ABSTRACT
BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. OBJECTIVE: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies. METHODS: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. RESULTS: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, ß-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.
Subject(s)
B-Lymphocyte Subsets/metabolism , Common Variable Immunodeficiency/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Multiple Sclerosis/metabolism , Receptors, Lysosphingolipid/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Agammaglobulinaemia Tyrosine Kinase , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/immunology , Antigens, Differentiation, T-Lymphocyte/metabolism , Arrestins/genetics , Arrestins/immunology , Arrestins/metabolism , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Cell Line , Cell Movement , Common Variable Immunodeficiency/genetics , Common Variable Immunodeficiency/immunology , Common Variable Immunodeficiency/pathology , Gene Expression Regulation , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/immunology , Guanine Nucleotide Exchange Factors/metabolism , Humans , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Lectins, C-Type/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Primary Cell Culture , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/immunology , Signal Transduction , Time-Lapse Imaging , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/immunology , Wiskott-Aldrich Syndrome Protein/metabolism , beta-Arrestin 2 , beta-ArrestinsABSTRACT
The authors report on a fatal case of severe tetanus in a 74-year old woman. Despite comprehensive intensive care management they could not achieve a satisfying control of the autonomic dysfunction caused by tetanus. By now there is no established therapy for the treatment of the autonomic dysfunction. This report demonstrates the dismal prognosis of severe tetanus in the older people, which is often complicated by cardiovascular comorbidity and underlines the importance of tetanus prevention by sufficient vaccination.
Subject(s)
Tetanus , Aged , Fatal Outcome , Female , Humans , Skin/injuries , Tetanus/diagnosisABSTRACT
OBJECTIVE: Integrins are attractive therapeutic targets. Inhibition of integrin alphaIIb beta3 effectively blocks platelet aggregation. However, limitations with intravenous alphaIIb beta3 antagonists and failure of oral alphaIIb beta3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. METHODS AND RESULTS: Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic alphaIIb beta3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of alphaIIb beta3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic alphaIIb beta3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic alphaIIb beta3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits alphaIIb beta3 antagonist-induced platelet activation. CONCLUSION: These findings can explain limitations of ligand-mimetic anti-alphaIIb beta3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.
Subject(s)
Models, Biological , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Cell Adhesion/drug effects , Fluorescent Antibody Technique , Humans , Ligands , Platelet Activation/physiology , Sensitivity and Specificity , Signal TransductionABSTRACT
Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS-145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/IIIa blockers.