William A. Frazier

CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer

In atherosclerosis and Alzheimers disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-β triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-β trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-β stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.

Integrin-associated protein (CD47) and its ligands

Integrin-associated protein (IAP or CD47) is a receptor for thrombospondin family members, a ligand for the transmembrane signaling protein SIRP alpha and a component of a supramolecular complex containing specific integrins, heterotrimeric G proteins and cholesterol. Peptides containing a VVM motif in the C-terminal domain of thrombospondins are agonists for CD47, initiating heterotrimeric Gi protein signaling that augments the functions of integrins of the beta 1, beta 2 and beta 3 families, thus modulating a range of cell activities including platelet activation, cell motility and adhesion, and leukocyte adhesion, migration and phagocytosis.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.

Nerve Growth Factor

Publisher Summary This chapter discusses the structure of nerve growth factor (NGF). The amino acid sequence of mouse NGF exhibited significant structural relatedness with the family of insulins and proinsulins. The sequence of mouse NGF could be aligned with human proinsulin with only five deletions required to yield the maximum homology of 21% identical residue positions. This is indicative of distant, but significant evolutionary relatedness. The majority of the identical residues were seen to be clustered in the segments of NGF, which aligned with the insulin A and B chain regions of the proinsulin sequence, separated by exactly the 35 residues required to accommodate the C activation peptide of proinsulin. Thus, the order of similar regions of NGF is B chain-C peptide-A chain, the same order in which they occur in proinsulin. Because NGF is 118 residues and human proinsulin is 86 residues, 37 residues of the C terminal region of NGF extend beyond the proinsulin molecule. This region is, however, similar to insulin B chain, suggesting a repetitive structure arising from a gene duplication event. It is also noteworthy that three out of the six half cystinyl residues that occur in both proteins are in identical positions, and two of these remain paired in identical fashion in insulin and NGF.

Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis

Tissue macrophages comprise a heterogeneous group of cell types differing in location, surface markers and function. Red pulp macrophages are a distinct splenic subset involved in removing senescent red blood cells. Transcription factors such as PU.1 (also known as Sfpi1) and C/EBPα (Cebpa) have general roles in myelomonocytic development, but the transcriptional basis for producing tissue macrophage subsets remains unknown. Here we show that Spi-C (encoded by Spic), a PU.1-related transcription factor, selectively controls the development of red pulp macrophages. Spi-C is highly expressed in red pulp macrophages, but not monocytes, dendritic cells or other tissue macrophages. Spic-/- mice have a cell-autonomous defect in the development of red pulp macrophages that is corrected by retroviral Spi-C expression in bone marrow cells, but have normal monocyte and other macrophage subsets. Red pulp macrophages highly express genes involved in capturing circulating haemoglobin and in iron regulation. Spic-/- mice show normal trapping of red blood cells in the spleen, but fail to phagocytose these red blood cells efficiently, and develop an iron overload localized selectively to splenic red pulp. Thus, Spi-C controls development of red pulp macrophages required for red blood cell recycling and iron homeostasis.

CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1 *

CD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations.

The Thrombospondin Receptor Integrin-associated Protein (CD47) Functionally Couples to Heterotrimeric Gi

Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with β3 integrins resulting in enhanced αvβ3-dependent cell spreading and chemotaxis and, in platelets, αIIbβ3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its β3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Giα is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPγ35S to membranes from cells expressing IAP and αvβ3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems.

Identification of integrin α3β1 as a neuronal thrombospondin receptor mediating neurite outgrowth

Summary Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these Interactions have not previously been Identified. In this study, monoclonal antibodies raised to the integHn (][31~1 heterodlmer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondln-1.0~1~1 is found to be expressed on the cell body, neurltes, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-Iigand binding assay is used to demonstrate the direct binding of Immunopurified 0~1 to thrombospondin-1. These results demonstrate a direct Interection between the integrin (131~1 and thrombospondln-1, which mediates neurite outgrowth in vitro and is likely to mediate the same Interactions in vivo.

CD47 Blockade Triggers T cell-mediated Destruction of Immunogenic Tumors

Macrophage phagocytosis of tumor cells mediated by CD47-specific blocking antibodies has been proposed to be the major effector mechanism in xenograft models. Here, using syngeneic immunocompetent mouse tumor models, we reveal that the therapeutic effects of CD47 blockade depend on dendritic cell but not macrophage cross-priming of T cell responses. The therapeutic effects of anti-CD47 antibody therapy were abrogated in T cell–deficient mice. In addition, the antitumor effects of CD47 blockade required expression of the cytosolic DNA sensor STING, but neither MyD88 nor TRIF, in CD11c+ cells, suggesting that cytosolic sensing of DNA from tumor cells is enhanced by anti-CD47 treatment, further bridging the innate and adaptive responses. Notably, the timing of administration of standard chemotherapy markedly impacted the induction of antitumor T cell responses by CD47 blockade. Together, our findings indicate that CD47 blockade drives T cell–mediated elimination of immunogenic tumors.

Increasing Survival of Ischemic Tissue by Targeting CD47

Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.