence of {beta}-glucan caused a 45 ± 2.4% and 48 ± 7.7% drop in resistance, respectively, thereby blocking the protection afforded by {beta}-glucan. Neutrophils treated with the cell surface control Ab against HLA class I molecules did not alter the protection by {beta}-glucan. Activated neutrophils treated with LM2.1, an Ab specific to the CR3 I-domain, were not able to cause significant endothelial cell retraction (5 ± 1.3%) due to the ability of the Ab to prevent neutrophil recognition of endothelial ICAM. Activated PMNs, pretreated with TS1/18, a CD18-specific Ab, also blocked PMN-induced endothelial barrier dysfunction (data not shown). The above findings suggest that the I-domain of CR3 mediates PMN-dependent endothelial cell retraction whereas the LLD alone does not play a role in regulating neutrophil-induced endothelial barrier dysfunction.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. Ab blocking of the CR3 LLD blocks the {beta}-glucan effect on endothelial barrier function and PMN transmigration. a, fMLP-activated (10–6 M) PMNs were pretreated with Abs and added onto confluent monolayers of RPAECs in the presence or absence of {beta}-glucan (10 µg/ml). Data are shown as mean ± SEM from six independent experiments; *, #: p < 0.01 vs control anti-HLA with {beta}-glucan. b, Percentage of PMNs migrated toward fMLP or PBS after 3 h at 37°C is shown. PMNs were pretreated, or not, with fMLP (10–9 M) and Ab before transmigration. Data are expressed as mean ± SEM from three independent experiments; *, p < 0.01 vs VIM12 with {beta}-glucan.

 
Transmigration experiments were performed to determine whether Ab blockade of the CR3 LLD on neutrophils affects their migratory capacity through an intact RPAEC monolayer. As seen in Fig. 5b, {beta}-glucan decreased the migration of neutrophils treated with control anti-HLA Ab but not of neutrophils treated with the CR3 LLD-blocking Ab VIM12 (24 ± 7% vs 64 ± 17% transmigrated cells, respectively). These results suggest that {beta}-glucan inhibits neutrophil diapedesis through a mechanism requiring its binding to the neutrophil CR3 LLD.

VIM12 activation of CR3 LLD mimicked the {beta}-glucan response by preserving endothelial cell barrier function and reducing neutrophil transmigration

To verify that signaling through the LLD of neutrophil CR3 is sufficient to protect endothelial barrier function, the effect of site-specific Ab cross-linking was examined. Cross-linking an anti-integrin Ab with a secondary Ab leads to clustering and activation of the target integrin (29). As shown in Fig. 6a, blocking CR3 with VIM12 did not alter the ability of activated PMNs to impair endothelial barrier function. However, cross-linking VIM12 with goat anti-mouse F(ab')2 Ab to activate the CR3 LLD resulted in maintenance of barrier function. Cross-linking anti-HLA class I mAb had no effect. Fig. 6b demonstrates that VIM12 cross-linked activated PMNs caused a 10 ± 2.5% decrease in resistance vs HLA cross-linked activated PMNs which caused a 29 ± 4% decrease. Cross-linking CR3 was able to mimic the protective role of {beta}-glucan on the endothelial barrier function thereby suggesting that signaling through the LLD of CR3 can preserve the integrity of the endothelial monolayer in the presence of activated neutrophils.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Ab cross-linking of the CR3 LLD mimicked the {beta}-glucan effect by preserving endothelial barrier function and decreasing PMN diapedesis. fMLP-activated (10–6 M) PMNs were preincubated, or not, with VIM12 and subsequently cross-linked (x-link) with goat anti-mouse F(ab')2 as described in Materials and Methods. a, Representative tracing of normalized transendothelial electrical resistance across RPAEC monolayers in response to fMLP-activated PMN addition either in the presence of {beta}-glucan or with VIM12 cross-linked. b, Values are mean ± SEM of the percent change of normalized resistance at 60 min of PMN addition from six independent experiments; *, p < 0.01 vs HLA x-link. c, Transendothelial migration of fMLP-activated (10–9 M) PMNs that were cross-linked, or not, with VIM12 or HLA Ab. Transmigration was measured relative to migration of naive PMNs toward fMLP and data are expressed as mean ± SEM from three independent experiments; *, p < 0.01 vs HLA x-link.

