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Originally published online as doi:10.1189/jlb.0904494 on June 16, 2005 Originally published online as doi:10.1189/jlb.0904494 on April 4, 2005

Published online before print April 4, 2005
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(Journal of Leukocyte Biology. 2005;78:51-61.)
© 2005 by Society for Leukocyte Biology

Human neutrophil gene expression profiling following xenogeneic encounter with porcine aortic endothelial cells: the occult role of neutrophils in xenograft rejection revealed

F. Al-Mohanna1, S. Saleh, R. S. Parhar, K. Khabar and K. Collison

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

1 Correspondence: Biological & Medical Research, MBC 03, King Faisal Specialist Hospital & 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
 
The role of innate immune cells in the recognition and activation of xenogeneic endothelium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the absence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact-dependent activation causes a transient calcium rise leading to increased reactive oxygen metabolite (ROM) production. Neutrophil gene-expression profiling using an adenylate uridylate-rich element-based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC-dependent neutrophil transcriptional activity was further confirmed by real-time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a number of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose {alpha}1,3-galactose (Gal{alpha}1,3-gal) structures, as inclusion of saturating concentrations of anti-Gal{alpha}1,3-gal l antibodies had no significant effect. Furthermore, this activation was inhibited in the presence of the calcium chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and human innate immune cells.

Key Words: Gal{alpha}1 • 3-gal • calcium • ROM • PAECs


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The profound lack of donor organs suitable for transplantation into humans makes animal donors (xenotransplantation) a viable alternative. However, the reality of hyperacute rejection (HAR) of the transplanted xenograft poses a formidable barrier to the use of animal organs. HAR is attributed to the presence of xenoreactive natural antibodies (XNA) and complement activation following encounter of vascularized xenograft with human blood [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 [7 8 9 10 11 12 13 14 ]. It is the binding of XNA to Gal{alpha}1,3-gal-decorated proteins and lipids that leads to the activation of the complement cascade and the formation of membrane attack complexes [15 , 16 ]. This converts the xenoendothelium from an antithrombic to a prothrombic state, resulting in the generation of inflammatory mediators, which in turn, recruit cellular components of the immune system to the site of complement activation. The process is fast and violent, leading to virtual strangulation and the ultimate demise of the transplanted xenograft. Many strategies have been used to prevent HAR and ensure the survival of vascularized xenograft. These include depletion of XNA [17 18 19 ], accommodation [20 21 22 23 ], and transgenesis [24 25 26 27 28 ]. Although these approaches may reduce HAR, the presence of other barriers has to be negotiated. In previous publications [29 30 31 ], we demonstrated the ability of innate immune cells, namely neutrophils and natural killer (NK) cells, to activate the xenoendothelium in the absence of XNA and complement and under conditions where the Gal{alpha}1,3-gal-binding sites are blocked completely. In this paper, we extend our finding and demonstrate a calcium and reactive oxygen metabolite (ROM)-dependent, direct activation of human naïve neutrophils by porcine aortic endothelial cells (PAECs). We used cDNA microarray containing 1500 probes representing adenylate uridylate (AU)-rich mRNA, which code for early and transient response gene products including cytokines, chemokines, and other proinflammatory mediators [32 ] to investigate differential gene expression in human neutrophils following xenogeneic encounter. We demonstrate that human naïve neutrophils produce a number of key inflammatory mediators at the message and protein levels in response to a challenge with PAECs. This production is relatively rapid, intracellular calcium- and ROM-mediated, and independent of Gal{alpha}1,3-gal structures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
1-(2,5-Carboxyoxazol-2-yl)-6-aminobenzfuran-5-oxyl-2-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetra-acetoxymethyl ester (Fura-2 AM), 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (2,7 DCFDA) were purchased from Molecular Probes (Eugene, OR). LightCycler instrument and LightCycler-DNA Master SYBR Green 1 were purchased from Roche Diagnostics (Mannheim, Germany). Diphelylene iodonium (DPI) was purchased from ICN (Irvine, CA). All other reagents were Analar grade and were purchased from Sigma Chemical Co. (St. Louis, MO) and BDH Chemicals (UK). Fura-2 AM, BAPTA-AM, 2,7 DCFDA, 5-amino-2, 3-dihydro-1,4-phthalazinedione (luminol), and DPI were dissolved in dimethyl sulfoxide (DMSO) and delivered to the cells at a final concentration of 1 µM, 2 µM, 1 µM, 11 µM, and 10 µM, respectively, in a final DMSO concentration of 0.1%.

