ABSTRACT
Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases.
Subject(s)
Chemotaxis, Leukocyte , Immunologic Tests/methods , Microfluidic Analytical Techniques/methods , Cell Polarity , Cell Tracking/instrumentation , Cell Tracking/methods , Chemotaxis, Leukocyte/drug effects , Equipment Design , HL-60 Cells , Humans , Immunologic Deficiency Syndromes/diagnosis , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Immunologic Tests/instrumentation , In Vitro Techniques , Infant , Microfluidic Analytical Techniques/instrumentation , Models, Biological , Mutation , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/physiology , rac GTP-Binding Proteins/genetics , RAC2 GTP-Binding ProteinABSTRACT
CXCR4 is a G protein-coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.
Subject(s)
Neutropenia/genetics , Neutropenia/pathology , Neutrophils/physiology , Agammaglobulinemia/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cell Movement , Chemokine CXCL12/genetics , Chemotaxis, Leukocyte , Disease Models, Animal , Gene Expression , Hematopoiesis , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/pathology , Molecular Sequence Data , Mutation , Neutrophils/pathology , Receptors, CXCR4/genetics , Signal Transduction , Syndrome , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-alpha-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-alpha-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-alpha-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-alpha-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-alpha stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-alpha-induced inflammatory responses.
