Ranjit Parhar,* Aaron Kwaasi,* BrianMeyer,*. Soad Saleh,* Sean Allen,t Sultan Al-Sedairy,* ...... JM Harlan, DY Liu. New York, WH Freeman, 1992, pp. 189-193.
American Journal ofPathology, Vol. 151, No. 1, July 1997 Copyright © American Societyfor Investigative Pathology
Activation of Naive Xenogeneic but Not Allogeneic Endothelial Cells by Human Naive Neutrophils A Potential Occult Barrier to Xenotransplantation
Futwan Al-Mohanna,* Kate Collison,* Ranjit Parhar,* Aaron Kwaasi,* Brian Meyer,* Soad Saleh,* Sean Allen,t Sultan Al-Sedairy,* David Stern,t and Magdi Yacoubt From the Department of Biological and Medical Research,* King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; the National Heart and Lung Institute and Harefield Heart Science Centre,t Harefield
Hospital, Harefield, United Kingdom, and the Departments of Physiology and Surgery,* Columbia University, College of Physicians and Surgeons, New York, New York
Here we demonstrate that human neutrophils, the predominant circulating leukocytes in intimate contact with endotheial cells lining the vasculature, directly recognize xenogeneic endothelium independently of xenoreactive natural antibody and complement A rapid and calcium-dependent activation of naive (unstimulated) xenogenic endothelial cells by human neutrophils leads to 1) translocation of P-selectin from the Wiebel-Palade bodies to the surface of xenogeneic endotheial ceUls, 2) increased synthesis and expression of vascular cel adhesion molecule-i on the xenogeneic endothelial ceUls, and 3) enhanced kiling of the xenogeneic endotheium by natural killer cells. Our data directly implicate naive neutrophils as major early participants in xenograft recognition and endothelal activation independent of xenoreactive natural antibodies and complenent (Am J Pathol 1997, 151:111-120)
sight. Xenoreactive natural antibodies (XNAs) and complement activation play a fundamental role in hyperacute vascularized xenograft rejection. 1-8 Central to xenograft rejection is the activation of donor endothelium leading to the loss of barrier function and the induction of a state of procoagulation in an otherwise anticoagulant quiescent endothelium.9 10 Whereas most of the attention has been focused on the humoral mechanisms leading to xenograft rejection, the extent of participation of the innate immune system remains poorly understood. Neutrophil involvement in the rejection process has been suggested by the rapid induction of neutrophil adhesion to endothelium after complement fixation,11 the prolonged survival of porcine hearts perfused with leukocyte-depleted human blood,12 and the prolonged survival of kidney xenografts in leukopenic rabbits.13 Although neutrophil involvement has always been considered a late event in the course of xenograft rejection, the ability of neutrophils to distinguish between xeno- and alloendothelium has not been established and neither has their ability to activate naive xenoendothelium. In this study, we demonstrate the novel finding that human naive neutrophils directly activate naive xenogeneic but not allogeneic endothelial cells in the absence of XNAs complement. This activation, which occurs in a calcium-dependent manner, renders the endothelium a more susceptible target for NK cells and may constitute an occult innate barrier to xenotransplantation. Supported by the Cardiovascular Collaborative Programme be-
Successful xenogeneic transplantation offers an attractive solution to the world-wide shortage of donor organs. Xenotransplantation has received much attention in recent years because of the general contention that solutions to xenograft rejection are in
tween King Faisal Specialist Hospital and Harefield Hospital and
Research Centre Funds. Accepted for publication April 17, 1997. Address reprint requests to Dr. Futwan Al-Mohanna, Biological & Medical Research, MBC 03, King Faisal Specialist Hospital & Research Centre, PO Box 3354, Riyadh 1 121 1, Saudi Arabia.
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Materials and Methods Antibodies Monoclonal mouse anti-human ICAM-1 antibody (BBA4; R&D Systems, Minneapolis, MN), monoclonal mouse anti-human CD1 1a, CD1 1b, and P-selectin (BCA1, BCA2, and BBA30, respectively; R&D Systems) were extensively dialyzed against phosphate-buffered saline (pH 7.4) to remove preservative. The extent of removal was tested using an azide-sensitive Escherichia coli strain (ATCC-25922). Cross-reactivity of anti-human ICAM-1 to porcine ICAM-1 was confirmed by increased immunofluorescence of human and porcine endothelial cells after treatment with tumor necrosis factor (TNF)-a and staining with BBA4. Fluorescence-labeled mouse anti-human P-selectin antibody (CD62/GMP 140) was purchased from Becton Dickinson, Mountain View, CA. Monoclonal antibodies to VCAM-1 (lM 1244 and BBA6) were purchased from Immunotech, Marseille, France, and R&D Systems, Minneapolis, MN, respectively.
Radioiodination and Binding of Anti P-Selectin Antibody Preservative free monoclonal anti P-selectin antibody (BBA30; R&D Systems, MN) was iodinated as described previously.14 The labeled protein was isolated on a Sephadex G25 column, and specific activity was determined (2.35 ,uCi/,tg of protein). For binding experiments, 2.5 x 104 human or porcine aortic endothelial cells (HAECs or PAECs) were plated on 96-well, flat-bottom microtest Ill tissue culture plates and 2.5 x 105 freshly isolated human neutrophils were added to each culture well at 18 hours after plating. Wells were washed 5 minutes after addition to remove unbound neutrophils as described under binding experiments, and 20 ,ul of radiolabeled anti-P-selectin antibody was added to each well to a total volume of 250 ,ul. The cells were incubated at 37°C for 1 hour after which cells were washed again as described above. The radioactivity associated with harvested cells was measured in a gamma counter (LKB-Wallac, CliniGamma 1272, Turku, Finland) and expressed as a percentage of the total added radioactivity obtained from 20 ,ul of labeled protein.
Isolation of Endothelial Cells PAECs, ovine aortic endothelial cells (SAECs), and human umbilical vein endothelial cells (HUVECs)
were isolated from adult pig aorta, adult sheep aorta and human umbilical vein, respectively, essentially as described by Jaffe et al.15 All three cell types were cultured in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, endothelial cell growth factor (Sigma Chemical Co., St. Louis, MO), penicillin (100 U/mi), streptomycin (25 gg/ml), and heparin (100 U/ml). HAECs were isolated from the thoracic aorta of donor hearts 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) supplemented with penicillin (100 U/ml), streptomycin (100 ,tg/ml), L-glutamine (1 mmol/L), bovine brain extract (25 ,ug/ml), heparin (15 U/ml), and 20% fetal bovine serum. 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) and to acetylated low-density Iipoprotein (Dil-Ac-LDL; Biogenesis, Bournemouth, UK). Cells were used from passages 2 to 10 in all experiments at a split ratio of 1:3. To test that endothelial cells were not activated during isolation and culture, ILl-a levels in conditioned medium were measured using enzyme-linked immunosorbent assay (R&D Systems, MN) and were consistently found to be negligible (le z
0 0
1
anti-VCAM-I
5
20
(ug/mi)
Figure 4. Induction of VCAM-1 synthesis in xenogeneic endothelial cells. RT-PCR of VCAM-1 transcripts from quiescent and human neutrophiltreated PAECs (a, lanes 1 and 2, respectively) and PAECs that were treated with BAPTA-AM( 10p.mmollLfor30 minutes at room temperature) before neutrophil contact (lane 3). Absence of VCAM-1 amplicons from quiescent and neutrophil-treated HAECs and HLWECs is shown in lanes 5 and 6 and lanes 8 and 9, respectively. Positive controls are shown in lanes 4, 7, and 10 where PAECs, HAECs, and HUVECs were treated with TNF-a (100 U1ic0 cells; Pharmingen) for 4 hours. B and C: Confocal fluiorescence micrographs of VCAM-1 expression in PAECs (B) and SAECs (C:) quiescent (i) neutrophil-treated (ii) endothelial cells. Also shown are inhibition of VCAM-1 expression in cells treated with BAPTA-AM before neutrophil contact (iii) and the induction of VCAM-1 expression after treatment of PAECs and SAECs with ionomycin (2 ,umol/L for 5 minutes) (iv). D: Enhanced cytotoxicity of human naive NK cells to xenogeneic but not allogeneic endothelial cells. E: Dose-dependent inhibition of neutrophil-induced enbancement of NK cell cytotoxicity by antibodies to VCAM-1.
Do Neutrophils Recognize Xenoendothelium? 119 AJPJuly 1997, Vol. 151, No. 1
Binding of human neutrophils to xenogeneic endothelial cells is associated with a transient calcium rise in the xenoendothelial cells. That direct contact is necessary for the calcium rise is suggested by 1) its inhibition by blocking antibodies to ICAM-1 and 2) its induction by a single neutrophil deposition on a xenogeneic endothelial cell, whereas supernatant from isolated naive neutrophils had no effect. The calcium rise was due to release from intracellular store(s) and was observed only in the xenoendothelium and not in the alloendothelium. Huang et a132 demonstrated that contact of human naive neutrophils with HUVEC monolayers had no apparent effect on intracellular calcium of either cell type and that a calcium rise was observed only after neutrophil activation with the chemotactic peptide fMLP. In contrast, an increase in intracellular calcium was found in each cell type after contact of bovine naive aortic endothelial cells or HAECs with human neutrophils.33 The apparent anomaly in the latter endothelial cells might be explained by the possibility of inadvertent neutrophil activation during the isolation procedure. In the present study, every neutrophil preparation (98 to 99% pure by FACS analysis) was tested for activation by measurement of reactive oxygen metabolite production using LDCL.16 Neutrophils were considered naive when no increase in resting LDCL was detected over a period of at least 10 minutes. Freshly isolated neutrophils displaying increased LDCL elicited a calcium rise in both xenogeneic and allogeneic endothelial cells, suggesting that these leukocytes were indeed activated. In contrast, naive neutrophils elicited a calcium rise in xenogeneic endothelial cells only. Pivotal to the rejection of vascularized xenograft is the activation of xenoendothelium, which has been categorized as either type or type 11 depending on whether protein synthesis is necessary.34'35 Human naive neutrophils induce activation of xenoendothelium, as seen by the rapid translocation of P-selectin (type 1) and the de novo synthesis of VCAM-1 (type 11). The fact that attenuating intraendothelial cell calcium transients inhibits naive neutrophil effects suggests a major role for calcium transients in xenoendothelial cell activation. We propose that surface expression of P-selectin would further augment leukocyte binding to the endothelium and that the expression of VCAM-1 renders the vasculature a more susceptible target for NK cells. The involvement of NK cells in xenograft rejection is becoming more apparent as demonstrated by the recent finding36 that human NK cells are capable of activating porcine endothelial cells independent of complement. We demonstrate that
naive NK cells recognize and kill xenogeneic endothelium and that this cytotoxicity is enhanced by previous treatment of xenoendothelium with neutrophils isolated from the same human donor. This enhancement is inhibited by antibodies to VCAM-1. Although XNAs and complement are currently the major contributors to xenograft rejection, removal of this barrier (through transgenes or otherwise) may serve to reveal an additional barrier to xenografting produced by neutrophil-induced activation of the xenoendothelium. Furthermore, the effect of neutrophils may also be detrimental in reperfusion injury, which might be accentuated in a xenograft. Together, our data suggest that naive neutrophils may represent an occult innate barrier to xenotransplantation.
Acknowledgments We thank S. Hafizi, P. Batten, 1. M. Moran-Verbeek, and P. S. Manogaran for technical assistance and M. A. Hannan, M. N. Al-Ahdal, C. M. G. Duran, and M. Paterson for valuable discussions.
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