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(Journal of Leukocyte Biology. 2002;71:932-940.)
© 2002 by Society for Leukocyte Biology

Rapid static adhesion of human naïve neutrophil to naïve xenoendothelium under physiologic flow is independent of Gal{alpha}1,3-gal structures

Sajila Sheikh, Ranjit Parhar and Futwan Al-Mohanna

Biological and Medical Research Department, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

Correspondence: Futwan Al-Mohanna, Ph.D., Biological and Medical Research, MBC 03, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. E-mail: futwan{at}kfshrc.edu.sa


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesion interactions under flow have long been known to depend on applied wall shear stress. We investigated the ability of human naïve neutrophils to adhere to xenogeneic endothelial cells under static and flow conditions. We demonstrate that human naïve neutrophils bind to xenogeneic endothelial cells under flow conditions. This binding is dependent on the applied stress and is independent of Gal{alpha}1,3-gal structures, ICAM-1, or its counter ligands LFA-1{alpha} and Mac-1. The binding was rapid and is characterized by stationary attachment with no obvious rolling or change in morphology. This binding leads to a transient increase in intracellular-free calcium levels in xenogeneic but not allogeneic-endothelial cells with occasional oscillations that persist long after the initial contact between the two cell types. Previous activation of xenoendothelium by autologous serum or human TNF-{alpha} augments binding of human naïve neutrophils to the endothelial cells. Our data suggest novel interaction sites between the xenogeneic endothelial cells and human naïve neutrophils.

Key Words: xenotransplantation • calcium • flow stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Porcine organs are considered the most viable source of xenografts for humans. One major barrier to discordant xenotransplantation is hyperacute rejection (HAR), which is mediated by the binding of recipient xenoreactive natural antibodies (XNA) to antigens present on the endothelium of the xenogeneic donor organ [1 2 3 4 5 6 ]. In the pig-to-primate model, XNA are directed against galactose {alpha}1,3-galactose (Gal{alpha}1,3-gal) epitopes on the pig endothelium [1 , 6 7 8 9 10 11 12 13 14 ]. Interaction of XNA with Gal{alpha}1,3-gal leads to the activation of the complement cascade to form membrane attack complexes (MAC) resulting in endothelial injury [15 , 16 ]. This leads to the conversion of xenoendothelium from an antithrombic to a prothrombic state and the generation of inflammatory mediators that recruit cellular components of the immune system to the site of complement activation. The former leads to virtual strangulation of the xenograft (HAR), and the latter precipitates in acute vascular rejection [17 , 18 ]. Participation of the innate immune cells in xenograft rejection has always been assumed secondary to HAR [19 ]. We have demonstrated previously that innate immune cells, namely human naïve neutrophils and natural killer (NK) cells, bind and activate xenoendothelium [20 , 21 ]. Several studies have supported the concept that adhesive phenomena are influenced strongly by applied flow stress [22 23 24 25 ]. It is now well established that leukocyte migration from the blood flow into inflamed or ischemic tissues is part of a complex series of adhesive interactions involving adhesion molecules on the leukocyte and the endothelial cell (EC) [26 27 28 29 30 ].

With the plethora of studies on xenograft rejection, very few have considered the rheological factors influencing interactions between host innate immune cells and donor endothelium [31 , 32 ]. The nature of the adhesive interactions occurring under flow conditions is clearly different from those found under static conditions [33 ]. Many transcription factors have been shown to bind to a shear stress response element and activate transcription of key endothelial genes such as tissue plasminogen activator, transforming growth factor-ß, monocyte chemoattractant protein-1, endothelin, adhesion molecules, and endothelial nitric oxide synthase [34 , 35 ].

