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Biomedical subjects

D L Simmons

Publications and source records attributed to D L Simmons.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Alzheimer Disease↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Neutrophils sense flow-generated stress and direct their migration through alphaVbeta3-integrin.

During inflammation neutrophils are recruited from the blood onto the surface of microvascular endothelial cells. In this milieu the presence of soluble chemotactic gradients is disallowed by blood flow. However, directional cues are still required for neutrophils to migrate to the junctions of endothelial cells where extravasation occurs. Shear forces generated by flowing blood provide a potential alternative guide. In our flow-based adhesion assay neutrophils preferentially migrated in the direction of flow when activated after attachment to platelet monolayers. Neutralizing alphaVbeta3-integrin with monoclonal antibodies or turning the flow off randomized the direction of migration without affecting migration velocity. Purified, immobilized alphaVbeta3-integrin ligands, CD31 and fibronectin, could both support flow-directed neutrophil migration in a concentration-dependent manner. Migration could be randomized by neutralizing alphaVbeta3-integrin interactions with the substrate using antibodies or Arg-Gly-Asp-containing peptide. These results exemplify mechanical signal transduction through integrin-ligand interactions and reveal a guidance system that was hitherto unknown in neutrophils. In more general terms, it demonstrates that cells can use integrin molecules to "sample" their physical microenvironment through adhesion and use this information to modulate their behavior.

Blood Circulation↗
PubMed1999

Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration.

PECAM-1 (CD31) is a highly abundant cell surface glycoprotein expressed on haemopoietic and endothelial cells. As well as mediating homophilic (PECAM-1/PECAM-1) adhesion, PECAM-1 can also bind the integrin alphavbeta3. Both PECAM-1 and alphavbeta3 have been shown to have roles in regulating angiogenesis, endothelial tube formation and in the case of alphavbeta3, endothelial cell apoptosis. In this study we show that despite being expressed at equivalent levels, endothelial alphavbeta3 is not a ligand for PECAM-1. Rather, PECAM-1 supports homophilic binding on HUVEC with similar characteristics to those we have previously reported for leukocytes and becomes tyrosine phosphorylated after homophilic PECAM-1 and integrin/fibronectin engagement. Immunoprecipitation studies show that in addition to SHP-2, tyrosine phosphorylated PECAM-1 can interact with at least four other phosphoproteins in pervanadate stimulated HUVEC. While PECAM-1/PECAM-1 interactions support robust endothelial cell adhesion, they do not support cell spreading or migration. In addition PECAM-1 homophilic adhesion rescues HUVEC from serum deprivation-induced apoptosis. Taken together our results indicate that PECAM-1 homophilic interactions play an important role in interendothelial cell adhesion, survival and signalling.

Apoptosis↗
PubMed1998

The role of the cysteine-rich region of the beta2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, alphaLbeta2, CD11a/CD18) heterodimer formation and ligand binding.

The cysteine-rich region (CRR) of the beta2 integrin subunit was replaced by that of beta1 to give the chimera beta2NV1. Beta2NV1 can combine with alphaL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the beta2 interaction with alphaL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing alphaL beta2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic beta2 CRR.

Amino Acid Sequence↗
PubMed1998

Signal-regulatory protein is selectively expressed by myeloid and neuronal cells.

Signal-regulatory proteins (SIRP) are transmembrane glycoproteins with three extracellular Ig-like domains, closely related to Ag receptors Ig, TCR, and MHC, and a cytoplasmic domain with two immunoreceptor with tyrosine-based inhibition motifs that can interact with src homology 2 domain-containing phosphatases. SIRP have previously been shown to inhibit signaling through receptor tyrosine kinases, but their physiologic function is unknown. Here we demonstrate by expression cloning that the mAbs ED9, ED17, and MRC-OX41 recognize rat SIRP. In addition, we show for the first time that rat SIRP is selectively expressed by myeloid cells (macrophages, monocytes, granulocytes, dendritic cells) and neurons. Moreover, SIRP ligation induces nitric oxide production by macrophages. This implicates SIRP as a putative recognition/signaling receptor in both immune and nervous systems.

Amino Acid Sequence↗
PubMed1998

The leukocyte function-associated antigen-1 (LFA-1)-binding site on ICAM-3 comprises residues on both faces of the first immunoglobulin domain.

