The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium-host interaction.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

Relationships between stem cell exhaustion, tumour suppression and ageing.

Ageing is a complex process characterised by a variety of disorders associated with general organismal decline and an inability to maintain tissue homoeostasis. As described in this review, recent studies indicate that ageing may be caused, in part, by the depletion of stem and progenitor cells that govern tissue renewal. The potential causes of stem and progenitor cell attrition are numerous; however, a commonly accepted theory is that these cells are lost as a result of naturally occurring DNA damage and the obligate checkpoint responses that follow. Failure to launch appropriate responses to DNA damage is strongly associated with cancer initiation and progression. Therefore, it is at this nexus, the response to DNA damage, that an important organismal fate may be determined: to degrade regenerative potential for the purpose of preventing cancer. According to this viewpoint, ageing may be the unfortunate mark of successful cancer suppression in stem cells and other cell types. In this review, we will describe how degeneration of tissue renewal capacity links ageing and cancer suppression.

Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis.

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers.

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia.

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae-induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo, by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens.

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation.

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.

Renal artery stenosis in minority patients undergoing diagnostic cardiac catheterization: prevalence and risk factors.

BACKGROUND: Atherosclerotic renal artery stenosis (RAS) is a frequently overlooked clinical entity that can cause progressive renal failure and uncontrolled hypertension. Revascularization of a stenosed renal artery is associated with improved clinical outcomes including the prevention of renal failure. Thus, it is important to recognize all potential candidates for renal artery revascularization. In a general population referred for diagnostic cardiac catheterization, RAS of any severity was found in 30% of patients and significant stenosis (> or = 50% diameter narrowing) was found in 15% of patients. The number of minority groups is increasing in the US population, and RAS in this population is not well investigated. Our purpose was to determine the prevalence and risk factors associated with RAS in minority patients referred for diagnostic cardiac catheterization. METHODS: Abdominal aortography was performed in 171 consecutive minority patients referred for diagnostic cardiac catheterization (hispanics = 115, African Americans = 56). The association of clinical and angiographic variables with RAS was examined using univariate and multivariate logistic regression analyses. RESULTS: Renal artery stenosis of any severity was identified in 13.5% of patients (unilateral 7.7%, bilateral 5.8%). Significant RAS was found in 7.7% of patients (unilateral 4.8%, bilateral 2.9%). Independent predictors of RAS included age (mean +/-1SD, 68 +/-10 vs 57 +/-12 yr, P < 0.001, for patients with vs without RAS), coronary artery disease, and elevated serum creatinine levels (> 115 micromol/L). Race/ethnicity (hispanics vs African Americans), sex, smoking, congestive heart failure, diabetes mellitus, peripheral vascular disease, and hypertension were not independent predictors. CONCLUSIONS: Renal artery stenosis in minority patients undergoing diagnostic cardiac catheterization is less common than reported in white patients, is similar in hispanics and African Americans, and is similar in women and men. The clinical and angiographic features are helpful in predicting its presence.

Normal ligand binding and signaling by CD47 (integrin-associated protein) requires a long range disulfide bond between the extracellular and membrane-spanning domains.

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys(33) and Cys(263). Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPalpha1 binding, and reduced CD47/SIRPalpha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca(2+) responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with alpha(v)beta(3) integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.

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.

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly.

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.

A family of tissue-specific resistin-like molecules.

We have identified a family of resistin-like molecules (RELMs) in rodents and humans. Resistin is a hormone produced by fat cells. RELMalpha is a secreted protein that has a restricted tissue distribution with highest levels in adipose tissue. Another family member, RELMbeta, is a secreted protein expressed only in the gastrointestinal tract, particularly the colon, in both mouse and human. RELMbeta gene expression is highest in proliferative epithelial cells and is markedly increased in tumors, suggesting a role in intestinal proliferation. Resistin and the RELMs share a cysteine composition and other signature features. Thus, the RELMs together with resistin comprise a class of tissue-specific signaling molecules.

The hormone resistin links obesity to diabetes.