Recently, we reported that naïve, human NK cells are capable of recognizing, binding, and killing xenogeneic endothelium independent of Gal{alpha}1,3-gal [21 ]. Attachment of NK cells to porcine or ovine endothelium under static, nonflow conditions resulted in activation of the ECs, represented by an increase in intracellular calcium. Here, we investigate the adhesive interactions between human naïve neutrophils and porcine and human aortic ECs (PAECs and HAECs, respectively) under physiological flow stress. We demonstrate that human naïve neutrophils adhere more to PAECs than HAECs and that adhesion was of the static type and did not appear to be preceded by rolling adhesion. This adhesion occurred in the absence of xenoreactive natural antibodies and complement and was unaffected by the presence of blocking antibodies against Gal{alpha}1,3-gal or soluble Gal{alpha}1,3-gal. Activation of PAECs by human serum or tumor necrosis factor {alpha} (TNF-{alpha}) resulted in a dramatic augmentation of neutrophil adhesion. Our data confirm the early involvement of neutrophils in xenograft rejection and that interactions between human naïve neutrophils and xenogeneic endothelium are independent of the Gal{alpha}1,3-gal epitope.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Soluble {alpha}1,3-gal was obtained from Dextra Labs Ltd. (Reading UK). Anti-Gal{alpha}1,3-gal antibodies were prepared as described previously [21 ]. Monoclonal mouse anti-human intercellular adhesion molecule-1 (ICAM-1) antibody (BBA4) was purchased from R&D Systems (Minneapolis, MN) and dialyzed against phosphate buffered saline (pH 7.4) to remove preservative. Cross-reactivity of anti-human ICAM-1 to porcine ICAM-1 was confirmed by increased immunofluorescence of HAECs and PAECs following treatment with TNF-{alpha} and staining with BB4 as described previously [20 ]. Anti-Mac-1 antibody was from Bender MedSystems (Austria). TNF-{alpha} and anti-human lymphocyte function associated antigen-1 (LFA-1) antibody were purchased from Pharmingen (San Diego, CA). Saturation concentrations of anti-Gal{alpha}1,3-gal, anti-ICAM-1, anti-Mac-1, and anti-LFA-1 antibodies used in the present work were based on previous studies [20 , 21 ] and were confirmed using flow cytometry in saturation analysis assays.

Isolation of neutrophils
Human venous blood was drawn from healthy volunteers into preservative-free sodium heparin. After dextran sedimentation, neutrophils were purified by centrifugation through Ficoll-Paque as described previously [20 ]. Contaminating red blood cells were removed by hypotonic lysis with buffered isotonic NH4Cl. Neutrophils were harvested and washed twice in Krebs-HEPES medium containing 1.3 mM CaCl2, 1.2 mM MgSO4, 4.8 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, and 0.1% bovine serum albumin (BSA). The neutrophils were counted and resuspended to a concentration of 1 x 107 cells/ml. Purity of the neutrophil suspension was between 98 and 99%, as indicated by flow cytometry, and >98% viable, as judged by trypan blue exclusion tests. Neutrophils were tested routinely for production of reactive oxygen metabolites by luminol-dependent chemiluminescence (LDCL) [36 ], and only cells that showed no increase in LDCL were used for experimentation.

Isolation of ECs
PAECs were isolated from adult pig aorta, essentially as described previously [37 ]. ECs were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum, glutamine (1 mmol/L), penicillin (100 U/ml), and streptomycin (25 µg/ml). HAECs were isolated from the thoracic aorta of donor hearts as described previously [20 ]. ECs were characterized by their cobblestone morphology, positive staining with antibodies to von Willebrand factor, and to acetylated low-density lipoprotein. Cells were used from passages 2 to 10 in all experiments at a split ratio of 1:3. In some experiments, PAECs were stimulated with TNF-{alpha} (10 ng/ml; Pharmingen) or human serum (autologous to the neutrophils used) for up to 6 h at 37°C.