ICAM-3 (CD50), a member of the Ig superfamily, is a major ligand for the leukocyte integrin LFA-1 (CD11a/CD18). This interaction represents one of several Ig superfamily/integrin ligand-receptor pairs that have been described to date. ICAM-3 is highly expressed on resting leukocytes and on APCs. In addition to an adhesive function, ICAM-3 can act as a signal-transducing molecule on T cells, providing a costimulatory signal for cell proliferation. Eighteen point mutations in ICAM-3 were generated, and residues important for binding of functional blocking Abs were identified. Mutation of seven of the residues reduced or abrogated adhesion to LFA-1, including three residues that are located on strand A of the ABED face of domain 1. In contrast, extensive mutagenesis analysis of ICAM-1 has shown that only residues on the GFC face interact with LFA-1. Our results provide evidence for a more extensive binding interface between ICAM-3 and LFA-1 than has previously been described. ICAM-3 appears to be unique among the ICAMs in utilizing residues on both faces of domain 1 for interaction with its ligand LFA-1.

Amino Acid Sequence↗
PubMed1998

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 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.

Animals↗
PubMed1998

Neutrophils rolling on immobilised platelets migrate into homotypic aggregates after activation.

Interactions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10(-7) M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of approximately 500 microm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/CD18 and ICAM-3, between alpha(v)beta3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.

Antibodies, Monoclonal↗
PubMed1997

Specific interactions between human integrin alpha v beta 3 and chimeric hepatitis B virus core particles bearing the receptor-binding epitope of foot-and-mouth disease virus.

Purified integrin alpha v beta 3 was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+ cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin alpha v beta 3. Electron micrographs showed several individual alpha v beta 3 molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to alpha v beta 3 and provide support for identification of alpha v beta 3 as a potential cellular receptor for FMDV.

Antigens, Viral↗
PubMed1997

Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31.

CD31 (PECAM-1) is a member of the immunoglobulin superfamily whose extracellular domain is comprised of six immunoglobulin-like domains. It is widely expressed on endothelium, platelets, around 50% of lymphocytes, and cells of myeloid lineage. CD31 has been shown to be involved in interendothelial adhesion and leukocyte-endothelial interactions, particularly during transmigration. CD31-mediated adhesion is complex, because CD31 is capable of mediating both homophilic and multiple heterophilic adhesive interactions. Here we show that the NH2-terminal (membrane-distal) immunoglobulin domain of CD31 is necessary but not sufficient to support stable homophilic adhesion. Key residues forming the binding site within this domain have been identified by analysis of 26 single point mutations, representing the most systematic analysis of a fully homophilic interaction between immunoglobulin superfamily family members to date. This revealed five mutations that affect homophilic binding. Uniquely, the residues involved are exposed on both faces of the immunoglobulin fold, leading us to propose a novel mechanism for CD31 homophilic adhesion.

Amino Acid Sequence↗
PubMed1997

The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis.

The aged garlic extract 'Kyolic' lowers serum cholesterol levels in humans and experimental animals and thus is presumed to have a protective effect against atherosclerosis. However, to date no studies have examined the effect of this substance on the actual development of the disease. In the present study, the right carotid artery of 24 rabbits was de-endothelialized by balloon catheterisation in order to produce a myointimal thickening. After 2 weeks the rabbits were randomly assigned to four groups: Group I received a standard diet; Group II received the standard diet supplemented with 800 microl/kg body weight/day 'Kyolic'; Group III received a 1% cholesterol supplemented standard diet; and Group IV received a 1% cholesterol supplemented standard diet plus 'Kyolic'. After 6 weeks, the cholesterol diet caused a 6-fold increase in serum cholesterol level (Group III; 6.4 +/- 0.6 mmol/l) compared to normal diet (Group I; 1.2 +/- 0.4 mmol/l) (P < 0.05) with only a minor, non-significant reduction seen by the addition of 'Kyolic' (Group IV; 6.2 +/- 0.7 mmol/l). Group III rabbits developed fatty streak lesions covering approximately 70 +/- 8% of the surface area of the thoracic aorta, which was significantly reduced to 25 +/- 3% in the 'Kyolic'-treated Group IV. No lesions were present in Groups I and II. The hypercholesterolaemic diet caused an increase in aortic arch cholesterol (2.1 +/- 0.1 mg cholesterol/g tissue) which was significantly reduced by 'Kyolic' supplementation (1.7 +/- 0.2 mg cholesterol/g tissue) (P < 0.05). 'Kyolic' significantly inhibited the development of thickened, lipid-filled lesions in the pre-formed neointimas produced by balloon-catheter injury of the right carotid artery in cholesterol-fed rabbits (intima as percent of artery wall, Group III 42.6 +/- 6.5% versus Group IV 23.8 +/- 2.3%, P < 0.01), but had little effect in rabbits on a standard diet (Group II 18.4 +/- 5.0% versus Group I 16.7 +/- 2.0%). In vitro studies showed that 'Kyolic' has a direct effect on inhibition of smooth muscle proliferation. In conclusion, 'Kyolic' treatment reduces fatty streak development, vessel wall cholesterol accumulation and the development of fibro fatty plaques in neointimas of cholesterol-fed rabbits, thus providing protection against the onset of atherosclerosis.