 
Transmigration assays were performed to determine the migratory capacity of LLD cross-linked neutrophils through an intact endothelial monolayer. Fig. 6c shows that VIM12 cross-linking significantly diminished neutrophil transmigration as compared with HLA cross-linking. Thus, cross-linking of CR3 was able to mimic the {beta}-glucan effects on both maintenance of barrier function and reduction of neutrophil transmigration.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulated migration of leukocytes through an endothelial cell barrier is a universal aspect of host defense and inflammation. This process is mediated by leukocyte integrins such as CR3, the importance of which is recognized by the reduced ability of cells to extravasate under conditions where CR3 function is blocked or is genetically inactive (2, 30). Conversely, excessive integrin-mediated adhesion to vascular endothelium has been implicated in disease states such as myocardial infarction and ischemia/reperfusion injury (3, 4, 5). Therefore, the ability to regulate the function of CR3 may offer opportunities to intervene therapeutically in clinical circumstances wherein excessive inflammation contributes to pathogenesis, rather than resolution of the disease. CR3 contains two distinct ligand-binding sites: the I-domain and the LLD. Studies from this and other laboratories suggest that dual ligation of the lectin-like domain and the I-domain may alter neutrophil function differently than ligation of either site alone (23). The current study tested and confirmed the hypothesis that LLD ligation would affect CR3 activity as noted by alterations in neutrophil:endothelial cell interactions. Agonists of the CR3 LLD prevented loss of endothelial barrier function caused by activated neutrophils and identified the LLD as a novel target site for the development of anti-inflammatory therapeutics.

The present studies demonstrate that the protective effect of endothelial barrier function by {beta}-glucan is due to its action on the neutrophil, and not on the endothelial cell. The relevance of this finding is heightened by a recent report demonstrating the capacity of endothelial cells to directly bind {beta}-glucan (31), although the functional significance of this observation is not yet defined. LLD-specific Ab blocking and activation experiments, performed on neutrophils before addition to endothelial monolayers, were found to regulate barrier function. These experiments demonstrate that {beta}-glucan does not exert a direct effect on endothelial cells in the context of protecting barrier function from neutrophil-induced disruption.

We demonstrated that LLD ligation can uncouple neutrophil adhesion from maintenance of barrier function and transmigration. The mechanism underlying this novel finding is not yet known, however Sans et al. (32) found that deletion of the ICAM cytoplasmic domain permitted neutrophil adhesion to engineered Chinese hamster ovary cells, but abrogated transmigration. The authors concluded that neutrophil migration is not a direct consequence of enhanced adhesion mediated by ICAM-1, but requires ICAM signaling. Whether occupancy of LLD alters ICAM signaling in endothelial cells remains to be determined. This mechanism may not be applicable to epithelial cell targets that are also sensitive to PMN-induced disruption of barrier function. Fucoidin binding to the CR3 I-domain was shown to alter adhesive interactions between PMN and epithelial cells (33), an observation that was not found in our present studies.

{beta}2 integrins have been shown to be required for neutrophil:endothelial adhesion and transmigration in several in vitro experimental systems (32, 34, 35). Of direct relevance to the physiological response to inflammation, the role of the {beta}2 integrin CR3 in mediating ICAM-1 binding is more pronounced in chemokine-activated neutrophils than in resting cells, which rely on LFA-1 for their binding onto the endothelial cells (36). Recent findings from Luscinskas and coworkers (37) have shown that maintenance of endothelial barrier function does not necessarily equate with reduced neutrophil transmigration. Studies showed that overexpression of vascular endothelial-cadherin in HUVECs was sufficient to increase endothelial barrier function without altering neutrophil migration. Therefore, finding that {beta}-glucan maintained barrier function of endothelial cells in the presence of activated neutrophils could not predict whether diapedesis would be similarly affected. Thus, we determined experimentally the effect of the CR3-ligated LLD on neutrophil transmigration. Results indicate that the presence of {beta}-glucan decreased extravasation and migration of activated neutrophils through an intact endothelial monolayer. Specifically, Fig. 4 shows a 50% reduction in transmigration of neutrophils in the presence of {beta}-glucan which directly correlated with the extent of protection of barrier function shown in Fig. 1. This observation was repeated with the VIM12 cross-linking experiments, thereby confirming that the decrease in neutrophil transmigration is mediated through signaling of the CR3 LLD.

