David L. Simmons

Human CD14 mediates recognition and phagocytosis of apoptotic cells

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 (refs 2, 3) on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome,. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with ‘non-self’ components (LPS) and ‘self’ components (apoptotic cells) produce distinct macrophage responses.

Inhibitor of neurite outgrowth in humans.

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

RGD peptides induce apoptosis by direct caspase-3 activation

Synthetic peptides containing the arginine–glycine–aspartate (RGD) motif have been used extensively as inhibitors of integrin–ligand interactions in studies of cell adhesion, migration, growth and differentiation,,, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of pro-caspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate–aspartate–methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD–DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent pro-apoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis,,.

Neurobiology: Inhibitor of neurite outgrowth in humans

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

Identification and characterisation of human Junctional Adhesion Molecule (JAM)

It is widely believed that migrating immune cells utilise the intercellular junctions as routes of passage, and in doing so cause the transient disruption of junctional structures. Thus there is much interest in the molecules that have been identified at cell-cell contact points and their potential involvement in the control of leukocyte diapedesis. In this report we describe the human orthologue to Junctional Adhesion Molecule (JAM), a recently identified member of the immunoglobulin superfamily expressed at intercellular junctions (Martin-Padura et al., 1998). The human protein shares a highly conserved structure and sequence with the murine protein. However it is distinct in that it is constitutively expressed on circulating neutrophils, monocytes, platelets and lymphocyte subsets. This broad expression pattern is similar to another IgSF molecule, CD31, expressed at intercellular junctions, and may indicate further complexities in the control of leukocyte/ endothelial interactions.

CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPα 1

The rat OX41 antigen is a cell surface protein containing three immunoglobulin superfamily domains and intracellular immunoreceptor tyrosine‐based inhibitory motifs (ITIM). It is a homologue of the human signal‐regulatory protein (SIRP) also known as SHPS‐1, BIT or MFR. Cell activation‐induced phosphorylation of the intracellular ITIM motifs induces association with the tyrosine phosphatases SHP‐1 and SHP‐2. To identify the physiological OX41 ligand, recombinant OX41‐CD4d3+4 fusion protein was coupled to fluorescent beads to produce a multivalent cell binding reagent. The OX41‐CD4d3+4 beads bound to thymocytes and concanavalin A‐stimulated splenocytes. This interaction was blocked by the monoclonal antibody (mAb) OX101. Affinity chromatography with OX101 mAb and peptide sequencing revealed the rat SIRP ligand to be CD47 (integrin‐associated protein). A direct interaction between human SIRP and human CD47 was demonstrated using purified recombinant proteins and surface plasmon resonance ruling out the involvement of other proteins known to be associated with CD47. The affinity of the SIRP/CD47 interaction was Kd ≈ 8 μM at 37°C with a koff ≥2.1 s–1. The membrane‐distal SIRP V‐like domain was sufficient for binding to CD47.

Galectin 3 Induces a Distinctive Pattern of Cytokine and Chemokine Production in Rheumatoid Synovial Fibroblasts via Selective Signaling Pathways

OBJECTIVE High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts. METHODS Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay. RESULTS Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5. CONCLUSION Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

The mid-developmental transition and the evolution of animal body plans

Animals are grouped into ~35 ‘phyla’ based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development—known as the phylotypic period—is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent ‘mid-developmental transition’ that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

Extracellular matrix-mediated chemotaxis can impede cell migration

Cells use a combination of changes in adhesion, proteolysis and motility (directed and random) during the process of migration. Proteolysis of the extracellular matrix (ECM) results in thecreation of haptotactic gradients which cells use to move in a directed fashion. The proteolytic creation of these gradients also results in the production of digested fragments of ECM. In this study we show that in the human fibrosarcoma cell line HT1080, matrix metalloproteinase–2(MMP–2)–digested fragments of fibronectin exert a chemotactic pull stronger than that of undigested fibronectin. During invasion, this gradient of ECM fragments is established in the wake of an invading cell, running counter to the direction of invasion. The resultant chemotactic pull is anti–invasive, contrary to the traditional view of the role of chemotaxis in invasion. Uncontrolled ECM degradation by high concentrations of MMP can thus result in steep gradients of ECM fragments, which run against the direction of invasion. Consequently, the invasive potential of a cell depends on MMP production in a biphasic mannerimplying that MMP inhibitors will upregulate invasion in high–MMPexpressing cells. Hence the therapeutic use of protease inhibitors against tumours expressing high levels of MMP could produce an augmentation of invasion.

Cell adhesion: a new target for therapy

Intercellular adhesive events are involved in a wide range of biological processes, including pattern formation and morphogenesis during development, immune responses, leukocyte recirculation, wound repair, tumour growth and metastasis. In the multicellular state, signals from cell adhesion molecules, along with those from growth factor and cytokine receptors, provide a range of information to the cell that is integrated to yield a final message, perhaps to maintain the cell cycle (if it is a stem cell) or follow a path towards terminal differentiation. Aberrant cell adhesion plays a key role in many developmental defects, acute and chronic inflammatory disease and cancer.