Isolation of endothelial cells
PAECs were isolated from adult pig aorta, essentially as described by Jaffe et al. [33 ]. The cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum (FBS), endothelial cell growth factor (Sigma Chemical Co.), penicillin (100 U/ml), streptomycin (25 µg/ml), and heparin (100 U/ml). Human aortic endothelial cells (HAECs) were isolated from the thoracic aorta of donor hearts (obtained with Institutional Review Board approval) as follows: A section of the thoracic aorta was collected during transplantation, incised longitudinally, and placed flat in a petri dish with the intraluminal surface exposed. The endothelium was gently scraped off using a sterile scalpel blade and placed in HEPES-buffered medium 199 (Sigma Chemical Co.) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (1 mM), bovine brain extract (25 µg/ml), heparin (15 U/ml), and 20% FBS. Cells were centrifuged and resuspended in the same medium, plated onto sterile culture dishes coated with 0.5% gelatin, and cultured as above. Endothelial cells were characterized by their cobblestone morphology and positive immunostaining with antibodies to von Willebrand factor (F3520; Sigma Chemical Co.) and to acetylated low-density lipoprotein (Biogenesis, Bournmouth UK). Cells were used from passages two to 10 in all experiments at a split ratio of 1:3. To test that endothelial cells were not activated during isolation and culture, interleukin (IL)-1{alpha} levels in conditioned medium were measured using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) and were consistently found to be negligible (<4 pg/ml). Lipopolysaccharide was routinely tested in culture media before and after experimentation using limulus amebocyte lysate pyrogent (BioWhittaker, Walkersville, MD) and found to be below the detection limit of the test kit.

Isolation of neutrophils
Human peripheral blood neutrophils were prepared by dextran sedimentation of heparinized whole blood obtained from healthy donors and centrifuged through Ficoll-Paque as described previously [31 ]. Contaminating red blood cells were removed by hypotonic lysis with isotonic NH4Cl. The remaining cells were suspended 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% bovine serum albumin and were further purified through neutrophil isolation medium (Cardinal Associates, Santa Fe, NM). Final purity and viability were between 98% and 99%, as indicated by flow cytometry and trypan blue dye exclusion tests. Neutrophils were tested routinely for production of ROM by luminol-dependent chemiluminescence (LDCL) for 10 min. Cells were considered naive and therefore suitable for experimentation only when no increase in LDCL was observed.

Preparation of anti-Gal{alpha}(1,3)-gal antibodies
Anti-gal{alpha}(1,3)-gal antibodies were prepared essentially as described previously [30 ]. Briefly, 10–15 kg baboons were immunized by intramuscular injection of emulsified, soluble Gal{alpha}(1,3)-gal with Hunter’s TiterMax adjuvant (Sigma Chemical Co.). The animals were given booster injections 3 weeks later. Samples of blood were taken at 5 weeks postimmunization and tested for binding to porcine thyroglobulin, soluble Gal{alpha}(1,3)-gal, and PAECs as described previously [30 ]. Booster injections were given at 4–6 weeks thereafter and continued for 6–9 months. To obtain Gal{alpha}(1,3)-gal, antiserum blood was collected in 50 ml sterile Falcon tubes and allowed to clot at room temperature for 30 min. Serum was centrifuged (3000 g, 4°C, 30 min), heat-inactivated (30 min, 56°C), and recentrifuged (1000 g, 4°C, 30 min). Antiserum immunoglobulins (IgG, IgA, and IgM) were quantified using the Cobas Mira Plus system (Hoffmann-La Roche, Basel, Switzerland) before extensive dialysis against 5 mM sodium phosphate buffer (pH 6.5). The dialysate was applied to equilibrated anion resin [Sephadex diethylaminoethyl (DEAE) A-50, Pharmacia, Uppsala, Sweden] at a ratio of 2:1 (resin:supernatant). The use of this anion resin ensured that essentially all serum protein components except IgG bind to the resin, leaving an eluate rich in IgG. The eluate was fractionated by ion-exchange chromatography on Sephadex DEAE A-50. Purified fractions were pooled, dialyzed against phosphate-buffered saline (PBS), and concentrated.

F(ab)2 fragments of anti-Gal{alpha}(1,3)-gal IgG were prepared by papain digestion. Papain solution (50 µL; 10 mg/ml in 0.1 M sodium phosphate buffer, pH 7.0) was added to 50 mg/ml IgG in 3 ml PBS. The digestion mixture was incubated at 37°C for 4 h. The hydrolyzed product was dialyzed first against Mill-Q water and then against 0.01 M sodium acetate buffer (pH 5.5). Samples were removed and fractionated on a Sephacryl-S-200 column (Pharmacia).

Blocking experiments with Gal{alpha}(1,3)-gal IgG were performed as follows: PAECs were trypsinized by incubation with Trypsin-EDTA solution for endothelial cell culture (Sigma Chemical Co.) for 2 min at 37°C. The cells were washed three times with RPMI-1640 medium (Sigma Chemical Co.). The washed cells were then incubated with 100 µg/ml Gal{alpha}(1,3)-gal IgG or F(ab)2 fragments of the antibodies at room temperature for 30 min. The suspension was then added to adherent neutrophils at a final ratio of 10:1 (neutrophils to PAECs). This concentration was previously shown to block all Gal{alpha}(1,3)-gal-binding sites sufficiently [30 ].