Work by Ross and colleagues (38, 39) showed that binding of the CR3 LLD by {beta}-glucan induces the high affinity metal ion-dependent activation site reporter epitope which is recognized by the CBRM1/5 activation reporter mAb. However, despite the high affinity conversion, {beta}-glucan inhibited adhesion of neutrophils to immobilized recombinant ICAM. These authors propose that CR3 must form complexes with uPAR for clustering to take place leading to adhesion. Binding of {beta}-glucan to the same lectin-like site as would be occupied by urokinase-type plasminogen activator receptor (uPAR) prevents complex formation and thereby CR3 binding to ICAM. Findings in this report show a lack of a significant effect of {beta}-glucan on neutrophil adhesion to endothelial cells. What the two studies have in common is the finding that {beta}-glucan does not promote excessive leukocyte adhesion to ICAM or to endothelial cells despite maintaining CR3 in a high affinity conformation. In that regard, the activation state of CR3 would not be expected to impose an impediment to neutrophil diapedesis.

An important physiological function of the CR3 LLD lies in its ability to form membrane complexes with GPI-anchored receptors such as Fc{gamma}RIIIB (CD16b), uPAR (CD87), or LPS (CD14). Because these receptors do not transit the plasma membrane, signaling functions are accomplished as a result of partnering with CR3 via the LLD. Occupancy of LLD with CD87 was shown by Ross and colleagues (40) to activate CR3 into a high affinity state for ICAM binding. In these studies, soluble {beta}-glucan dissociated CR3 from CD87 and reduced the affinity of CR3 for ICAM. The authors predicted that use of small m.w. polysaccharides might cause a steric blockade of the I-domain, thus preventing PMN diapedesis and collateral tissue damage. Current studies, using multiple endothelial cell lines, did not demonstrate alterations in adhesive interactions between {beta}-glucan-treated activated PMNs and the endothelium, but did in fact support that prediction. Our findings indicate that the LLD does not mediate PMN:endothelial cell adhesion because activated neutrophils pretreated with blocking Abs against the lectin site still caused endothelial barrier dysfunction. In comparison, neutrophils pretreated with a blocking Ab to the CR3 I-domain maintained the integrity of the endothelial monolayer presumably by obviating ICAM recognition. These findings correlate with previous studies that binding of soluble {beta}-glucan can induce the high affinity conformation of the CR3 I-domain without promoting neutrophil spreading and adhesion (39).

Protection of the integrity of the monolayer via LLD cross-linking confirms that signaling events through the CR3 lectin site alter PMN CR3-dependent functions normally associated with occupancy of the I-domain. This finding supports and extends previous reports from this laboratory that demonstrated a conversion of random to directed neutrophil migration when extracellular matrix is supplemented with {beta}-glucan (22). A series of Ab blocking experiments attributed this shift in migration to corecognition of fibronectin by the CR3 I-domain and {beta}-glucan by the LLD. Dual ligation of CR3 by {beta}-glucan and the I-domain ligand iC3b has also been shown to result in NK cell cytolysis of iC3b-opsonized tumor targets that would otherwise resist this mechanism of cell-mediated cytotoxicity (39). This suggests that regulation of CR3 I-domain function through the LLD is not restricted to neutrophils and may find therapeutic applicability in heightening host defense against tumor cells.

The nature of the {beta}-glucan receptor has been of great interest over the years and several strong candidates have been reported, in addition to the {beta}2 integrin CR3 (41, 42, 43). For example, dectin-1, a newly described {beta}-glucan receptor has been reported to be responsible for the recognition of unopsonized zymosan (44, 45, 46) and shown to be expressed on cells of macrophage and neutrophil lineage. In our experimental system, the mechanism of {beta}-glucan protection of the endothelial monolayer was found to be completely accountable by its binding to the LLD of CR3. This suggests that the preservation of endothelial barrier function by {beta}-glucan treatment of activated neutrophils may not involve alternate receptors.

