Using multivalent protein probes, an evolutionarily conserved endogenous ligand for EMR2, a human myeloid cell-restricted EGF-TM7 receptor, was identified on the surface of a number of adherent cell lines. In addition, in situ staining of the ligand has revealed specific in vivo patterns consistent with a connective tissue distribution. The interaction is conserved across species and mediated exclusively by the largest EMR2 isoform containing 5 epidermal growth factor (EGF)-like modules. Antibody-blocking studies subsequently revealed that the fourth EGF-like module constitutes the major ligandbinding site. The largest isoform of CD97, a related EGF-TM7 molecule containing an identical EGF-like module, also binds to the putative EMR2 ligand. Through the use of mutant Chinese hamster ovary (CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as the enzymatic removal of specific cell surface GAGs, the molecular identity of the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS GAGs blocked the EMR2-ligand interaction in a dose-dependent manner. EMR2-CS interaction is Ca 2؉ -and sulphation-dependent and results in cell attachment. This is the first report of a GAG ligand for the TM7 receptors extending the already vast repertoire of stimuli of the GPCR superfamily.

The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopeniaassociated autoimmunity in humans.T lymphocytes | reconstitution | immunotherapy T he anti-CD (cluster of differentiation molecule) 52 monoclonal antibody alemtuzumab has proven efficacy in relapsing remitting multiple sclerosis (RRMS) (1-3). Each cycle of alemtuzumab leads to profound panlymphopenia, but relatively infrequent dosing allows reconstitution to occur: B cells recovery rapidly, whereas CD4 and CD8 cells take 35 and 20 mo, respectively, to reach normal values (4). For 5 y after alemtuzumab and maximally, at 2 y, secondary autoimmune conditions may develop: 30% of individuals experience thyroid autoimmunity, and 1% of individuals have idiopathic thrombocytopenic purpura (ITP); there are rare cases of autoimmune hemolytic anemia, autoimmune neutropenia, and Goodpasture syndrome (1-3). One-third of patients develop asymptomatic autoantibodies.An association between lymphopenia and autoimmunity is recognized; in humans, T lymphopenia is a feature of systemic lupus erythematosus, rheumatoid arthritis, and Crohn and Sjogren syndromes (5), and animal models of autoimmunity often involve the induction of lymphopenia. The mechanism driving autoimmunity in these situations is unclear. In some cases it has been attributed to loss of regulatory cells; however, although treatment of lymphopenic hosts with CD4 +

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu2Ser 518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P ؉1 cleavage site (Ser 518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P ؊2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cellfree system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cisproteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.

A successful pregnancy depends on the intricate and timely interactions of maternal and fetal cells. Placental extravillous cytotrophoblast invasion involves a cellular transition from an epithelial to mesenchymal phenotype. Villous cytotrophoblasts undergo a partial epithelial to mesenchymal transition (EMT) when differentiating into extravillous cytotrophoblasts and gain the capacity to migrate and invade. This review summarizes our current knowledge regarding known regulators of EMT in the human placenta, including the inducers of EMT, upstream transcription factors that control EMT and the downstream effectors, cell adhesion molecules and their differential expression and functions in pregnancy pathologies, preeclampsia (PE) and fetal growth restriction (FGR). The review also describes the research strategies that were used for the identification of the functional role of EMT targets in vitro. A better understanding of molecular pathways driven by placental EMT and further elucidation of signaling pathways underlying the developmental programs may offer novel strategies of targeted therapy for improving feto-placental growth in placental pathologies including PE and FGR.

No abstract

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/ CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.

At present, approximately 150 different members of the adhesion-G protein-coupled receptor (GPCR) family have been identified in metazoans. Surprisingly, very little is known about their function, although they all possess large extracellular domains coupled to a seven-transmembrane domain, suggesting a potential role in cell adhesion and signaling. Here, we demonstrate how the human-restricted adhesion-GPCR, EMR2 (epidermal growth factor-like module-containing mucin-like hormone receptor), regulates neutrophil responses by potentiating the effects of a number of proinflammatory mediators and show that the transmembrane region is critical for adhesion-GPCR function. Using an anti-EMR2 antibody, ligation of EMR2 increases neutrophil adhesion and migration, and augments superoxide production and proteolytic enzyme degranulation. On neutrophil activation, EMR2 is rapidly translocated to membrane ruffles and the leading edge of the cell. Further supporting the role in neutrophil activation, EMR2 expression on circulating neutrophils is significantly increased in patients with systemic inflammation. These data illustrate a definitive function for a human adhesion-GPCR within the innate immune system and suggest an important role in potentiating the inflammatory response. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.

We retrospectively reviewed the surgical treatment of 12 patients (nine female, mean age 16.1+/-8.7 years) with sub-mitral aneurysms managed in our institution between 1991 and 2002. We identified three groups of patients in accordance with the degree of posterior mitral annular involvement by the aneurysm. A single aneurysm neck was found in seven patients, multiple necks in two and involvement of the entire posterior mitral annulus in three patients. Involvement of the entire posterior annulus by the aneurysmal process has not been previously described. The mean age of this latter group 29+/-5.1 years was significantly older than the former (P=0.001), suggesting a possible progressive nature of sub-mitral aneurysms. An intracardiac surgical approach was used in six patients and a combined intra and extracardiac approach in the remainder. There was no operative mortality. The mitral valve was initially repaired in eight patients. Failure of closure of the aneurysm necessitating reoperation occurred in four patients (33.3%). An understanding of the inter-relationship between the aneurysm and mitral valve is essential for successful surgical repair. Histology of the aneurysm tissue showed rheumatic heart disease in two patients and tuberculosis in two patients. Hence, although sub-valvar aneurysms are thought to be congenital, a third of our patients had evidence of co-existent rheumatic heart disease or tuberculosis.