Timothy A. Springer

Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins

Rolling of leukocytes on vascular endothelial cells, an early event in inflammation, can be reproduced in vitro on artificial lipid bilayers containing purified CD62, a selectin also named PADGEM and GMP-140 that is inducible on endothelial cells. Neutrophils roll on this selectin under flow conditions similar to those found in postcapillary venules. Adhesion of resting or activated neutrophils through the integrins LFA-1 and Mac-1 to ICAM-1 in a lipid bilayer does not occur at physiologic shear stresses; however, static incubation of activated neutrophils allows development of adhesion that is greater than 100-fold more shear resistant than found on CD62. Addition of a chemoattractant to activate LFA-1 and Mac-1 results in the arrest of neutrophils rolling on bilayers containing both CD62 and ICAM-1. Thus, at physiologic shear stress, rolling on a selectin is a prerequisite for activation-induced adhesion strengthening through integrins.

Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1).

Lymphocyte function-associated antigen 1 (LFA-1) is a leukocyte cell surface glycoprotein that promotes intercellular adhesion in immunological and inflammatory reactions. It is an alpha beta complex that is structurally related to receptors for extracellular matrix components, and thus belongs to the integrin family. ICAM-1 (intercellular adhesion molecule-1) is a distinct cell surface glycoprotein. Its broad distribution, regulated expression in inflammation, and involvement in LFA-1-dependent cell-cell adhesion have suggested that ICAM-1 may be a ligand for LFA-1. We have purified ICAM-1 and incorporated it into artificial supported lipid membranes. LFA-1+ but not LFA-1- cells bound to ICAM-1 in the artificial membranes, and the binding could be specifically inhibited by anti-ICAM-1 treatment of the membranes or by anti-LFA-1 treatment of the cells. The cell binding to ICAM-1 required metabolic energy production, an intact cytoskeleton, and the presence of Mg2+ and was temperature dependent, characteristics of LFA-1- and ICAM-1-dependent cell-cell adhesion.

Global Conformational Rearrangements in Integrin Extracellular Domains in Outside-In and Inside-Out Signaling

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families

Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence.

THE CHEMOKINE RECEPTOR CXCR4 IS REQUIRED FOR THE RETENTION OF B LINEAGE AND GRANULOCYTIC PRECURSORS WITHIN THE BONE MARROW MICROENVIRONMENT

We report that the chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within fetal liver and bone marrow microenvironment. In CXCR4-deficient embryos, pro-B cells are present in blood but hardly detectable in liver; myeloid cells are elevated in blood and reduced in liver compared to wild-type embryos. Mice reconstituted with CXCR4-deficient fetal liver cells have reduced donor-derived mature B lymphocytes in blood and lymphoid organs. The numbers of pro-B and pre-B cells are reduced in bone marrow and abnormally high in blood. Granulocytic cells are reduced in bone marrow but elevated and less mature in the blood. B lineage and granulocytic precursors are released into the periphery in absence of CXCR4.

Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation

Both the integrins LFA-1 and Mac-1 bind to ICAM-1, an immunoglobulin superfamily member. Previously, we localized the binding sites of LFA-1 and the major group of human rhinoviruses to the first NH2-terminal immunoglobulin-like domain of ICAM-1. Here, we show that the binding site on ICAM-1 for Mac-1 is unexpectedly distinct from that for LFA-1 and maps to the third NH2-terminal immunoglobulin-like domain. These findings provide a function for the tandem duplication of immunoglobulin-like domains in ICAM-1 and have implications for other immunoglobulin superfamily members. Mutations at two sites in the third domain that destroy consensus sequences for N-linked glycosylation enhance binding to purified Mac-1. Agents that interfere with carbohydrate processing provide evidence that the size of the N-linked oligosaccharide side chains on ICAM-1 affects binding to Mac-1 but not to LFA-1. Thus, we suggest that the extent of glycosylation on ICAM-1 may regulate adhesion to LFA-1 or Mac-1 in vivo.

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses

Rhinoviruses, which cause common colds, possess over 100 serotypes, 90% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.

Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury.

Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics.

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin α and β subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin αIIbβ3. Allostery in the β3 I domain alters three metal binding sites, associated loops and α1- and α7-helices. Piston-like displacement of the α7-helix causes a 62° reorientation between the β3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper β3 leg causes a 70 Å separation between the knees of the α and β legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.

The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus

Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1.