Animals↗
PubMed1997

Cross-talk between cell adhesion molecules regulates the migration velocity of neutrophils.

BACKGROUND: Although the adhesive mechanisms underlying the capture and immobilization of circulating neutrophils in inflamed blood vessels have been well described, factors controlling the subsequent migration of neutrophils over and through the blood vessel endothelium are poorly understood. Directional rearrangement of the actin cytoskeleton within the neutrophil, along with modulation of integrin-mediated adhesion, are necessary for neutrophil migration. Signals from chemotactic agents and from the adhesive substrate may regulate these processes, but little is known about their relative importance or their mode of integration. RESULTS: We examined the kinetics of neutrophil migration after formyl tripeptide or platelet-activating factor was perfused over neutrophils that were already rolling on the adhesion molecule P-selectin, which was presented either on the surface of immobilized platelets or in purified form coated on glass capillaries. Upon activation, neutrophils stopped rolling, spread and began to migrate; each of these processes was dependent on beta2 integrin (CD11b/CD18). The rate of migration increased over a period of about 8 minutes and was modulated directly by both the P-selectin and the CD31 surface receptors. Antibody blockade of either CD31 or P-selectin on platelets resulted in a reduction in the velocity of migration, and simultaneous blockade of both receptors reduced velocity further. Purified CD31 and P-selectin (but not a control adhesion molecule, ICAM-1) increased migration velocity in a concentration-dependent and additive manner that reconstituted the migratory behaviour observed on platelets. CONCLUSIONS: These studies show that binding of ligands to CD31 and/or P-selectin modifies the rate of integrin-supported neutrophil migration. This novel example of 'cross-talk' between surface receptors suggests that cell adhesion molecules might generally transduce accessory signals between adjacent cells to modify their migratory responses to chemotactic signals.

Animals↗
PubMed1997

Role of ligands in the activation of LFA-1.

Lymphocyte function-associated-antigen-1 (LFA-1) is able to bind selectively to its ligands intercellular adhesion molecules 1 and 3 (ICAM-1 and ICAM-3), suggesting that LFA-1 can exist in distinct ligand-specific binding states. In the case of ICAM-1, apart from ligand itself and the recently cloned molecule cytohesin-1, the natural physiological regulators of LFA-1-mediated binding to ICAM-1 are unknown. We have investigated the role of ligands (ICAM-1 and ICAM-3) in LFA-1 activation by using ICAM-blocking monoclonal antibodies and a fixation protocol for "freezing" LFA-1 on the surface of cells after prior exposure to ICAM-1 and ICAM-3. These studies not only confirm that LFA-1 exists in distinct ICAM-specific activation states, but also demonstrate that ICAM-1 plays a role in the activation of LFA-1 binding to ICAM-3.

Antigens, CD↗
PubMed1997

NSAID-induced apoptosis in Rous sarcoma virus-transformed chicken embryo fibroblasts is dependent on v-src and c-myc and is inhibited by bcl-2.

Mounting epidemiological and experimental evidence implicates non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

Animals↗
PubMed1997

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.

Animals↗