Calcium measurements
Neutrophils were loaded with Fura-2 AM (1 µM) as described previously [31 ]. The cells were washed, placed on glass coverslips, and allowed to adhere for 15 min at room temperature. Coverslips were washed and then secured between two plates of a custom-designed coverslip holder and placed onto a heated microscope stage (37°C), and [Ca+ +]i images were acquired at 1- to 2-s intervals using the Openlab system (Improvision, Coventry, UK). For each coverslip, 100 µl PAECs, suspended in Krebs-Hepes buffer (pH 7.4), containing 104 cells, were added, and image acquisition was continued for at least 5 min. Control experiments were carried out using an equal number of HAECs/coverslip. Images were analyzed using Openlab software, and fluorescence intensity (from each cell) was transformed into absolute calcium levels as described previously [31 ].

Measurement of ROM production
The production of ROM by neutrophils was measured using LDCL, as described previously [34 35 36 ]. Briefly 1 ml neutrophil (suspended in Krebs-Hepes, pH 7.4) containing 106 cells was challenged with 100 µl containing 104 PAECs (suspended in Krebs-Hepes buffer, pH 7.4), and LDCL was followed for 15 min. For control experiments, PAECs were replaced with HAECs. ROM production was also followed fluorimetrically using 2,7 DCFDA, essentially as described previously [37 ]. Briefly, 106 neutrophils were incubated with 1 µM 2,7 DCFDA (room temperature for 15 min). Cells were washed and allowed to adhere to glass coverslips (15 min at room temperature). Coverslips were washed and secured between two plates of a custom-designed coverslip holder, placed onto a microscope stage, and viewed using a Leica TCS confocal microscope (Leica-Kaki, Riyadh, Saudi Arabia). Images were obtained at a temporal resolution of 3 s/image. For each coverslip, 100 µl PAECs, suspended in Krebs-Hepes buffer (pH 7.4), containing 104 cells, were added, and image acquisition was continued for at least 15 min. Control experiments were carried out using an equal number of HAECs/coverslip. Fluorescence intensity was measured using the Leica Scanware software and transferred to an Excel spreadsheet for further analysis.

Preparation of total RNA for microarray
Human neutrophils (5x107), suspended in 1 ml Krebs-Hepes buffer (pH 7.4), were cocultured with 1 ml PAEC suspension in the same buffer containing 5 x 106 PAECs for 1 h at 37°C. Total RNA was isolated from the coculture by the guanidine isothiocyanate method using Tri Reagent (Sigma Chemical Co.), according to the manufacturer’s instructions. Parallel experiments were carried out in which total RNA was isolated from unstimulated neutrophils (5x107 cells/ml) and unstimulated PAECs (5x106 cells/ml) separately and then mixed and made up to the correct volume of the test samples. For each test and control microarray experiment, pooled RNA from five different experiments performed using neutrophils from five individual donors was used in triplicate. The whole experiment was done twice on two separate microarrays using two independently pooled RNA from different donors.

Microarray expression and analysis
We used cDNA microarray, which contained 1500 cDNA distinct probes and a total of 4000 elements [32 ]. The cDNA probes were generated via polymerase chain reaction (PCR) amplification of the clone-containing glycerol bacterial culture stocks using M13 primers and spotted (100 µm diameter) on poly-L-lysine-coated glass slides using the OmniGrid robot (GeneMachines, San Carlos, CA). Total RNA samples (20 µg, 5 µg/µl) were reverse-transcribed using SuperScript II (Gibco) and a primer provided by the Genisphere kit (Genisphere, Hatfield, PA), and the cDNAs were ethanol-precipitated. The hybridization buffer supplied by the Genisphere kit was heated to 55°C for 10 min and mixed with two blocking solutions, oligo(dT) and locked nucleic acid (LNA(dT)), for nonspecific hybridization of labeled cDNA to elements containing poly(dA) sequences and for poly A-containing elements, including spotted poly(dA) sequences, respectively. The cDNA pellets were incubated in the hybridization buffer with the blockers as "hybridization mixes" of 25% formamide, 4x saline sodium citrate, 1% sodium dodecyl sulfate, and 2x Denhardt’s solution and were then applied to the microarray, which was covered with coverslips for 18 h at 55°C in a sealed, humidified chamber. After hybridization, the slides were washed briefly by a series of washes to remove unbound cDNA. Subsequently, the microarrays were subjected to dendrimer hybridization using Genisphere DNA dendrimer capture reagent labeled with the Cye-Dyes Cy3 or Cy5 samples. The hybridization mixes were first incubated at 75–80°C for 10 min and then at 50°C for 20 min and then added to prewarmed microarray slides. The microarrays were covered with coverslips and incubated for 3 h in a dark, humidified chamber at 45–50°C. After hybridization, the slides were washed briefly several times to remove unbound dendrimer molecules, and the microarrays were dried in a centrifuge.

Image acquisition and intensity extraction
The cDNA microarrays were scanned at 10 µm resolution using the ScanArray 5000 (GSI Lumonics, San Jose, CA) scanner, which excites the Cy3 (green) and Cy5 (red) fluorophores at optimal wavelengths of 532 nm and 635 nm, respectively. The median intensities of Cy3 and Cy5 fluorescent signals from each cDNA spot on the microarray image were extracted using adaptive circle algorithm (QuantArray software). The spot intensities with a large, local background were excluded if the median intensity of the spot is less than 1.4 of the median background intensity of the same spot. The negative values, if present, were also excluded from the data. The mean background intensities were subtracted from the spot intensities. The background-corrected intensities were then normalized to total signal intensities to account for different input RNA concentrations or labeling efficiency in the individual Cy3 and Cy5 reverse transcriptase (RT) reaction. The intensity ratios (Cy5/Cy3), which represent the relative expression profile of the genes in the two starting RNA samples, were calculated.

