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1.
Nat Immunol ; 13(1): 67-76, 2011 Dec 04.
Article in English | MEDLINE | ID: mdl-22138716

ABSTRACT

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.


Subject(s)
Chemokines/metabolism , Endothelial Cells/metabolism , Lymphocytes/immunology , Transendothelial and Transepithelial Migration/immunology , Transport Vesicles/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Endothelial Cells/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Hyaluronan Receptors/metabolism , Integrins/metabolism , Lymphocytes/metabolism , Lymphocytes/ultrastructure , Mice , Receptors, CCR2/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/ultrastructure , Tumor Necrosis Factor-alpha/pharmacology , Vasculitis/immunology , Vasculitis/metabolism
2.
Biochim Biophys Acta ; 1790(6): 431-44, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19289150

ABSTRACT

The ability of cells to attach to each other and to the extracellular matrix is of pivotal significance for the formation of functional organs and for the distribution of cells in the body. Several molecular families of proteins are involved in adhesion, and recent work has substantially improved our understanding of their structures and functions. Also, these molecules are now being targeted in the fight against disease. However, less is known about how their activity is regulated. It is apparent that among the different classes of adhesion molecules, the integrin family of adhesion receptors is unique in the sense that they constitute a large group of widely distributed receptors, they are unusually complex and most importantly their activities are strictly regulated from the inside of the cell. The activity regulation is achieved by a complex interplay of cytoskeletal proteins, protein kinases, phosphatases, small G proteins and adaptor proteins. Obviously, we are only in the beginning of our understanding of how the integrins function, but already now fascinating details have become apparent. Here, we describe recent progress in the field, concentrating mainly on mechanistical and structural studies of integrin regulation. Due to the large number of articles dealing with integrins, we focus on what we think are the most exciting and rewarding directions of contemporary research on cell adhesion and integrins.


Subject(s)
Cell Adhesion/physiology , Integrins/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Humans , Integrins/chemistry , Integrins/genetics , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment
3.
Blood ; 112(5): 1853-62, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18550856

ABSTRACT

Leukocyte integrins of the beta2 family are essential for immune cell-cell adhesion. In activated cells, beta2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the beta2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of beta2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated beta2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K(d), 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K(d), 0.5 mM). Phosphorylation did not regulate talin binding to beta2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated beta2 integrin. X-ray crystallographic data clearly explained how phosphorylation eliminated filamin binding and induced 14-3-3 protein binding. Filamin knockdown in T cells led to an increase in stimulated cell adhesion to ICAM-1-coated surfaces. Our results suggest that the phosphorylation of beta2 integrins on Thr758 acts as a molecular switch to inhibit filamin binding and allow 14-3-3 protein binding to the integrin cytoplasmic domain, thereby modulating T-cell adhesion.


Subject(s)
14-3-3 Proteins/metabolism , CD18 Antigens/chemistry , CD18 Antigens/metabolism , Contractile Proteins/metabolism , Microfilament Proteins/metabolism , 14-3-3 Proteins/chemistry , Amino Acid Substitution , Binding Sites , CD18 Antigens/genetics , Cell Adhesion , Contractile Proteins/chemistry , Filamins , Humans , In Vitro Techniques , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells , Microfilament Proteins/chemistry , Models, Molecular , Multiprotein Complexes , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , T-Lymphocytes/metabolism , Talin/metabolism , Threonine/chemistry
4.
Blood ; 111(7): 3607-14, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18239087

ABSTRACT

Inside-out signaling regulation of the beta2-integrin leukocyte function-associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b-/- mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b-/- bone marrow-derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b-deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b-/-LFA-1-/- mice. Consistently, LFA-1-mediated adhesion of BMDM to ICAM-1 but not VLA-4-mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the beta2-chain of LFA-1 and thereby in enhanced association of 14-3-3beta protein with the beta2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/beta2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b-/- BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1-mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 beta-chain and 14-3-3 proteins.


Subject(s)
14-3-3 Proteins/immunology , Cell Movement/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Macrophages, Peritoneal/immunology , Oncogene Protein v-cbl/immunology , Signal Transduction/immunology , 14-3-3 Proteins/genetics , Animals , CD18 Antigens/genetics , CD18 Antigens/immunology , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Line, Tumor , Cell Movement/genetics , Endothelium, Vascular/immunology , Humans , Inflammation/genetics , Inflammation/immunology , Integrin alpha4beta1/genetics , Integrin alpha4beta1/immunology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Lymphocyte Function-Associated Antigen-1/genetics , Mice , Mice, Knockout , Oncogene Protein v-cbl/genetics , Signal Transduction/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology
5.
J Biol Chem ; 282(2): 968-75, 2007 Jan 12.
Article in English | MEDLINE | ID: mdl-17107954