Various PMN-derived soluble factors have been implicated to cause changes in vascular monolayer permeability including adenosine, oxidants, lysosomal enzymes, and cationic proteins such as CAP37 (47, 48). We have identified the mechanism of the {beta}-glucan protection of the endothelium to be secondary neither to inhibition of adhesion nor to a paracrine event. Activated PMN supernatants, treated with {beta}-glucan or not, added onto endothelial monolayers did not cause endothelial barrier dysfunction (Fig. 1b), indicating that direct interaction of neutrophils with the endothelial cells is necessary for loss of barrier function. Levels of CAP37 (heparin-binding protein) known to cause increased vascular monolayer permeability were the same among fMLP-activated neutrophils that were treated with or without {beta}-glucan (data not shown). Following activation, neutrophils release 5'-AMP that can be metabolized to adenosine by 5'-ectonucleotidase (CD73) expressed on the endothelial cell surface resulting in increased barrier function in a paracrine loop (49). In contrast, our studies did not detect a gain in barrier function when supernatant was collect from fMLP neutrophils either in the presence or absence of {beta}-glucan. An explanation for this apparent discrepancy may lie in a technical difference between the experimental protocols. Lennon et al. (50) used supernatants from 108 fMLP-treated PMNs/ml to see an increase in barrier function due to 5'-AMP derived from the activated PMNs. This affect was no longer evident when supernatants were diluted 1/16 which approximates incubation with 6 x 106 cells/ml. We used 3 x 106 PMNs/ml supernatant as a control for our experimental system to match conditions in which permeability measurements were obtained in the presence of the same number of effector PMNs. Therefore, it is quite possible that we did not condition our media with a sufficient number of cells to reproduce the aforementioned mechanism of barrier function protection. Finally, addition of fixed neutrophils caused retraction of the endothelial monolayer which was not altered by {beta}-glucan, signifying that protection of the endothelial barrier function occurs through active signaling of the LLD and therefore requires viable effector cells.

The preparation of {beta}-glucan used in our current studies has already been shown to be well-tolerated by patients in clinical trials when used for an anti-infectious indication (51). It is suggested that {beta}-glucan or structurally related lectin site agonists may be useful in the design of therapeutic agents indicated for attenuation of excessive inflammation.


   Acknowledgments
 
We thank Dr. Anne Pereira (Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK) for measurements of CAP37 and for helpful advice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants GM-066194 (to J.S.R.), GM-42859 (to J.E.A.), and HL-067795 (to E.O.H.), the Carter Family Charitable Trust (Armand D. Versaci Research Scholar in Surgical Sciences Award (to V.L.T.)), and allocations to the Department of Surgery by Rhode Island Hospital. Back

2 Address correspondence and reprint requests to Dr. Jonathan S. Reichner, Department of Surgery, NAB-219, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. E-mail address: Reichner{at}Brown.edu Back

3 Abbreviations used in this paper: CR3, complement receptor 3; LLD, lectin-like domain; PMN, polymorphonuclear neutrophil; RPAEC, rat pulmonary aortic endothelial cell; uPAR, urokinase-type plasminogen activator receptor. Back