Quantification of specific transcripts with LightCycler RT-PCR
Neutrophils and PAECS or HAECs were cocultured at 37°C for 1 h at an effector:target ratio of 10:1. Total RNA was prepared from each coculture (referred to as PN and HN, respectively) by the guanidine isothiocyanate method using Tri Reagent (Sigma Chemical Co.), according to the manufacturer’s instructions. Control experiments were carried out in which total RNA was isolated from unstimulated neutrophils (5x107 cells/ml), unstimulated PAECs (5x106 cells/ml), and unstimulated HAECs (5x106 cells/ml) separately. mRNAs from neutrophils and PAECs or neutrophils and HAECs were mixed and made up to the correct volume of the test samples. These are referred to as CON and HCON, respectively. cDNA was synthesized from the total RNA using avian myeloblastosis virus RT (Promega, Madison, WI), according to the manufacturer’s protocol. cDNA synthesis was performed in a final volume of 20 µl. Briefly, 2 µl (1:10 dil) cDNA was used for amplification in the SYBR Green format using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Catalog No. 2 239 264).

Primers derived from the human gene sequence were designed using the Oligo 6 primer analysis software (Applied Biosystems, Foster City, CA). To prevent coamplification from porcine, cell sequence primers were chosen from the nonhomology regions between human and porcine sequences. Mastermixes were prepared according to the manufacturer’s instructions, using the following primer sets: Quantitative PCR was performed using the LightCycler system (Roche Diagnostics). Briefly, to the 8 µl LightCycler mastermix in the LightCycler glass-precooled capillaries, a maximum of 10 ng cDNA in a 2-µl vol was added as PCR template. A no-target control received 8 µl reaction mixture with 2 µl water. Sealed capillaries were centrifuged (5 s at 1000 rpm) using the LightCycler centrifuge adapters and placed into the LightCycler rotor. PCR amplification was performed in triplicate wells. The following temperature profile was used for amplification: denaturation for 1 cycle at 95°C for 30 s and 40 cycles at 95°C for 10 s (temperature transition, 20°C/s), 64–50°C (step size, 1°C; step delay, 5 cycle) for 15 s (temperature transition, 20°C/s), and 72°C for 15 s (temperature transition, 2°C/s) with fluorescence acquisition at 55–50°C in single mode. Melting-curve analysis was done at 45–90°C (temperature transition, 0.2°C/s) with continuous fluorescence acquisition. Sequence-specific standard curves were generated using tenfold serial dilutions (102–108 copies/µl) of known amounts of cDNA for each cytokine and for glyceraldehyde-3 phosphate dehydrogenase (G3PDH). The respective concentration for any given sample was calculated using crossing-cycle analysis provided by the LightCycler software. For realistic quantifications, the start amount of RNA was the same for all samples. Minor sampling errors were avoided by normalization with the housekeeping gene G3PDH.

Quantification of released IL-1{alpha}, IL-1ß, IL-6, and IL-8
Quantifications of IL-1, IL-6, and IL-8 were done by ELISA. Briefly, samples of human neutrophil suspension in Krebs-HEPES medium (5x106 cells/ml) were incubated with PAECs (5x105 cells/ml) or irradiated PAECs for various time intervals at 37°C and 5% CO2. Irradiation was done using a cobolt 60 source (MDS Nordion, Ontario, Canada) at 2000 rad. Cells were pelleted for 10 s, and supernatants were collected for ELISA analysis, which was performed using paired-capture and biotinylated detection of antibodies from Amersham BioScience UK Ltd. (Bucks), according to the manufacturer’s instructions. Standard curves were constructed using serial dilutions. ELISA plates were read using a CERES UV 900C spectrophotometer (Bio-TEK Instruments, Winooski, VT).

Statistical analysis
Where applicable, results are presented as means ± SEM. Statistical significance was performed using one-way ANOVA. The threshold for significance was adjusted by using the Bonferroni method to give a lower probability level (uncorrected P value divided by number of comparisons).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Xenogeneic but not allogeneic encounter evokes a transient calcium rise in human neutrophils
Resting human neutrophils adherent to glass coverslips exhibited resting intracellular calcium levels of 104 ± 5 nM (mean±SEM, n=5). Upon addition of PAECs (104 cell/ml), the neutrophil calcium level rose to 416 ± 43 nM within 10 s before decaying to prestimulatory levels (Fig. 1a and 1b ). The rise was asynchronous and heterogeneous with the number of neutrophils responding, dependent on the number of PAECs added. The transient calcium rise was mainly a result of release from intracellular stores, as pretreatment of neutrophils with EGTA (1 mM) in the absence of extracellular calcium reduced the calcium transient only marginally. Moreover, blocking the Gal{alpha}1,3-gal-binding sites on PAECs by anti-Gal{alpha}1,3-gal antibodies had no apparent effect on the PAEC-induced calcium rise in human neutrophils. In contrast, HAECs had no apparent effect on the resting calcium levels of human neutrophils (Fig. 1b) .