Diabetes mellitus is a chronic disease that leads to complications including heart disease, stroke, kidney failure, blindness and nerve damage. Type 2 diabetes, characterized by target-tissue resistance to insulin, is epidemic in industrialized societies and is strongly associated with obesity; however, the mechanism by which increased adiposity causes insulin resistance is unclear. Here we show that adipocytes secrete a unique signalling molecule, which we have named resistin (for resistance to insulin). Circulating resistin levels are decreased by the anti-diabetic drug rosiglitazone, and increased in diet-induced and genetic forms of obesity. Administration of anti-resistin antibody improves blood sugar and insulin action in mice with diet-induced obesity. Moreover, treatment of normal mice with recombinant resistin impairs glucose tolerance and insulin action. Insulin-stimulated glucose uptake by adipocytes is enhanced by neutralization of resistin and is reduced by resistin treatment. Resistin is thus a hormone that potentially links obesity to diabetes.

Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow.

Integrin-associated protein (CD47/IAP) is a pentaspan molecule that regulates integrin functions. We prepared a CD47-deficient Jurkat T cell line to assess its role in the arrest of T cells on inflammatory endothelium. Under flow conditions, constitutive arrest of CD47-deficient cells is strongly decreased as compared to the original cell line, whereas reexpression of CD47 reestablishes their ability to stop. Moreover, cells transfected with a chimera made with the extracellular portion of CD47 and the transmembrane domain of CD7 or several truncated forms of CD47 show that the first transmembrane domain and a short cytoplasmic loop are sufficient for this process. CD47 effect is indirect and depends mainly on the alpha4beta1/VCAM-1 pathway, as shown by blocking antibodies. We detected on endothelium the two CD47 counter receptors known to date: thrombospondin and SIRP1alpha. Blocking experiments show that both are involved. Overall, CD47 participates in the constitutive arrest of T lymphocytes on inflamed vascular endothelium by up-regulating alpha 4beta1 integrins.

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.

The macrophage fusion receptor (MFR), also called P84/BIT/SIRPalpha/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophage-macrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.

Role of a bacillus Calmette-Guérin fibronectin attachment protein in BCG-induced antitumor activity.

Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG-induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity.

ATR disruption leads to chromosomal fragmentation and early embryonic lethality.

Although a small decrease in survival and increase in tumor incidence was observed in ATR(+/-) mice, ATR(-/-) embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR(-/-) blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR(-/-) cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53.

The role of p53 in megakaryocyte differentiation and the megakaryocytic leukemias of Down syndrome.

Sequence analysis of the p53 tumor suppressor gene was performed on diagnostic blood and bone marrow samples from nine children with Down syndrome and acute megakaryoblastic leukemia. p53 sequence alterations were not observed in any of seven cases of transient leukemia; however, samples from two of three patients with acute megakaryoblastic leukemia harbored sequence alterations. The acquisition of both mutations (Gly245Val and Arg72Pro) in the transformation from transient leukemia to overt acute megakaryoblastic leukemia suggests a functional role of mutant p53 in the evolution of this disease.

Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47.

Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRPalpha1 and SIRPalpha2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34(+), AC133(+), CD90(+) hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRPalpha1 and SIRPalpha2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRPalpha1-reactive MoAbs. In addition, SIRPalpha1 and SIRPalpha2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRPalpha1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.

Ubiquitin-related proteins regulate interaction of vimentin intermediate filaments with the plasma membrane.

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxyterminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named proteins linking IAP with cytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

Role of cholesterol in formation and function of a signaling complex involving alphavbeta3, integrin-associated protein (CD47), and heterotrimeric G proteins.

Integrin-associated protein (CD47) is a multiply membrane spanning member of the immunoglobulin superfamily that regulates some adhesion-dependent cell functions through formation of a complex with alphavbeta3 integrin and trimeric G proteins. Cholesterol is critical for the association of the three protein components of the supramolecular complex and for its signaling. The multiply membrane spanning domain of IAP is required for complex formation because it binds cholesterol. The supramolecular complex forms preferentially in glycosphingolipid-enriched membrane domains. Binding of mAb 10G2 to the IAP Ig domain, previously shown to be required for association with alphavbeta3, is affected by both the multiply membrane spanning domain and cholesterol. These data demonstrate that cholesterol is an essential component of the alphavbeta3/IAP/G protein signaling complex, presumably acting through an effect on IAP conformation.