Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p185erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.

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The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1+ T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28−/− recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-γ and IFN-γ-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor–inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite–type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.

Injuries to the vessel wall and subsequent exposure of collagen from the subendothelial matrix result in thrombus formation. In physiological conditions, the platelet plug limits blood loss. However, in pathologic conditions, such as rupture of atherosclerotic plaques, platelet–collagen interactions are associated with cardiovascular and cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets, and people who are deficient in GPVI suffer from bleeding disorders. Based on the fact that GPVI is coupled to the Fc receptor (FcR)-γ chain and thus should share homology with the FcR chains, the genes encoding human and mouse GPVI were identified. They belong to the immunoglobulin (Ig) superfamily and share 64% homology at the protein level. Functional evidence demonstrating the identity of the recombinant protein with GPVI was shown by binding to its natural ligand collagen; binding to convulxin (Cvx), a GPVI-specific ligand from snake venom; binding of anti-GPVI IgG isolated from a patient; and association to the FcR-γ chain. The study also demonstrated that the soluble protein blocks Cvx and collagen-induced platelet aggregation and that GPVI expression is restricted to megakaryocytes and platelets. Finally, human GPVI was mapped to chromosome 19, long arm, region 1, band 3 (19q13), in the same region as multiple members of the Ig superfamily. This work offers the opportunity to explore the involvement of GPVI in thrombotic disease, to develop alternative antithrombotic compounds, and to characterize the mechanism involved in GPVI genetic deficiencies.

Skeletal muscle ACh receptors (AChRs) accumulate at neuromuscular junctions (nmjs) at least partly because of the selective induction of AChR subunit genes in subsynaptic myotube nuclei by the motor nerve terminal. Additionally, mammalian AChRs undergo a postnatal change in subunit composition from embryonic (alpha 2 beta gamma delta) to adult (alpha 2 beta epsilon delta) forms, a switch that also depends on innervation. ARIA, a protein purified from chicken brains based on its ability to induce AChR synthesis in primary chick muscle cells, is a strong candidate for being the molecule responsible for these early developmental events. ARIA mRNA has been detected in embryonic motor neurons during synapse formation, and the gene continues to be expressed postnatally. In this report, we provide evidence that ARIA-like immunoreactivity is concentrated in rat motor nerve terminals from early postnatal ages, and that it can be detected in motor neurons in E18 embryos. ARIA is also detectable in axons within colchicine-treated sciatic nerves, suggesting that the protein in the nerve terminal has been transported from the cell body. ARIA mRNA is present in, but not restricted to, cholinergic neurons. Likewise, we report here that ARIA-like immunoreactivity is present in some noncholinergic central synapses. We also present evidence that isoforms of ARIA are differentially distributed among functionally distinct classes of neurons.

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.