Preparation of microslides for EC coating
Microslides are flat glass microcapillary tubes with a cross section of 300 µm by 3 mm and a length of 50 mm with good optical qualities (Camlab, Cambridge, UK). The microslides were washed in a solution of 70% nitric acid in distilled water and then washed with copious amounts of distilled water. The microslides were dried by immersion in anhydrous acetone twice and then immersed twice in a 4% solution of 3-aminopropyl-triethoxysilane (APES; Sigma Chemical Co., UK). APES serves to produce a coating inside the microslide, which facilitates the adhesion and spreading of ECs on the glass surface. The microslides were rinsed with anhydrous acetone followed by four washes with distilled water. Finally, the microslides were placed at 37°C to allow evaporation of all liquid from the inside of the tubes.

Human or porcine cells were propagated to confluence in the glass microslides before use in the flow adhesion assay. Confluence of the ECs was confirmed by visualization under phase contrast microscopy along the microslide.

In vitro flow-based adhesion assay
Cellular interactions between flowing neutrophils and the endothelial monolayer were observed using an in vitro flow-based adhesion assay system as described previously [38 39 40 ]. ECs were grown to confluence in the microslide. The microslide was then glued across the width of a microscope slide using super loctite glue. The microscope slide was placed on the light microscope stage. One end of the microslide was attached to a three-way electronic valve using silicon rubber tubing. This electronic valve allowed exchange between neutrophil cell suspension or cell-free medium. The other end of the microslide was attached to a Harvard syringe pump (Harvard Apparatus, South Natic, MA). Neutrophil suspensions were drawn through the microslide for 5 min at a wall shear stress of 0.05 Pascal (Pa). The rate of flow (Q) of liquid through the microslide was controlled by the speed setting of the pump. The wall shear stress exerted on the endothelial surface was calculated using the following equation: t = (6Q · {eta})/(w · h2), where {eta} is the viscosity of the suspending medium, h is the internal depth of the microslide, and w is the microslide internal width. Neutrophil-endothelial interactions were video-recorded (Hyper HAD CCD-IRIS/RGB, Sony, Riyadh, Saudi Arabia). Quantitation and categorization of adhesion were determined by analysis of the videotapes using the OpenLab image processing system (Improvision, Coventry, UK). All experiments were conducted at room temperature.

Calcium measurements
Calcium in PAECs under static conditions was measured as described previously [20 ]. Briefly, PAECs or HAECs, which had been plated on glass coverslips at 106 cells/ml, 12–18 h previously, were loaded with Fura-2 AM (1 µM, room temperature, 30 min) in Krebs-HEPES medium (pH 7.4, containing 120 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 4.8 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, and 0.1% BSA). The coverslips were mounted on a temperature-controlled holder, and intracellular calcium was measured as described previously [41 ]. Calcium measurements were performed on individual cells using an Ionvision dual excitation system (Improvision). Emission at 510 nm after excitation at 340 nm and 380 nm with a xenon arc lamp was collected using a charge-coupled device video camera (Photonic Science, Sussex, UK) at 1- to 3-s intervals. The images were processed and ratioed using the ionVision software. The intensity levels at 510 nm after excitation at 340 nm and 380 nm were background subtracted and ratioed before transforming into absolute calcium values [42 ]. Absolute calcium levels were determined by using the measured Rmin and Rmax of 0.1 and 7.6, respectively. The K1/2 for Fura-calcium was determined to be 856 nM. ECs that showed a transient calcium increase of greater than twofold were considered responsive. For flow experiments, PAECs propagated to confluence in the glass microslides were loaded with Fluo-3 AM (1 µM/ml for 30 min at 37°C). Microslides were washed and then secured onto a Lieca TCS confocal microscope stage. Images were obtained as described previously [43 ]. Images were analyzed using Scanware software (Leica Microsystems, Heidleberg, Germany), and fluorescence intensity (from each cell) was transformed into absolute calcium levels as described previously [43 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Naïve neutrophil adhesion to xenogeneic ECs under flow conditions is independent of Gal{alpha}1,3-gal
When human naïve neutrophil suspension (1x107/ml) was perfused over PAEC monolayers under shear stress, neutrophils bound to PAECs by immediate stationary attachment in a manner that was dependent on the applied wall shear stress (Fig. 1a ). Maximum binding occurred at 0.05 Pa with higher shear stresses being inhibitory. This stationary attachment was not preceded by any obvious rolling adhesion or change in morphology over a period of time. In contrast, neutrophils did not bind by any significant levels to unstimulated HAEC monolayers.



