Red-cell ICAM-4 is a ligand for the monocyte/macrophage integrinCD11c/CD18 : characterization of the binding sites on ICAM-4

Intercellular adhesion molecule-4 (ICAM-4) is a unique member of the ICAM family due to its specific expression on erythroid cells and ability to interact with several types of integrins expressed on blood and endothelial cells. The first reported receptors for ICAM-4 were CD11a/CD18 and CD11b/CD18. In contrast to these two, the cellular ligands and the functional role of the third beta2-integrin, CD11c/CD18, have not been well defined. Here we show that ICAM-4 functions as a ligand for the monocyte/macrophage specific CD11c/CD18. Deletion of the individual immunoglobulin domains of ICAM-4 demonstrated that both its domains contain binding sites for CD11c/CD18. Analysis of a panel of ICAM-4 point mutants identified residues that affected binding to the integrin. By molecular modeling the important residues were predicted to cluster in two distinct but spatially close regions of the first domain with an extension to the second domain spatially distant from the other residues. We also identified two peptides derived from sequences of ICAM-4 that are capable of modulating the binding to CD11c/CD18. CD11c/CD18 is expressed on macrophages in spleen and bone marrow. Inhibition of erythrophagocytosis by anti-ICAM-4 and anti-integrin antibodies suggests a role for these interactions in removal of senescent red cells

Claudin 13, a Member of the Claudin Family Regulated in Mouse Stress Induced Erythropoiesis

Mammals are able to rapidly produce red blood cells in response to stress. The molecular pathways used in this process are important in understanding responses to anaemia in multiple biological settings. Here we characterise the novel gene Claudin 13 (Cldn13), a member of the Claudin family of tight junction proteins using RNA expression, microarray and phylogenetic analysis. We present evidence that Cldn13 appears to be co-ordinately regulated as part of a stress induced erythropoiesis pathway and is a mouse-specific gene mainly expressed in tissues associated with haematopoietic function. CLDN13 phylogenetically groups with its genomic neighbour CLDN4, a conserved tight junction protein with a putative role in epithelial to mesenchymal transition, suggesting a recent duplication event. Mechanisms of mammalian stress erythropoiesis are of importance in anaemic responses and expression microarray analyses demonstrate that Cldn13 is the most abundant Claudin in spleen from mice infected with Trypanosoma congolense. In mice prone to anaemia (C57BL/6), its expression is reduced compared to strains which display a less severe anaemic response (A/J and BALB/c) and is differentially regulated in spleen during disease progression. Genes clustering with Cldn13 on microarrays are key regulators of erythropoiesis (Tal1, Trim10, E2f2), erythrocyte membrane proteins (Rhd and Gypa), associated with red cell volume (Tmcc2) and indirectly associated with erythropoietic pathways (Cdca8, Cdkn2d, Cenpk). Relationships between genes appearing co-ordinately regulated with Cldn13 post-infection suggest new insights into the molecular regulation and pathways involved in stress induced erythropoiesis and suggest a novel, previously unreported role for claudins in correct cell polarisation and protein partitioning prior to erythroblast enucleation

FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

<div><p>The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (K_d) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the K_d between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the K_d determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine K_d as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.</p></div

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin

High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the β2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-κB in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here