Received for publication December 19, 2003. Accepted for publication May 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11.[Medline]
  2. Bunting, M., E. S. Harris, T. M. McIntyre, S. M. Prescott, G. A. Zimmerman. 2002. Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving {beta}2 integrins and selectin ligands. Curr. Opin. Hematol. 9:30.[Medline]
  3. Vedder, N. B., R. K. Winn, C. L. Rice, E. Y. Chi, K. E. Arfors, J. M. Harlan. 1988. A monoclonal antibody to the adherence-promoting leukocyte glycoprotein, CD18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J. Clin. Invest. 81:939.
  4. Mileski, W. J., R. K. Winn, N. B. Vedder, T. H. Pohlman, J. M. Harlan, C. L. Rice. 1990. Inhibition of CD18-dependent neutrophil adherence reduces organ injury after hemorrhagic shock in primates. Surgery 108:206.[Medline]
  5. Simpson, P. J., R. F. Todd, 3rd, J. K. Mickelson, J. C. Fantone, K. P. Gallagher, K. A. Lee, Y. Tamura, M. Cronin, B. R. Lucchesi. 1990. Sustained limitation of myocardial reperfusion injury by a monoclonal antibody that alters leukocyte function. Circulation 81:226.[Abstract/Free Full Text]
  6. Gallin John, S. R.. 1999. Inflammation Basic Principles and Clinical Correlates Lippincott Williams and Wilkins, Philadelphia.
  7. Lu, H., C. W. Smith, J. Perrard, D. Bullard, L. Tang, S. B. Shappell, M. L. Entman, A. L. Beaudet, C. M. Ballantyne. 1997. LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice. J. Clin. Invest. 99:1340.[Medline]
  8. Coxon, A., P. Rieu, F. J. Barkalow, S. Askari, A. H. Sharpe, U. H. von Andrian, M. A. Arnaout, T. N. Mayadas. 1996. A novel role for the {beta}2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653.[Medline]
  9. Diamond, M. S., J. Garcia-Aguilar, J. K. Bickford, A. L. Corbi, T. A. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J. Cell Biol. 120:1031.[Abstract/Free Full Text]
  10. Diamond, M. S., T. A. Springer. 1993. A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J. Cell Biol. 120:545.[Abstract/Free Full Text]
  11. Miller, J. L., A. Castella. 1982. Platelet-type von Willebrand’s disease: characterization of a new bleeding disorder. Blood 60:790.[Abstract/Free Full Text]
  12. Walzog, B., D. Schuppan, C. Heimpel, A. Hafezi-Moghadam, P. Gaehtgens, K. Ley. 1995. The leukocyte integrin Mac-1 (CD11b/CD18) contributes to binding of human granulocytes to collagen. Exp. Cell Res. 218:28.[Medline]
  13. Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, S. D. Wright. 1989. Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins. J. Cell Biol. 109:1341.[Abstract/Free Full Text]
  14. Schober, J. M., N. Chen, T. M. Grzeszkiewicz, I. Jovanovic, E. E. Emeson, T. P. Ugarova, R. D. Ye, L. F. Lau, S. C. Lam. 2002. Identification of integrin {alpha}M{beta}2 as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions. Blood 99:4457.[Abstract/Free Full Text]
  15. Yakubenko, V. P., V. K. Lishko, S. C. Lam, T. P. Ugarova. 2002. A molecular basis for integrin {alpha}M{beta}2 ligand binding promiscuity. J. Biol. Chem. 277:48635.[Abstract/Free Full Text]
  16. Russell, D. G., S. D. Wright. 1988. Complement receptor type 3 (CR3) binds to an Arg-Gly-Asp-containing region of the major surface glycoprotein, gp63, of Leishmania promastigotes. J. Exp. Med. 168:279.[Abstract/Free Full Text]
  17. Relman, D., E. Tuomanen, S. Falkow, D. T. Golenbock, K. Saukkonen, S. D. Wright. 1990. Recognition of a bacterial adhesion by an integrin: macrophage CR3 ({alpha}M{beta}2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 61:1375.[Medline]
  18. Forsyth, C. B., H. L. Mathews. 1996. Lymphocytes utilize CD11b/CD18 for adhesion to Candida albicans. Cell. Immunol. 170:91.[Medline]
  19. Ross, G. D., J. A. Cain, B. L. Myones, S. L. Newman, P. J. Lachmann. 1987. Specificity of membrane complement receptor type three (CR3) for {beta}-glucans. Complement 4:61.[Medline]
  20. Ross, G. D., V. Vetvicka, J. Yan, Y. Xia, J. Vetvickova. 1999. Therapeutic intervention with complement and {beta}-glucan in cancer. Immunopharmacology 42:61.[Medline]
  21. Sitrin, R. G., R. F. Todd, 3rd, H. R. Petty, T. G. Brock, E. Albrecht, M. R. Gyetko. 1996. The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. J. Clin. Invest. 97:1942.[Medline]
  22. Harler, M. B., E. Wakshull, E. J. Filardo, J. E. Albina, J. S. Reichner. 1999. Promotion of neutrophil chemotaxis through differential regulation of {beta}1 and {beta}2 integrins. J. Immunol. 162:6792.[Abstract/Free Full Text]
  23. Vetvicka, V., B. P. Thornton, T. J. Wieman, G. D. Ross. 1997. Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and {beta}-glucan-primed CR3 (CD11b/CD18). J. Immunol. 159:599.[Abstract]
  24. Wegener, J., C. R. Keese, I. Giaever. 2000. Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp. Cell Res. 259:158.[Medline]
  25. Keese, C. R., K. Bhawe, J. Wegener, I. Giaever. 2002. Real-time impedance assay to follow the invasive activities of metastatic cells in culture. BioTechniques 33:842.[Medline]
  26. Taooka, Y., J. Chen, T. Yednock, D. Sheppard. 1999. The integrin {alpha}9{beta}1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1. J. Cell Biol. 145:413.[Abstract/Free Full Text]
  27. Gautam, N., H. Herwald, P. Hedqvist, L. Lindbom. 2000. Signaling via {beta}2 integrins triggers neutrophil-dependent alteration in endothelial barrier function. J. Exp. Med. 191:1829.[Abstract/Free Full Text]
