Marten K. J. Schneider

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity.

The galactose-α-1,3-galactose (αGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcγRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of αGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an αGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the αGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on αGal expression. In conclusion, removal of αGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that αGal is not a dominant target for direct human NK cytotoxicity against porcine cells.

HLA-G Inhibits Rolling Adhesion of Activated Human NK Cells on Porcine Endothelial Cells

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.

Immortalized bone-marrow derived pig endothelial cells.

Abstract: Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti‐pig immune responses in vitro, pig microvascular bone‐marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM‐derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNγ. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xeno‐antigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium‐phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV.15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity.

Background. The expression of galactose-&agr;(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal−/− pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal−/− and Gal+/+ pigs, and (2) the susceptibility of such Gal−/− porcine endothelial cells to human natural killer (NK) cell cytotoxicity. Methods. Primary porcine aortic endothelial cells (PAEC) were isolated from Gal−/− (PAEC-Gal−/−) and Gal+/+ (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. Results. Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal−/− cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal−/− and PAEC-Gal+/+ was detected. Tumor-necrosis factor-&agr; stimulation of PAEC-Gal−/− and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal expression on PAEC-Gal−/− cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2–activated NK cells. Conclusions. Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

Chlamydia pneumoniae induces aponecrosis in human aortic smooth muscle cells

BackgroundThe intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.ResultsChlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria.ConclusionThis study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.

Rolling adhesion of human NK cells to porcine endothelial cells mainly relies on CD49d-CD106 interactions.

Background. Acute vascular rejection in pig-to-primate xenotransplantation involves recognition and damage of porcine (po) endothelial cells (EC) by human (hu) leukocytes, probably including natural killer (NK) cells. To study such interactions we analyzed rolling and static adhesion of hu NK cells to po EC. Methods. The effects of blocking hu and po adhesion molecules on the adhesion hu NK cells to po EC monolayers was analyzed under shear stress (10 min, 37°C, 0.7 dynes/cm2) or under static conditions (10 min, 37°C). All used cell populations were phenotypically characterized by flow cytometry. Results. Blocking of CD106 on po EC or its ligand CD49d on hu NK cells decreased rolling adhesion of both fresh and activated hu NK cells by more than 75%. Masking of CD62L on fresh but not activated hu NK resulted in a 44% decrease in rolling adhesion, in line with the diminished cell surface expression of CD62L upon activation. Antibodies to CD31, CD54, CD62E, and CD62P on EC or CD11a, CD18, and CD162 on NK cells had only minor effects on rolling adhesion. The adhesion of the Fc&ggr;RIII− hu NK cell line NK92 to po EC was inhibited by 95% after masking po CD106 whereas antibodies to po CD31, CD54, CD62E, or CD62P had no effect, thereby excluding effects of Fc-receptor-dependent binding of hu NK cells to po EC. Static adhesion of activated NK cells was reduced by approximately 60% by blocking either CD49d or CD106, by 47% by blocking CD11a, and by 82% upon simultaneous blocking of CD11a and CD49d. Conclusions. Interactions between hu CD49d and po CD106 are crucial for both rolling and firm adhesion of hu NK cells to po EC and thus represent attractive targets for specific therapeutic interventions to prevent NK cell-mediated responses against po xenografts.

Current cellular innate immune hurdles in pig-to-primate xenotransplantation.

Purpose of reviewThe generation of pigs lacking Galα1,3Gal (αGal) or overexpressing human regulators of complement has largely overcome the barrier of hyperacute xenograft rejection. Nevertheless, delayed xenograft rejection remains a major hurdle, including humoral responses against nonαGal epitopes, and cellular innate immune responses. This review addresses the early interactions between the porcine endothelium and human natural killer cells, neutrophils, and monocytes/macrophages. Recent findingsWhether human leukocyte recruitment to, and lysis of, porcine endothelial cells includes direct recognition of αGal remains a matter of debate. Although the human natural killer receptor natural killerRP1A may directly recognize αGal, several studies did not reveal significant differences regarding adhesion, transmigration, and cytotoxicity using porcine endothelial cells expressing or lacking αGal. The strong human monocyte response against pig cells partly relies on the inability of porcine ligands to ligate inhibitory human receptors, such as SIRPα. Strategies to overcome cellular innate immune responses include transgenic expression in porcine cells of human ligands for inhibitory receptors, together with species-specific blocking of porcine ligands mediating human leukocyte interactions. SummaryCellular human innate immune responses are increasingly recognized as barriers to successful pig-to-human xenotransplantation, and only a detailed knowledge of the molecular mechanisms involved will allow us to overcome this barrier.

Adhesive Interactions between Human NK Cells and Porcine Endothelial Cells

Human natural killer (NK) cells are able to adhere to xenogeneic porcine endothelial cells (EC) and evidence from in vitro studies as well as animal models suggests a potential role for NK cells in the cellular recognition and damage of porcine xenogeneic tissues. One possible explanation for the observed NK cell‐mediated xenogeneic cytotoxicity against porcine EC is the molecular incompatibility between porcine major histocompatibility complex (MHC) class I molecules and MHC‐specific inhibitory receptors on human NK cells. In this review we attempt to summarize the current knowledge concerning adhesive interactions between human NK cells and porcine EC under special considerations of the cross‐species receptor–ligand interactions. Methodological differences in assessing adhesion between various studies are reviewed and comparisons to the syngeneic/allogeneic adhesion mechanisms are made. Finally, the therapeutic potential of blocking antibodies and transgenic HLA expression in preventing NK‐cell adhesion and xenogeneic cytotoxicity is discussed.

Porcine aortic endothelial cells transfected with HLA-G are partially protected from xenogeneic human NK cytotoxicity.

In this study we tested whether the expression of HLA-G protects porcine endothelial cells (PEC) from the lysis mediated by human natural killer (NK) cells. Because HLA-E is not present in PEC, this model provides an ideal tool to study the direct role of HLA-G in NK inhibition. Immortalized porcine aortic endothelial cells (PED) were stably transfected with a vector coding for the HLA-G1 protein and surface expression was demonstrated by flow cytometry analysis. Although the adhesion of human NK cells to PED was not compromised by HLA-G, the expression of HLA-G partially protected PED from the lysis mediated by polyclonal NK lines derived from different donors. A decrease of the surface expression of HLA-G on PED corresponded to a loss of the capacity of PED to inhibit NK cytotoxicity, indicating that the surface density of HLA-G molecules must exceed a certain threshold to protect target cells. In summary, these data show that HLA-G, independent from the presence of HLA-E, can only partially and inefficiently protect PED from human NK cell-mediated cytotoxicity. Because ILT-2/LIR-1 expression did not correlate with HLA-G mediated inhibition, we hypothesize that other yet unidentified receptors expressed by peripheral blood NK cells are involved in the recognition of HLA-G.

Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression.

Because of organ shortages in clinical allotransplantation, the potential of pig‐to‐human xenotransplantation is currently being explored showing a possible critical role for natural killer (NK) cells in the immune response against xenografts. Therefore, we analyzed the cytotoxic pathways utilized by human natural killer cells (hNK) against porcine endothelial cells (pEC). Transmission electron microscopy of pEC cocultured with hNK cells showed both apoptotic and necrotic cell death, whereas soluble factors such as Fas ligand or TNFα did not induce apoptosis in pEC. NK lysis of pEC was abrogated by concanamycin A and ammonium chloride, reagents inhibiting the perforin/granzyme B (grB) pathway, but only partially blocked by caspase inhibition with z‐VAD‐fmk. Overexpression of bcl‐2 protected pEC against apoptosis induced by staurosporine or actinomycin D, but failed to prevent hNK cell‐mediated lysis. In conclusion, pEC are lysed in vitro by hNK cells via the perforin/grB pathway and are not protected from NK lysis by overexpression of bcl‐2.