ABSTRACT

Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin beta2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion. Here, we have investigated the signaling effects of beta2 integrin Thr-758 phosphorylation. A penetratin-coupled phospho-Thr-758-beta2 peptide (mimicking the part of the integrin beta-chain surrounding Thr-758) stimulated adhesion of human T cells to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Additionally, the peptide activated the small GTPases Rac-1 and Cdc42 in T cells. Constitutively active forms of Rac-1 and Cdc42, but not Rho, could compensate for the reduction of cell adhesion to ICAM-1 caused by the T758A mutation in the beta2 integrin. Additionally, the active GTPases salvaged the cell-spreading defect of T758A integrin-transfected cells on coated ICAM-1. A dominant negative form of Cdc42, on the other hand, significantly reduced wild-type beta2 integrin-mediated cell adhesion and spreading. In a T cell stimulation system, the pThr-758 penetratin peptide acted in a similar manner to coated ICAM-1 to increase T cell receptor-induced CD69 expression. These results show that Thr-758-phosphorylated LFA-1 is upstream of Rac-1/Cdc42, cell adhesion, and costimulatory activation of human T cells, thus identifying phosphorylation of Thr-758 in beta2 as a proximal element in LFA-1 signaling.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , T-Lymphocytes/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Antibodies, Monoclonal/pharmacology , COS Cells , Carrier Proteins/metabolism , Cell Adhesion/immunology , Cell-Penetrating Peptides , Chlorocebus aethiops , Humans , Intercellular Adhesion Molecule-1/metabolism , Lectins, C-Type , Lymphocyte Activation/physiology , Lymphocyte Function-Associated Antigen-1/genetics , Phosphorylation , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology , T-Lymphocytes/cytology , Threonine/metabolism , Transfection , cdc42 GTP-Binding Protein/genetics
6.
Ann Med ; 38(7): 503-11, 2006.
Article in English | MEDLINE | ID: mdl-17101541

ABSTRACT

Inflammation is a crucial response against invading pathogens, in which immune cells, including neutrophils and T cells, are recruited into tissue from the bloodstream to help clear infection. However, a prevailing inflammatory response where the immune cells attack healthy tissue is associated with many diseases, including asthma, rheumatoid arthritis, atherosclerosis and multiple sclerosis. Integrins are key players in the recruitment of immune cells from the bloodstream into tissues, and are thus therapeutic targets for intervention with inflammatory responses. Thus far, mainly extracellularly acting therapeutics (monoclonal antibodies) have been developed against integrins, targeting ligand binding sites in these heterodimeric adhesion receptors. However, since these therapeutics nonselectively block all integrin functions, some side effects are expected and have been observed. Therefore, novel concepts need to be developed in the therapeutic targeting of integrins. Recently, major advances have been made in the understanding of integrin biology. Integrin structures have been solved by X-ray crystallography, revealing unexpected data about the activation mechanism of integrins in cells. Additionally, several intracellular factors in the integrin activation process have been identified, providing potential specific targets for therapeutic intervention. Here, we present key events and players in leukocyte integrin activation, and discuss potential new drug targets in the prevention of inflammatory disease.


Subject(s)
Anti-Inflammatory Agents , Antibodies, Monoclonal , Inflammation/therapy , Integrins/immunology , Leukocytes/immunology , Animals , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Cell Adhesion/immunology , Cell Adhesion Molecules , Cell Movement , Humans , Inflammation/immunology , Integrins/chemistry , Integrins/metabolism , Phosphorylation , Protein Conformation
7.
Ann N Y Acad Sci ; 1090: 318-25, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17384276

ABSTRACT

Engagement of the T cell receptor (TCR) initiates intracellular signaling cascades that result in T cell activation, differentiation, acquisition of effector functions, or apoptosis. The signals from the TCR are coupled to distal signaling pathways by adapter proteins leading to dramatic changes in the cytoskeleton, transcription, and activation of integrins, which mediate adhesion. LFA-1 (leukocyte function-associated antigen-1) integrin (alphaLbeta2 or CD11a/CD18) plays an important role in adhesion, for example, by linking extracellular ligands to the actin cytoskeleton. The intracellular tails of integrins contain several phosphorylation sites, making them candidate-binding partners for 14-3-3 proteins, which are adaptor proteins that bind to phosphorylated ligands. In a screen for 14-3-3 binding partners in T cells, we identified both beta2 integrins and filamin. The integrin beta2 chain binds to 14-3-3 proteins through phosphorylated Thr758 after TCR ligation and this association regulates integrin-mediated cell spreading, which is necessary for adhesion. Here, we show that filamin associates with 14-3-3 proteins in activated T cells. 14-3-3 association with T cell membrane and cytoskeleton proteins after cell stimulation may mediate numerous T cell functions.


Subject(s)
14-3-3 Proteins/metabolism , Contractile Proteins/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Microfilament Proteins/metabolism , T-Lymphocytes/metabolism , 14-3-3 Proteins/chemistry , Amino Acid Sequence , Cells, Cultured , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Filamins , Humans , Immunoprecipitation , Lymphocyte Activation , Molecular Sequence Data , Phorbol 12,13-Dibutyrate/pharmacology , Protein Binding , T-Lymphocytes/drug effects
8.
J Cell Biol ; 171(4): 705-15, 2005 Nov 21.
Article in English | MEDLINE | ID: mdl-16301335

ABSTRACT

Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. alpha Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the beta chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.


Subject(s)
Lymphocyte Function-Associated Antigen-1/physiology , 14-3-3 Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, CD/metabolism , COS Cells , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Line , Cell Line, Tumor , Chemokines/metabolism , Chlorocebus aethiops , Chromatography, Affinity , Cytoplasm/metabolism , Cytoskeleton/metabolism , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Humans , Immunoprecipitation , Integrins/metabolism , Intercellular Adhesion Molecule-1/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Proteins/metabolism , Recombinant Proteins/chemistry , Serine/chemistry , Talin/chemistry , Time Factors , Transfection
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