  28. Gautam, N., P. Hedqvist, L. Lindbom. 1998. Kinetics of leukocyte-induced changes in endothelial barrier function. Br. J. Pharmacol. 125:1109.[Medline]
  29. Wary, K., D. M. A. Mariotti, F. Giancotti. 1999. Integrin Protocols Humana Press, Totowa.
  30. Ross, G. D., R. A. Thompson, M. J. Walport, T. A. Springer, J. V. Watson, R. H. Ward, J. Lida, S. L. Newman, R. A. Harrison, P. J. Lachmann. 1985. Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1. Blood. 66:882.[Abstract/Free Full Text]
  31. Lowe, E. P., D. Wei, P. J. Rice, C. Li, J. Kalbfleisch, I. W. Browder, D. L. Williams. 2002. Human vascular endothelial cells express pattern recognition receptors for fungal glucans which stimulates nuclear factor {kappa}B activation and interleukin 8 production. Winner of the Best Paper Award from the Gold Medal Forum. Am. Surg. 68:508.[Medline]
  32. Sans, E., E. Delachanal, A. Duperray. 2001. Analysis of the roles of ICAM-1 in neutrophil transmigration using a reconstituted mammalian cell expression model: implication of ICAM-1 cytoplasmic domain and Rho-dependent signaling pathway. J. Immunol. 166:544.[Abstract/Free Full Text]
  33. Zen, K., Y. Liu, D. Cairo, C. A. Parkos. 2002. CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans. J. Immunol. 169:5270.[Abstract/Free Full Text]
  34. Furie, M. B., M. C. Tancinco, C. W. Smith. 1991. Monoclonal antibodies to leukocyte integrins CD11a/CD18 and CD11b/CD18 or intercellular adhesion molecule-1 inhibit chemoattractant-stimulated neutrophil transendothelial migration in vitro. Blood 78:2089.[Abstract/Free Full Text]
  35. Issekutz, A. C., D. Rowter, T. A. Springer. 1999. Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration. J. Leukocyte Biol. 65:117.[Abstract]
  36. Smith, C. W., S. D. Marlin, R. Rothlein, C. Toman, D. C. Anderson. 1989. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83:2008.
  37. Shaw, S. K., P. S. Bamba, B. N. Perkins, F. W. Luscinskas. 2001. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J. Immunol. 167:2323.[Abstract/Free Full Text]
  38. Ross, G. D.. 2000. Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/{alpha}M{beta}2-integrin glycoprotein. Crit. Rev. Immunol. 20:197.[Medline]
  39. Vetvicka, V., B. P. Thornton, G. D. Ross. 1996. Soluble {beta}-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J. Clin. Invest. 98:50.[Medline]
  40. Xia, Y., G. Borland, J. Huang, I. F. Mizukami, H. R. Petty, R. F. Todd, 3rd, G. D. Ross. 2002. Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion. J. Immunol. 169:6417.[Abstract/Free Full Text]
  41. Brown, G. D., S. Gordon. 2001. Immune recognition: a new receptor for {beta}-glucans. Nature 413:36.[Medline]
  42. Czop, J. K., J. Kay. 1991. Isolation and characterization of {beta}-glucan receptors on human mononuclear phagocytes. J. Exp. Med. 173:1511.[Abstract/Free Full Text]
  43. Zimmerman, J. W., J. Lindermuth, P. A. Fish, G. P. Palace, T. T. Stevenson, D. E. DeMong. 1998. A novel carbohydrate-glycosphingolipid interaction between a {beta}-(1–3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J. Biol. Chem. 273:22014.[Abstract/Free Full Text]
  44. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams, L. Martinez-Pomares, S. Y. Wong, S. Gordon. 2002. Dectin-1 is a major {beta}-glucan receptor on macrophages. J. Exp. Med. 196:407.[Abstract/Free Full Text]
  45. Willment, J. A., S. Gordon, G. D. Brown. 2001. Characterization of the human {beta}-glucan receptor and its alternatively spliced isoforms. J. Biol. Chem. 276:43818.[Abstract/Free Full Text]
  46. Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. Martinez-Pomares, S. Gordon, S. Y. Wong. 2002. The {beta}-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169:3876.[Abstract/Free Full Text]
  47. Gautam, N., A. M. Olofsson, H. Herwald, L. F. Iversen, E. Lundgren-Akerlund, P. Hedqvist, K. E. Arfors, H. Flodgaard, L. Lindbom. 2001. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat. Med. 7:1123.[Medline]
  48. Dallegri, F., L. Ottonello. 1997. Tissue injury in neutrophilic inflammation. Inflamm. Res. 46:382.[Medline]
  49. Narravula, S., P. F. Lennon, B. U. Mueller, S. P. Colgan. 2000. Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function. J. Immunol. 165:5262.[Abstract/Free Full Text]
  50. Lennon, P. F., C. T. Taylor, G. L. Stahl, S. P. Colgan. 1998. Neutrophil-derived 5'-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A2B receptor activation. J. Exp. Med. 188:1433.[Abstract/Free Full Text]
  51. Babineau, T. J., A. Hackford, A. Kenler, B. Bistrian, R. A. Forse, P. G. Fairchild, S. Heard, M. Keroack, P. Caushaj, P. Benotti. 1994. A phase II multicenter, double-blind, randomized, placebo-controlled study of three dosages of an immunomodulator (PGG-glucan) in high-risk surgical patients. Arch. Surg. 129:1204.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 INT J LOW EXTREM WOUNDSHome page
S. Kumar, D. Ghosh, Tuhin Kanti Biswas, U. Dutta, P. Das, and S. Kundu
Spermatheca Gland Extract of Snail (Telescopium telescopium) Has Wound Healing Potential: An Experimental Study in Rabbits
International Journal of Lower Extremity Wounds, December 1, 2008; 7(4): 204 - 209.
[Abstract] [PDF]