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  		Figure 1. Intracellular calcium changes in human neutrophils following xenogeneic encounter. (a) [Ca+ +]i maps in individual human neutrophils at the indicated time intervals (in seconds) after the addition of 104 PAECs/100 µl at time 0. The calcium changes are color-coded, such that warm colors indicate high calcium, and cold colors indicate low calcium. This is a representative experiment of at least 10 in which neutrophils were prepared from five different donors. (b) Average [Ca+ +]i in neutrophils treated with PAECs in control experiments ({diamondsuit}), in the presence of saturating concentrations (100 µg/ml) of anti-Gal{alpha}1,3-gal antibodies ({blacktriangleup}), and in the F(ab)2 portion of the antibodies ({blacksquare}). The lack of effect of HAECs is shown (x), and negative control of human neutrophils treated with medium is shown (*). The arrow indicates time of addition of PAECs, HAECs, or medium to human neutrophils. Each point represents the mean ± SEM of at least seven to nine cells obtained from one field of view.

 
Xenogeneic contact induces ROM production in human neutrophils
One of the major targets for the transient calcium rise in human neutrophils is the nonmitochondrial oxidase system responsible for ROM production. We used LDCL to investigate the effect of PAECs on human neutrophil ROM production. In a series of experiments, we found that PAECs caused a dose-dependent increase in ROM production (Fig. 2a ). Conversely, no effect was observed when human neutrophils were treated with HAECs. In addition, pretreatment of PAECs with blocking antibodies to the Gal{alpha}1,3-gal structures had no apparent effect on ROM production by human neutrophils. To confirm ROM production in response to PAECs, we used fluorescence confocal microscopy to image ROM production in 2,7 DCFDA-loaded neutrophils. We found that addition of PAECs to neutrophils increased the fluorescence intensity as a result of 2,7 DCFDA, the kinetics of which was comparable with that of LDCL (Fig. 2b) .



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  		Figure 2. Changes in ROM production in human neutrophils following xenogeneic encounter. (a) Dose-response curve for ROM production in human neutrophils at various concentrations of PAECs as measured by LDCL. The traces underneath the curve show the lack of effect of anti-Gal{alpha}1,3-gal antibodies and F(ab)2 on LDCL and the negative control of HAECs (1.3x106 HAECs) added to human neutrophils. (b) A representative experiment (of at least five with neutrophils isolated from five different donors) confirming ROM production by human neutrophils following encounter with PAECs. Images are confocal micrographs in 2,7 DCFDA-loaded neutrophils obtained at the indicated time intervals. The images are color-coded as indicated in the color bar. PAECs were added at time 0.

 
Microarray gene expression analysis of human neutrophils following xenogeneic encounter with PAECs
We used microarrays containing 1500 cDNA probes representing AU-rich element (ARE)-cDNA clones, the identities of which were obtained from an ARE-containing mRNA database (ARED) to detect gene expression responses as a result of the xenogeneic encounter. We have chosen this focused cDNA microarray because of the fact that many of the ARE genes encode proteins involved in early and transient responses, such as those in inflammation, intracellular communications, and innate immunity [32 ]. We found that exposure of human neutrophils to PAECs (10:1 ratio) for 1 h at 37°C caused a dramatic change in ARE-mRNA transcripts levels compared with the control levels of these transcripts. Of the 1500 ARE-mRNA transcripts tested in the microarray, 52 transcripts exhibited two and one-half or more fold increase than the control levels. This was concomitant with a decrease of 10 transcripts following xenogeneic encounter (Fig. 3a ). Analysis of these transcripts using Biological Process Ontology from the Gene Ontology Database (http://www.geneontology.org/doc/GO.tools.html) to annotate transcript (transcript product) revealed a number of functions associated with the PAEC-induced transcripts in neutrophils (Fig. 3b) . There were a number of transcripts that showed cross-functional activities. As the neutrophils constitute approximately 60% of circulating leukocytes, which puts them amongst the major decisionmakers within the immune system, we focused on the transcripts that are associated with immune response (Fig. 3b) . The major increase was that of proinflammatory cytokines including IL-1{alpha}, IL-1ß, IL-6, and IL-8. At this stage, it was not possible to determine whether the change in the microarray genetic profiles was attributed to either of the cell type or to both. For this, we used real-time PCR and primer pairs that were specific for human genes.



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  		Figure 3. Effect of PAECs on human neutrophil gene expression. (a) A summarized list of transcripts color-coded such that high expression is indicated by the hot colors, and low expression is indicated by cold colors. (b) Output of gene ontology analysis grouping the transcripts into the indicated ontologies on the right-hand side. (c) The various inflammatory cytokines grouped together by gene ontology analysis. The data shown were obtained using pooled RNA from five different experiments with neutrophils isolated from five different donors, and each data point was done in triplicate. The data are representative of two independent microarray slides using two independently pooled RNA.

 
Induction of inflammatory cytokines in human neutrophils in response to xenogeneic encounter
As cytokines constitute one of the major intercellular communicating systems amongst many cell types, we tested their involvement in the xenogeneic encounter. In a series of experiments using quantitative RT-PCR by LightCycler [38 ], we found that exposure (1 h at 37°C) of human neutrophils (107 cell/ml) to PAECs (106 cell/ml) induced the transcription of a number of cytokines, namely IL-1, IL-6, and IL-8 (Fig. 4a 4b 4c 4d ) in the human neutrophil. In quantitative assays using the housekeeping enzyme and G3PDH as standard, we found that PAECs significantly raised the ratio of IL-1, IL-6, and IL-8 relative to the standard G3PDH (Fig. 4a 4b 4c 4d) . In a series of experiments, we found that the ratio of IL-1{alpha} to G3PDH transcripts was 0.22 ± 0.02 (mean±SEM, n=3) in unstimulated human neutrophils rising to 9.13 ± 0.67 (P<0.001) following exposure to PAECs. Similarly, the ratios for IL-ß, IL-6, and IL-8 have increased from the resting levels of 0.31 ± 0.02, 0.57 ± 0.05, and 0.06 ± 0.01 to 44.30 ± 1.03 (P<0.001), 2.95 ± 0.13 (P<0.001), and 1.01 ± 0.06 (P<0.001), respectively. To ascertain that human neutrophils were the only source of these cytokines, we designed primer sets from areas of maximum mismatch between human and porcine genes encoding these cytokines. At the RT-PCR conditions used, there was no amplification of porcine cytokines by the human cytokine-specific primers (Fig. 4e) . In contrast to PAECs, HAECs had no apparent effect on IL-1, IL-6, and IL-8 transcripts (Fig. 4a 4b 4c 4d) .



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  		Figure 4. Semiquantitative analysis of neutrophil gene expression following xenogeneic encounter as revealed by LighCycler experiments. PAEC-induced rise in mRNA levels of IL-1{alpha} (a), IL-1ß (b), IL-6 (c), and IL-8 (d), where CON is control, prepared as indicated in Materials and Methods; PN is neutrophils after encounter with PAECs; HCON is HAECs and human neutrophil control prepared as described in Materials and Methods; and HN is neutrophil after encounter with HAECs. (e) An example is RT-PCR illustrating that primers designed from areas of maximum mismatch between human and porcine IL-1{alpha} gene recognize human but not porcine IL-1{alpha}. Lane 1, Molecular weight markers; lane 2, RT-PCR with water replacing mRNA using primers designed from human IL-1{alpha} sequence; lane 3, amplicons of IL-1{alpha} obtained from human neutrophils stimulated with IL-12 (1 ng/ml) using human primer sets; lane 4, the absence of amplicons of porcine IL-1{alpha} in tumor necrosis factor {alpha} (TNF-{alpha})-stimulated PAECs using human primer sets; and lane 5, amplicons of porcine IL-1{alpha} in TNF-{alpha}-stimulated PAECs detected by porcine primer sets. PAEC-induced rise in mRNA levels of IL-1{alpha} (f), IL-1ß (g), IL-6 (h), and IL-8 (i) in the presence of BAPTA, DPI, anti-Gal{alpha}1,3-gal antibodies (GAL), and F(ab)2 portion of anti-Gal{alpha}1,3-gal antibodies (Fab). Each histogram represents the mean ± SEM of the ratio of the specified cytokine:G3PDH. Experiments were done in triplicates, and neutrophils were obtained from three different donors.

 
Pretreating human neutrophils with the intracellular calcium chelator (BAPTA-AM) or the ROM production inhibitor DPI tested that the PAEC-induced increase in cytokine transcription is mediated through calcium and ROM production. We found that PAEC-induced increase in transcripts for the above cytokines was significantly reduced by pretreatment of the neutrophils with BAPTA-AM (Fig. 4f 4g 4h 4i) . It is interesting, however, that whereas PAEC-induced IL-6 and IL-8 transcripts were inhibited by the presence of the ROM production inhibitor DPI, neither IL-1{alpha} nor IL-1ß transcripts exhibited any sensitivity to DPI (Fig. 4f and 4g) . Moreover, saturating concentrations of blocking antibodies to anti-Gal{alpha}1,3-gal (100 µg/ml) had no apparent effect on the PAEC-induced increase in inflammatory cytokine transcription (Fig. 4f 4g 4h 4i) .

Detection of IL-1, IL-6, and IL-8 by ELISA
We used ELISA to detect the amount of secreted cytokines in response to a challenging load of PAECs. In a series of experiments, we found that the secreted levels of IL-1{alpha}, IL-1ß, IL-6, and IL-8 have significantly increased following xenogeneic encounter (Fig. 5 ). We found that PAEC-activated neutrophils produced 8.04 ± 0.49 pg/106 neutrophils (P<0.05) of IL-{alpha} 4 h post initial exposure. This was paralleled by increased levels of secreted IL-1ß, IL-6, and IL-8, with levels rising to 10.98 ± 0.91 pg/106 neutrophils (P<0.01), 82.21 ± 5.04 pg/106 neutrophils (P<0.05), and 318 ± 71 pg/106 neutrophils (P<0.05), respectively. The question as to the source of the secreted cytokine was addressed by irradiating either cell type before encounter. In a series of experiments, we found that irradiated PAECs (2000 rad) increased secretion of these cytokines to similar levels evoked by nonirradiated PAECs (Fig. 5) . Conversely, nonirradiated PAECs failed to evoke any cytokine release from irradiated neutrophils (2000 rad), suggesting that human neutrophils were the only source for the released cytokines following xenogeneic encounter.



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  		Figure 5. Release of inflammatory cytokines IL-1{alpha} (a), IL-1ß (b), IL-6 (c), and IL-8 (d) by human neutrophils following encounter with PAECs. Histograms are means ± SEM of three experiments done in triplicates. The histograms show release from unstimulated neutrophils (Neuts), unstimulated PAECs, neutrophils after encounter with PAECs (Neut+PAECs), neutrophils after encounter with irradiated (2000 rad from cobalt 60 source) PAECs (Neut+IrPAECs), and irradiated (2000 rad from cobalt 60 source) neutrophils after encounter with PAECs (IrNeut+PAECs).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The paucity of donor organs makes xenotransplantation a viable option; even if not to replace human organs totally, it may be used as a bridge for a selected population of transplant patients until suitable human organs become available. The problem as it stands is not only HAR and XNA [1 2 3 4 5 6 7 8 9 10 11 12 13 14 ] but also the acute vascular rejection [39 40 41 ] and the innate immune system, which is presenting yet another barrier to xenotransplantation. We have argued that as neutrophils, the major circulating leukocytes are designed to rid the body of infection, debris, and foreign material; they must be involved intimately in the demise of the xenograft. Their involvement has always been considered secondary to HAR. However, we have reported previously that naïve human neutrophils and NK cells activate xenogeneic but not allogeneic endothelium in the total absence of XNA and complement [30 , 31 ].

By virtue of their nonmitochondrial oxidase system, the neutrophils produce ROM in a well-coordinated and controlled manner. ROM have a number of functions, namely, direct toxicity toward the ingested material and/or providing the "right" environment for a cascade of degrading enzymes and/or acting as a tertiary signaling pathway for the cells to pursue their ultimate function: the effective clearance of infection, debris, and ingested foreign material. It is therefore not surprising that these cells would recognize xenogeneic cells directly. Whereas most of the studies have focused on what happens at the endothelial cell level, few have considered the effects on the recipient innate cells. Here, we describe the direct activation of human naïve neutrophils by PAECs. This contact-dependent activation is rapid and leads to a transient increase in intracellular calcium, which is accompanied by transient production of ROM. It is unlikely that this activation was Gal{alpha}1,3-gal-dependent, as PAECs were still able to activate calcium transients and ROM production in neutrophils under conditions where Gal{alpha}1,3-gal structures were blocked and in the total absence of XNA and complement. As neutrophils do not bind to Gal{alpha}1,3-gal epitopes [42 ], the question arises as to the identity of the Gal{alpha}1,3-gal-independent binding sites. In a previous publication [31 ], we demonstrated that under static conditions, human naïve neutrophil binding to PAECs was significantly reduced in the presence of antibodies to intercellular adhesion molecule-1 or its counter ligands lymphocyte function-associated antigen-1 and membrane-activated complex-1. It is, however, unlikely that these adhesion molecules are the only Gal{alpha}1,3-gal-independent interaction sites between human neutrophils and PAECs. It is now established that the neutrophils express a number of Toll-like receptors that are crucial for the detection of foreign pathogens [43 ]. The involvement of this highly conserved family of molecular pattern-recognizing proteins in xenogeneic recognition is yet to be delineated.

ARE-mRNA are short-lived and functionally associated with a number of cellular responses such as inflammation and intercellular communications [32 ]. As neutrophils play a key role in inflammation, the possibility existed that ARE-mRNA may be involved intimately in responses of human neutrophils to xenogeneic encounter. This was addressed by using microarray of ARE-mRNA containing 1500 cDNA probes representing ARE-cDNA clones obtained from ARED [32 ]. Using a cut-off ratio of 2.5, we identified a number of transcripts associated with xenogeneic encounter, of which proinflammatory cytokines such as IL-1{alpha}, IL-1ß, IL-6, and IL-8 were prominent. Verification of the microarrays results was carried out by real-time, semiquantitative PCR using primer sets designed from areas of maximum mismatches between human and porcine genes of interest and using the housekeeping enzyme G3PDH as the reference gene. Under such conditions, all four cytokines exhibited levels that were several-fold higher than control-unstimulated neutrophils following xenogeneic encounter. The possibility existed that the increase in gene expression was dependent on the initial calcium rise observed in human neutrophils following xenogeneic encounter, which was tested by using the intracellular calcium chelater BAPTA-AM. Under conditions where the PAEC-induced calcium transient was inhibited, all tested cytokine transcripts were reduced significantly, suggesting a pivotal role for calcium transients in increased transcript levels following xenogeneic encounter. However, as calcium transients are necessary for ROM production by neutrophils and as ROM are intimately involved in cytokine transcription through the transcription factor nuclear factor (NF)-{kappa}B [44 ], the possibility existed that an increase in ROM production was responsible for the PAEC-induced increase in cytokine transcripts, which was tested by using the ROM production inhibitor DPI. Under such conditions, two of the cytokines induced, namely, IL-6 and IL-8, were significantly inhibited by DPI but not IL-1{alpha} or IL-1ß. This suggests that IL-6 and IL-8 are under NF-{kappa}B regulation, which is in accordance with previous work demonstrating that IL-6 production by mouse splenic macrophages [45 ] and IL-8 production by human neutrophils [44 ] were indeed regulated by NF-{kappa}B.

Although [Ca+ +]i is implicated in IL-1 transcription, the mechanism of such involvement is yet to be delineated. There is a plethora of information suggesting that control of IL-1{alpha} and -ß transcription may be cell-specific [46 ]. A number of transcription factors, including NF-{kappa}B [47 ], cyclic adenosine monophosphate-response element [48 ], signal transducer and activator of transcription-like factor [49 ], PU.1 [50 ], and a number of POU domain transcription factors including Oct-1 [51 ] have been associated with IL-1{alpha} and -ß transcription, depending on the cell type.

Although the role of inflammatory cytokines in xenograft rejection has been associated with delayed vascular rejection [52 ], in this study, we demonstrate that neutrophils are one of the major sources of cytokine production, even in the absence of XNA and complement, and under conditions in which Gal{alpha}1,3-gal-binding sites were totally blocked by excess concentrations of whole anti-Gal{alpha}1,3-gal antibodies or their F(ab)2 region.

IL-1 is one of the major multifunctional cytokines with a direct role in inflammatory responses and hematopoiesis. IL-1{alpha} involvement in xenograft rejection has been suggested in studies using xenogeneic islets transplanted into nonobese diabetic mice [53 ]. Although early investigation suggested that islet xenograft rejection in mice was associated with a T helper cell type 2 (Th2) immune response [54 ], more recent studies demonstrated that this rejection has the hallmark of delayed hypersensitivity reaction, which is associated with a Th1 immune response [55 ]. Krook et al. [55 ] have demonstrated the presence of proinflammatory cytokines IL-1ß, IL-2, IL-12p40, interferon-{gamma}, and TNF-{alpha} transcripts in the rejected xenograft and in the host’s lymph nodes.

The role of IL-6 in xenograft rejection has been demonstrated in primate cardiac xenotransplants, where the severity of vascular rejection was associated with high levels of IL-6. Porcine hCD55 transgenic cardiac xenografts transplanted into a baboon exhibited higher levels of IL-6 associated with delayed xenograft rejection [40 ]. IL-6, a pleiotropic cytokine, exhibits a number of functions including augmentation of humoral and cellular immune responses [56 ]. It has been demonstrated that IL-6 induces B cell maturation and enhances IgM and IgG production [57 , 58 ]. In addition, IL-6 effects on haemostasis have been shown to occur through promotion of coagulation independently of fibrinolysis and through stimulation of platelet production and activation [59 60 61 ]. It is therefore tempting to speculate that PAEC-induced IL-6 production by human neutrophils may play a crucial role in activation of other effector cells and in promoting haemostatic sequelae at the xenograft milieu.

A role for IL-8 in xenograft rejection was suggested by the finding that exposure of NK cells to PAECs causes the release of IL-8 from NK cells [62 ]. Furthermore, inhibition of NF-{kappa}B in endothelial cells suppresses the inflammatory responses associated with endothelial cell activation [39 ]. IL-8 is a powerful {alpha}-chemokine with potent chemoattractant properties regulating leukocyte trafficking and recruitment. It has been suggested that IL-8 plays a dual autocrine/paracrine signaling to spatially limit and amplify the inflammatory response [44 ]. It is interesting, however, that antibodies to IL-8 were found to induce platelet activation and suggested the existence of IL-8 receptors [CXC chemokine receptor A (CXCRA) and/or CXCRB] on the platelet plasma membrane [63 ].

Taken together, it is possible to speculate that the PAEC-induced inflammatory cytokine released from human neutrophils may serve as potential "converters" of the normally antithrombic to a prothrombic xenogeneic endothelium in the absence of XNA and complement, making the xenoendothelium a more attractive target for rejection by humoral and cellular components of the immune system.


   ACKNOWLEDGEMENTS
 
This work was supported by the Cardiovascular Research Program Funds and Research Centre Funds. We are indebted to Dr. M. Inan for helping with the gene ontology analysis and to Miss R. Bakheet and Ms. H. Khaleel for technical help. We also thank Mr. Mohammad Dhalla for his technical assistance in the microarray experiments.

Received September 6, 2004; revised March 5, 2005; accepted March 6, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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