Immortalized bone-marrow derived pig endothelial cells.

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 IFNgamma. 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 xenoantigens, 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.

Identification and gene organization of three novel members of the IL-1 family on human chromosome 2.

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.

HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.

Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(alpha1,3)Galactose epitope.

BACKGROUND: The cell surface carbohydrate moiety, Gal(alpha1,3)Galactose (alphaGal), has been implicated as the major determinant recognized by more than 80% of human anti-porcine natural antibodies (NAb). An ELISA system was developed for the detection of this subpopulation of porcine cell-reactive NAb using synthetic alphaGal conjugated to bovine serum albumin. METHODS: A screen of 95 human serum samples by this method demonstrated marked variability in the alphaGal reactivity of unrelated donors. The percentage of alphaGal-reactive NAb relative to total immunoglobulin was determined for 10 donors. RESULTS: alphaGal-reactive NAb comprised 1.0-2.4% of total serum IgG, whereas the range was from 3.9% to 8.0% for IgM. CONCLUSIONS: The higher level of alphaGal-reactive IgM suggests that xenoreactive NAbs may be the product of germ-line genes. Two-dimensional gel analysis of affinity-purified alphaGal-reactive NAb from two donors provided evidence suggesting that IgM from this subpopulation of NAb were restricted in protein charge heterogeneity.

Relationship between ABO blood group and levels of Gal alpha,3Galactose-reactive human immunoglobulin G.

BACKGROUND: The terminal Gal alpha1,3Galactose (alphaGal) determinant is present on all porcine glycoproteins and glycolipids, but is not expressed by human cells. Consequently human sera contain anti-alphaGal natural antibodies. The human blood group B antigen [Gal alpha1,3(Fuc1,2)Galactose] is differentiated from the alphaGal epitope by the presence of a fucosyl group. METHODS: To determine whether the expression of the B antigen has any effect on the level of alphaGal-reactive natural antibodies, equal numbers (n=12) of A, B, AB, and O serum samples were evaluated by ELISA and flow cytometry. RESULTS: A significant reduction in IgG alphaGal reactivity was observed with serum samples from B antigen-expressing donors (B, AB) relative to non-B antigen-expressing donors (A, O). CONCLUSIONS: These results are consistent with the possibility that anti-alphaGal antibodies in non-B antigen-expressing individuals include a subset that is reactive with the structurally related B antigen and that this subset is absent in B and AB individuals.

Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.