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  		Figure 1. Effect of wall shear stress on adhesion of flowing naïve neutrophils to unstimulated PAECs. (a) Neutrophils adhered to PAECs in a shear stress-dependent manner. Data represent the mean ± SE from three experiments. (b) Lack of effect of pretreatment of neutrophils with antibodies against LFA-1 or Mac-1 or pretreatment of PAECs with antibodies against Gal{alpha}1,3-gal or ICAM-1. Data represent the mean ± SE of at least three experiments.

 
Because Gal{alpha}1,3-gal-containing structures are the major recognition sites for human XNA binding to xenogeneic endothelium, the possibility existed that similar Gal{alpha}1,3-gal-containing structures might be involved in the neutrophil/PAECs adhesion. We tested this possibility by blocking Gal{alpha}1,3-gal structures under flow stress (0.05 Pa). We found that treating PAECs with blocking anti-Gal{alpha}1,3-gal antibody (up to 0.5 mg/ml) had no apparent effect on the binding of human neutrophils to PAECs. Such concentrations of Gal{alpha}1,3-gal have been shown to totally block the Gal{alpha}1,3-gal-containing structures [21 ] (Fig. 1b) . Since the adhesion molecule ICAM-1 is known to partially mediate neutrophil adhesion to activated human umbilical vein EC (HUVEC) under flow [44 ] and to naïve PAECs under static conditions [20 ], we tested the effect of blocking ICAM-1 binding sites on neutrophil adhesion to naive PAEC using blocking anti-ICAM-1 antibody (BBA4, 0.1 mg/ml). We found that anti ICAM-1 antibody at concentrations sufficient to block all the binding sites on PAECs (as determined by flow cytometry) did not affect the level of neutrophil binding to PAECs, nor did blocking antibodies to the ICAM-1 counter ligand on the neutrophil membranes LFA-1{alpha} (100 ng/ml) and Mac-1 (10 µg/ml) (Fig. 1b) .

Human neutrophil induces calcium rise in PAECs under static and flow conditions
Direct contact between human naïve neutrophils and PAECs under static conditions caused a transient calcium rise in the ECs from a resting level of 76 ± 1 nM to 386 ± 26 nM within 30 s (Fig. 2 ). PAECs that were not in direct contact with the neutrophils exhibited no change in intracellular calcium. Single-cell intracellular calcium maps revealed a global, initial, cytosolic rise, followed by a more localized punctate that seem to fuse together with time, followed by a slow decay that takes the calcium levels to the precontact levels over several minutes (Fig. 2a and 2b) . Although the calcium response was heterogeneous and asynchronous, the extent of the calcium rise was not dependent on the number of neutrophils in contact with PAECs. In contrast, direct contact between human naïve neutrophils and HAECs showed no transient calcium rise. We next tested the effect of soluble Gal{alpha}1,3-gal or anti-Gal{alpha}1,3-gal immunoglobulin G (IgG) on human neutrophil-induced calcium transients in PAECs. Neither soluble Gal{alpha}1,3-gal nor anti-Gal{alpha} 1,3-gal affected the extent of the calcium rise induced in PAECs by human neutrophils (P>0.05 for both; Fig. 2c and 2d , respectively).



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  		Figure 2. Effect of human naïve neutrophils on intraendothelial calcium in PAECs under static conditions. (a) Calcium maps in PAECs before (time 0) and at the indicated time intervals after contact with human naïve neutrophils. Calcium changes are color-coded such that high calcium is indicated by warm colors. Original scale bar is 20 µm. (b) Dynamic of calcium changes in a single PAEC after exposure to human naïve neutrophils. (c and d) Calcium maps in PAECs before and at the indicated time intervals after exposure to human naïve neutrophils in the presence of soluble Gal{alpha}1,3-gal (1 µg/ml) and anti-Gal{alpha}1,3-gal IgG (100 ng/ml), respectively. Calcium changes are color-coded as in (a).

 
Confocal laser scanning microscopy, used to investigate calcium transients under flow conditions, revealed a transient rise in intracellular-free calcium in PAECs upon encountering human naïve neutrophils under flow stress (0.05 Pa; Fig. 3a ). This calcium rise occurred only in PAECs that were in direct contact with human neutrophils. PAECs that have not contacted neutrophils did not exhibit any calcium rise. Maximum calcium reached was 790 ± 90 nM, representing an approximate tenfold increase from the resting level of 74 ± 11 nM. The calcium response was asynchronous and heterogeneous with some cells exhibiting distinct oscillations long after their contact with human neutrophils (Fig. 3b) . The average number of spikes was 2.2 ± 0.3/s, displaying an amplitude of 311 ± 23 nM calcium with oscillations seizing at approximately 100 s post contact with human neutrophils. Pretreatment of PAECs with anti-Gal{alpha}1,3-gal antibodies had no apparent effect on the extent of the calcium transient; however, the time of onset of the calcium transient was delayed (Fig. 3c) .



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  		Figure 3. Intracellular calcium changes in PAECs following contact with naïve human neutrophils under flow conditions (0.05 Pa). (a) Calcium changes in a single PAEC in response to neutrophil encounter. (b) Calcium oscillations in a single PAEC after encounter with human naïve neutrophils. (c) Changes in Fluo-3 fluorescence in PAECs following encounter with human naïve neutrophils. Intensity is coded as indicated in the color bar. Top and bottom panels indicate fluorescence maps in the presence and absence of anti-Gal{alpha}1,3-gal IgG (100 ng/ml), respectively.

 
Enhanced adhesion of human naïve neutrophils to TNF-{alpha}- and serum-activated PAECs
Human neutrophil adhesion to PAECs was augmented greatly after treatment of PAECs with TNF-{alpha} (10 ng/ml) in a time-dependent manner (Fig. 4a ). In contrast to unstimulated PAECs, the adhesion observed under these conditions was a combination of static and rolling adhesion (11% rolling). The additional neutrophils appeared to be recruited from flow onto the endothelium surface, partake in a period of rolling adhesion, and then to eventually stop rolling in order to remain statically attached on the endothelial substrate. Maximum adhesion occurred at 4 h following TNF-{alpha} treatment, when the number of adherent cells increased from 9.7 ± 0.9 to 131.7 ± 24.6 cells per field of view (P<0.01), representing an approximate 13-fold increase in adhesion. To test the hypothesis that human serum can stimulate xenogeneic endothelium in a similar manner to potent activating agents such as TNF-{alpha}, we treated the PAECs with freshly prepared human serum before the adhesion assay. We found that the number of adherent cells/field of view increased from 9.7 ± 0.9 to 29 ± 0.9 cells (P=0.08) within 2 h of serum treatment, representing a threefold increase in the level of neutrophil binding to PAECs (Fig. 4b) . After 4 h, this level of binding had increased further to 48 ± 7 cells per field of view, representing fivefold the original level (P<0.05). However, serum treatment caused an obvious time-dependent retraction of PAECs from the glass microslide, suggesting the demise of xenogeneic ECs (Fig. 4c) . In contrast to TNF-{alpha}, serum-induced neutrophil adhesion to PAECs remained predominantly static (only 2% rolling) after 4 h of treatment. The presence of anti- Gal{alpha}1,3-gal antibodies had no apparent effect on adhesion of human neutrophils to TNF-{alpha}- or serum-activated PAECs (P>0.05).



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  		Figure 4. Neutrophil adhesion to (a) TNF-{alpha}-stimulated PAECs at flow stress of 0.05 Pa. (b) Serum-treated PAECs at flow stress of 0.05 Pa. Treatment was not extended beyond 4 h because retraction of the PAEC monolayer was evident at this stage. (c) Effect of human serum on PAEC monolayer retraction and subsequent detachment at the indicated time intervals at flow stress of 0.05 Pa.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Xenogeneic antibodies directed against the Gal{alpha}1,3-gal epitope are considered to be the major obstacle for successful cardiac xenotransplantation. Recent investigations have focused mainly on strategies to overcome the process of HAR mediated by XNA [45 46 47 48 ]. Approaches such as intravenous administration of synthetic Gal{alpha}1,3-gal [49 ], Gal{alpha}1,3-gal antigen modification using coffee bean {alpha}-galactosidase [50 ], transgenesis, gene transfection [51 ], and {alpha}1,3 galactosyltransferase knockout animal models [52 ] have all been used in search of inhibition of HAR.

Limitations in overcoming HAR have resulted in poor characterization of the cell-mediated mechanisms underlying xenograft rejection. Few studies have investigated cellular responses to discordant xenografts, although infiltration of leukocytes into xenogeneic organs suggests an inevitable role of cellular components in the demise of the transplanted organ. Kirk et al. [53 ] demonstrated infiltrates of human leukocytes in the pig heart, consisting predominantly of neutrophils, macrophages, T cells, and few B cells and NK cells, whereas Inveradi et al. [54 ] demonstrated that perfusion of human cells through the coronary system of a rat heart resulted in preferential retention of NK cells in the xenogeneic organ. Using an in vitro porcine-to-human model, human peripheral blood lymphocytes were shown to adhere to resting and activated PAECs [55 ]. Collectively, these studies suggest a role for cell-mediated mechanisms in the rejection of vascularized xenografts. However, an important question arises as to whether innate immune cells recognize and interact with the xenograft independent of Gal{alpha}1,3-gal structures. We have shown previously that human naïve neutrophils do recognize and activate PAECs and sheep aortic ECs (SAECs) in the absence of XNA and complement [20 ]. Furthermore, human naïve NK cells recognize and activate PAECs and SAECs in the absence of XNA and complement, and under conditions in which Gal{alpha}1,3-gal structures are blocked [21 ]. These studies revealed that innate cells can interact with the xenoendothelelium and that the interaction is not a consequence of XNA/complement activation, suggesting that innate immune cells may be involved intimately in the early events leading to HAR.

It is well established that adhesive phenomena are affected greatly by hydrodynamic parameters [33 ]. Indeed, adhesive interactions under flow depend on the kinetics of the interaction as well as the affinity between a receptor and its ligand [22 , 23 , 56 ]. Hence, investigators concerned with xenograft cellular rejection have recently started to address the rheological constraints encountered in the circulation [31 , 32 ].

In the present study, we demonstrate that human naïve neutrophils adhere to resting or activated xenogeneic ECs more avidly than allogeneic endothelium under flow conditions. Unlike allogeneic interactions, where rolling is necessary for adhesion, adhesion to PAECs was static with no rolling. Linke et al. [57 ] have described a similar form of adhesion during xenogeneic perfusion using heparinized rat blood. In their study, the adherent leukocytes were described as "stagnant." The molecular mechanism(s) of this static (stagnant) form of adhesion is yet to be delineated.

In contrast to the results presented in this study, Robinson et al. [31 ] observed no adhesion of neutrophils to resting xenogeneic endothelium under flow. This discrepancy may be attributed to the high shear stress used in their study, 1.85 dynes/cm2 (0.18 Pa), which is almost fourfold higher than that used in the present study. In that study, the authors state that quiescent PAECs supported minimal neutrophil binding under static or rotating conditions. The question then arises as to how minimum is minimum binding. Previous work from our laboratory has demonstrated that a single neutrophil was able to cause a transient calcium rise in the individual xenogeneic EC with which it was brought into contact [20 ]. We thus argue that even minimal attachment of neutrophils to PAECs may be sufficient to activate the endothelium.

Here, we illustrate that recognition of xenogeneic endothelium by naïve human neutrophils is independent of Gal{alpha}1,3-gal structures. Our data demonstrate that the presence of blocking antibodies to Gal{alpha}1,3-gal or the presence of soluble Gal{alpha}1,3-gal had no significant effect on binding of naïve human neutrophils to PAECs.

The possibility of involvement of ICAM-1 and its counter ligands LFA-1{alpha} and Mac-1 in the PAEC/neutrophil interactions was dismissed by the finding that antibodies to these adhesion molecules and their respective isotypes had no apparent effect on this form of adhesion. This is in contrast to the adhesion characteristics under nonflow conditions in which ICAM-1, LFA-1{alpha}, Mac-1, and very late antigen-4 are clearly involved [20 , 55 ]. The possibility existed that the concentrations of blocking antibodies used in the present study were not sufficient to block their respective binding sites totally. This possibility seemed unlikely because indirect immunofluorescence using flow cytometry showed maximum intensity of anti-ICAM-1, anti-LFA-1, and anti-Mac-1-labeled cells, occurring at concentrations that were much less than the concentrations used in the blocking experiments.

To confirm our finding that neutrophil binding under flow was causing activation of the xenoendothelial cells, we used an in vitro flow system and confocal laser scanning microscopy to directly visualize and measure calcium transients under flow. We found that PAECs, which came into contact with flowing neutrophils, exhibited a transient calcium rise that was asynchronous and heterogeneous with a number of cells displaying obvious calcium oscillations. It is interesting that the extent and kinetics of the calcium transient evoked by neutrophils differed under the different experimental conditions. Caution must therefore be exercised when interpreting such data, especially because different calcium reporters were used for the static and flow conditions (Fura-2 and Fluo-3, respectively) and because of the different optics that flow experiments demanded. It is, however, interesting to note that under flow conditions, some cells exhibited repeated calcium spikes that differed in frequency and amplitude. It is generally accepted that calcium signals can be frequency and/or amplitude coded [58 ]. Many cells have evolved mechanisms for decoding these calcium signals through a number of proteins "systems" including protein kinase C [59 ] and Ca++/calmodulin-dependent protein kinase II [60 ]. The question therefore arises as to the function of these calcium transients in PAECs. Previous studies have demonstrated that human naïve neutrophils activated naïve PAECs in a calcium-dependent manner. This activation led to expression of a number of adhesion molecules, namely P-selectin and vascular cell adhesion molecule-1 on the surface of the PAECs, which made these cells more susceptible targets for killing [20 ]. In the present study, we show that the calcium transient is independent of Gal{alpha}1,3-gal structures. Evidence for this is drawn from the fact that blocking antibodies to Gal{alpha}1,3-gal at 100 ng/ml, which was sufficient to saturate all the Gal{alpha}1,3-gal-binding sites [21 ], and the presence of soluble Gal{alpha}1,3-gal failed to cause any significant change in the amplitude of the calcium signal. The question as to whether higher concentrations of antibody would yield substantial blockade or slowing of PAECs activation seemed unlikely because in a previous study, concentrations of up to 960 µg/ml anti-Gal{alpha}1,3-gal IgG failed to inhibit activation and subsequent killing of PAECs by human naïve NK cells [21 ]. Although in several flow experiments the onset of the calcium rise was delayed compared with human isotype-matched IgG-treated cells, this delay may be due to nonspecific antibody effect, especially since the negative control was done with human rather than baboon isotype-matched IgG because of the unavailability of the latter.

It is well established that TNF-{alpha} activation of ECs augments adhesion of allogeneic leukocytes under static [61 ] and flow conditions [62 ]. In the xenogeneic system, the ability of human TNF-{alpha} to stimulate PAECs [55 , 63 , 64 ] and the cross-reactivity of human TNF-{alpha} with PAECs have been documented [64 , 65 ]. Furthermore, elevated levels of TNF-{alpha} have been shown following allograft transplantation [66 , 67 ] and in xenogeneic studies, including an ex vivo kidney perfusion model [68 ] and discordant fetal pig islet xenografts in mice [69 ]. Therefore, the possibility existed that TNF-{alpha} might be implicated in the development of xenograft rejection. This was addressed by investigating the adhesion of human neutrophils to TNF-{alpha}-stimulated PAECs. We found that under physiological flow, TNF-{alpha} increased human naïve neutrophil adhesion to PAECs significantly, in a time-dependent fashion. Optimum adhesion was achieved after 4 h of stimulation. This observation is consistent with others who have demonstrated similar conditions for optimal binding of allogeneic neutrophils to TNF-{alpha}-stimulated HUVECs [70 ].

TNF-{alpha} activation caused a qualitative as well as a quantitative effect on neutrophil adhesion. An increase in adhesion levels was accompanied by an increase in the population of cells, which was participating in rolling adhesion rather than stationary attachment. The rolling nature of the increased adhesion suggested up-regulation of the selectin family of adhesion molecules on the activated PAECs. Indeed, previous studies have described the up-regulation of E-selectin on PAECs after stimulation with TNF-{alpha} [31 ].

As human serum contains XNA and complement, we studied the effect of pretreatment of xenoendothelium with freshly prepared serum on neutrophil binding. Our data demonstrate that treatment of PAECs with xenogeneic human serum augmented the binding of human naïve neutrophils significantly as compared with untreated PAEC controls. It is interesting that adhesion levels after serum treatment were lower than those observed after TNF-{alpha} treatment. However, microscopic observations showed an obvious retraction and loss of the endothelium monolayer from the glass microslide. This suggests endothelial injury and loss of barrier function following exposure to human serum and, presumably, activation of the complement system. These results are consistent with studies by Morigi et al. [32 ] in which increased adhesion of neutrophils to serum-treated PAECs was observed.

Although serum treatment caused an increase in absolute adhesion, the interactions remained predominantly static. This gives rise to the hypothesis that there are varying levels of adhesion molecule expression on xenoendothelium after serum or TNF-{alpha} activation. These two stimulating agents may indeed regulate different families of adhesion molecules on the xenoendothelium in order to create an inflammatory environment where neutrophils and other leukocytes may be recruited from flow. It is interesting, however, that the presence of anti-Gal{alpha}1,3-gal IgG had no significant effect on the binding of human neutrophils to TNF-{alpha}- or serum-activated PAECs.

In conclusion, our work demonstrates that naïve human neutrophils are able to recognize, activate, and bind to xenogeneic endothelium under physiological flow conditions in the absence of XNA and complement, and under conditions in which Gal{alpha}1,3-gal structures are blocked. Further studies are required to determine the Gal{alpha}1,3-gal-independent interaction site(s), mediating the process of innate-immune cell binding to xenoendothelium.


   ACKNOWLEDGEMENTS
 
This work was supported by the CardioVascular Research Programme and Research Centre funds. The authors are indebted to Drs. Kate Collison, M. C. Paterson, and B. F. Meyer for stimulating discussions and critical review of the manuscript. We give our special gratitude to Drs. S. Nakeeb, K. Al-Hussain, Miss S. Saleh, and Mr. P. Manogaran for technical help.

Received October 2, 2001; revised January 16, 2002; accepted January 31, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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F. Al-Mohanna, S. Saleh, R. S. Parhar, K. Khabar, and K. Collison
Human neutrophil gene expression profiling following xenogeneic encounter with porcine aortic endothelial cells: the occult role of neutrophils in xenograft rejection revealed
J. Leukoc. Biol., July 1, 2005; 78(1): 51 - 61.
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