Home page
 Clin. Cancer Res.Home page
C. Salvador, B. Li, R. Hansen, D. E. Cramer, M. Kong, and J. Yan
Yeast-Derived {beta}-Glucan Augments the Therapeutic Efficacy Mediated by Anti-Vascular Endothelial Growth Factor Monoclonal Antibody in Human Carcinoma Xenograft Models
Clin. Cancer Res., February 15, 2008; 14(4): 1239 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. M. Lavigne, X. M. O'Brien, M. Kim, J. W. Janowski, J. E. Albina, and J. S. Reichner
Integrin Engagement Mediates the Human Polymorphonuclear Leukocyte Response to a Fungal Pathogen-Associated Molecular Pattern
J. Immunol., June 1, 2007; 178(11): 7276 - 7282.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
B. W. LeBlanc, J. E. Albina, and J. S. Reichner
The effect of PGG-{beta}-glucan on neutrophil chemotaxis in vivo
J. Leukoc. Biol., April 1, 2006; 79(4): 667 - 675.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Braun, J. Hamacher, D. R. Morel, A. Wendel, and R. Lucas
Dichotomal Role of TNF in Experimental Pulmonary Edema Reabsorption
J. Immunol., September 1, 2005; 175(5): 3402 - 3408.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsikitis, V. L.
Right arrow Articles by Reichner, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsikitis, V. L.
Right arrow Articles by Reichner, J. S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS