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Vascular endothelial growth factor (VEGF) is a dimeric angiogenic factor that is overexpressed by many tumors and stimulates tumor angiogenesis. VEGF initiates signaling by dimerizing the receptors VEGFR-1 and VEGFR-2. The Fas receptor stimulates apoptosis, and artificial dimerization of the Fas cytoplasmic domain has been shown to induce apoptosis. We constructed a chimeric receptor (VEGFR2Fas) combining the extracellular and transmembrane domains of VEGFR-2 with the cytoplasmic domain of Fas receptor. When VEGFR2Fas was stably expressed in endothelial cells in vitro, treatment with VEGF rapidly induced cell death with features characteristic of Fas-mediated apoptosis. These findings demonstrate that VEGFR2Fas functions as a VEGF-triggered death receptor and raise the possibility that introduction of VEGFR2Fas into tumor endothelium or tumor cells in vivo may convert tumor-derived VEGF from an angiogenic factor into an antiangiogenesis agent.

Natural killer (NK) cells express families of homologous receptors, members of which either activate or inhibit NK cells. We demonstrate that mouse Ly-49D is an activating receptor for the MHC antigen H2-Dd, which is also a ligand for the related inhibitory receptor Ly-49A. To compare and contrast their interactions with class I MHC ligand, we studied each of these receptors expressed in a rat NK-cell hne, RNK-16, for their capacity to recognize wild-type or mutated H2-Dd. Our studies with Ly-49A reveal that functional interaction with H2-Dd depends on residues in the floor of the H2-Dd peptide-binding groove. The recent co-crystal of Ly-49A with H2-Dd indicates that these are not contact residues, thus they may contribute to allelic specificity through conformational changes in H2-Dd. We found that structural requirements for functional recognition of H2-Dd by Ly-49D differ markedly from those for recognition by Ly-49A. We note that H2-Dd expression on certain target cells is not sufficient to activate lysis mediated by Ly-49D, though the additional requirements for functional interaction are not yet identified. Here we review recent studies of Ly-49 receptor ligand specificities and their molecular basis. The functions of these related receptors with opposing functions and shared allospecificity remains unclear.

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

Introduction of the MHC class I transgene H-2Dd on C57BL/6 (B6) background conveys NK cell-mediated "missing self" reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2Dd/Ld MHC class I transgenes, and show that the alpha1/alpha2 domains of H-2Dd were necessary and sufficient to induce "missing self" recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the alpha1/alpha2 domains of H-2Ld induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the alpha1/alpha2 domains of H-2Ld on NK cell specificity was surprising, considering that both H-2Ld and H-2Dd have been reported to interact with Ly49G2. Therefore, the role of H-2Ld for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2Dd, but not H-2Ld, abolished killing by sorted Ly49G2+ NK cells, indicating that H-2Ld does not inhibit NK cells via the Ly49G2 receptor.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cgamma1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

Mouse NK lymphocytes express Ly-49 receptors, which inhibit cytotoxicity upon ligation by specific MHC I molecules on targets. Different members of the lectin-like mouse Ly-49 receptor family recognize distinct subsets of murine H-2 molecules, but the molecular basis for the allelic specificity of Ly-49 has not been defined. We analyzed inhibition of natural killing by chimeric MHC I molecules in which the alpha1, alpha2, or alpha3 domains of the Ly-49A-binding allele H-2Dd were exchanged for the corresponding domains of the nonbinding allele H-2Db. Using the Ly-49A-transfected rat NK cell line, RNK-mLy-49A.9, we demonstrated that the H-2Dd alpha2 domain alone accounts for allelic specificity in protection of rat YB2/0 targets in vitro. We also showed that the H-2Dd alpha2 domain is sufficient to account for the allele-specific in vivo protection of H-2b mouse RBL-5 tumors from NK cell-mediated rejection in D8 mice. Thus, in striking contrast to the alpha1 specificity of Ig-like killer inhibitory receptors for human HLA, the lectin-like mouse Ly-49A receptor is predominantly restricted by the H-2Dd alpha2 domain in vitro and in vivo.

Studies of allogeneic lymphocyte cytotoxicity have shown that the rat NK allorecognition repertoire is controlled by genetic elements in both the MHC (RT1) and the NK gene complex (NKC). DA rats, possessing NK cells that are unable to lyse allogeneic lymphoblasts, were immunized with alloreactive NK cells from MHC-matched PVG.1AV1 rats, and two mAb, STOK1 and STOK2, were generated. STOK1 and STOK2 stained identical subsets of NKR-P1+ T and NK cells from certain strains of rats. Relative numbers varied markedly in a panel of MHC congenic strains, however, implicating a role for self MHC genes in their development. Both STOK1 and STOK2 immunoprecipitated a 110-kDa disulfide-linked homodimeric molecule, with extensive N-linked glycosylations, encoded by a gene that mapped to the NKC. NK cells expressing this glycoprotein displayed an increased ability to lyse allogeneic lymphoblasts, while syngeneic targets were spared. However, blockade of the STOK2 Ag with F(ab')2 of STOK2 permitted the NK lysis of syngeneic targets, but did not affect NK allorecognition. These results indicate that mAb STOK1 and STOK2 identify an NKC-encoded MHC receptor in the rat that acts as a negative regulator of cytotoxicity.

DAP12 and or FcRγ ITAM signals are required during normal osteoclast (OC) development in vivo and in vitro. To determine whether ITAM deficient mice are resistant to bone loss induced by estrogen deficiency, we performed ovariectomy (OVX) or SHAM surgery on 10–12 week female DAP12−/−, FcRγ−/−, DAP12−/−FcRγ−/− and control C57BL/6 (B6) mice. MicroCT of the distal femur showed statistically significant losses of trabecular bone in DAP12−/−FcRγ−/−, DAP12−/−, FcRγ−/− and B6 OVX groups compared to SHAM groups, BV/TV dropping from 56% to 35% (p