ABSTRACT
Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMß2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.
Subject(s)
Phagocytosis/immunology , Phagocytosis/physiology , Actin Cytoskeleton/metabolism , Animals , Biophysical Phenomena , Cell Movement/immunology , Cell Movement/physiology , Cell Surface Extensions/immunology , Cell Surface Extensions/physiology , Humans , Ligands , Macrophage-1 Antigen/chemistry , Macrophage-1 Antigen/immunology , Macrophage-1 Antigen/physiology , Models, Immunological , Myosin Type II/immunology , Myosin Type II/physiology , Phagosomes/immunology , Phagosomes/physiology , Protein Conformation , Pseudopodia/immunology , Pseudopodia/physiology , Receptors, IgG/chemistry , Receptors, IgG/immunology , Receptors, IgG/physiologyABSTRACT
Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.
Subject(s)
Bacteria/metabolism , CX3C Chemokine Receptor 1/metabolism , Cell Surface Extensions/metabolism , Intestine, Small/cytology , Intestine, Small/microbiology , Lactic Acid/metabolism , Pyruvic Acid/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Bacteria/immunology , CX3C Chemokine Receptor 1/deficiency , CX3C Chemokine Receptor 1/genetics , Cell Surface Extensions/drug effects , Cell Surface Extensions/immunology , Female , HEK293 Cells , Humans , Intestine, Small/drug effects , Intestine, Small/immunology , Lactic Acid/pharmacology , Lactobacillus helveticus/metabolism , Male , Methanol , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Pyruvic Acid/pharmacology , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Salmonella/immunology , Salmonella/metabolismABSTRACT
Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Communication/immunology , Cell Surface Extensions/immunology , Fibroblast Growth Factor 2/immunology , Myocytes, Smooth Muscle/immunology , CD4-Positive T-Lymphocytes/cytology , Humans , Mitochondria/immunology , Myocytes, Smooth Muscle/cytology , Receptor, Fibroblast Growth Factor, Type 1/immunology , Respiratory System/cytology , Respiratory System/immunologyABSTRACT
Mast cells (MC) and myeloid dendritic cells (DC) act proximally in detecting and processing antigens and immune insults. We sought to understand their comparative dynamic behavior with respect to the airway epithelium in the steady state and in response to an allergic stimulus in mouse trachea. We devised methods to label MC in living trachea and to demonstrate that MC and DC occupy distinct layers of the tracheal mucosa, with DC being closer to the lumen. DC numbers doubled after allergen challenge, but MC numbers remained stable. MC and DC migrated minimally in either steady state or allergen-challenge conditions, and their interactions with one another appeared to be stochastic and relatively infrequent. While DC, unlike MC, exhibited probing behaviors involving dendrites, these projections did not cross the epithelium into the airway lumen. MC typically were located too far from the epithelial surface to contact the tracheal lumen. However, MC had protrusions toward and into blood vessels, likely to load with IgE. Thus, DC and MC occupy distinct niches and engage in sessile surveillance in the mouse trachea. Little or no access of these cell types to the airway lumen suggests that trans-epithelial transport of proteins in the steady state would be required for them to access luminal antigens.
Subject(s)
Allergens/immunology , Blood Vessels/immunology , Blood Vessels/pathology , Cell Surface Extensions/immunology , Mast Cells/cytology , Mast Cells/immunology , Trachea/immunology , Animals , Cell Movement , Dendritic Cells/immunology , Imaging, Three-Dimensional , Immunoglobulin E/immunology , Mice, Inbred C57BL , Ovalbumin/immunology , Reproducibility of Results , Staining and LabelingABSTRACT
Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.
Subject(s)
Cell Communication , Cell Surface Extensions/metabolism , Hodgkin Disease/metabolism , Ki-1 Antigen/metabolism , Reed-Sternberg Cells/metabolism , Secretory Vesicles/metabolism , Signal Transduction , Tumor Microenvironment , Biomarkers, Tumor/metabolism , CD30 Ligand/metabolism , Cell Line, Tumor , Cell Surface Extensions/immunology , Cell Surface Extensions/ultrastructure , Cryoelectron Microscopy , Eosinophils/immunology , Eosinophils/metabolism , Flow Cytometry , Granulocytes/immunology , Granulocytes/metabolism , Hodgkin Disease/immunology , Hodgkin Disease/pathology , Humans , Interleukin-8/metabolism , Mass Spectrometry , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , Organelle Size , Reed-Sternberg Cells/immunology , Reed-Sternberg Cells/ultrastructure , Secretory Vesicles/immunology , Secretory Vesicles/ultrastructureABSTRACT
Communication between neurons and glia in the dorsal root ganglia (DRG) and the central nervous system is critical for nociception. Both glial activation and proinflammatory cytokine induction underlie this communication. We investigated whether satellite glial cell (SGC) and tumor necrosis factor-α (TNF-α) activation in DRG participates in a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat model of visceral hyperalgesia. In TNBS-treated rats, TNF-α expression increased in DRG and was colocalized to SGCs enveloping a given neuron. These SGCs were activated as visualized under electron microscopy: they had more elongated processes projecting into the connective tissue space and more gap junctions. When nerves attached to DRG (L6-S1) were stimulated with a series of electrical stimulations, TNF-α were released from DRG in TNBS-treated animals compared with controls. Using a current clamp, we noted that exogenous TNF-α (2.5 ng/ml) increased DRG neuron activity, and visceral pain behavioral responses were reversed by intrathecal administration of anti-TNF-α (10 µg·kg(-1)·day(-1)). Based on our findings, TNF-α and SGC activation in neuron-glial communication are critical in inflammatory visceral hyperalgesia.
Subject(s)
Cell Communication , Colitis/metabolism , Colon/innervation , Ganglia, Spinal/metabolism , Hyperalgesia/metabolism , Inflammation Mediators/metabolism , Neuroglia/metabolism , Neurons/metabolism , Pain Threshold , Tumor Necrosis Factor-alpha/metabolism , Animals , Animals, Newborn , Antibodies, Monoclonal/administration & dosage , Behavior, Animal , Cell Communication/drug effects , Cell Surface Extensions/immunology , Cell Surface Extensions/metabolism , Cells, Cultured , Colitis/chemically induced , Colitis/immunology , Colitis/physiopathology , Disease Models, Animal , Electric Stimulation , Ganglia, Spinal/drug effects , Ganglia, Spinal/immunology , Ganglia, Spinal/physiopathology , Gap Junctions/immunology , Gap Junctions/metabolism , Hyperalgesia/chemically induced , Hyperalgesia/immunology , Hyperalgesia/physiopathology , Hyperalgesia/prevention & control , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/immunology , Male , Neuroglia/immunology , Neurons/drug effects , Neurons/immunology , Pain Threshold/drug effects , Pressure , Rats , Rats, Sprague-Dawley , Trinitrobenzenesulfonic Acid , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/immunology , Up-RegulationABSTRACT
Persistent inflammation is a well-known determinant of progressive tissue fibrosis; however, the mechanisms underlying this process remain unclear. There is growing evidence indicating a role of the cytokine IL-1ß in profibrotic responses. We previously demonstrated that fibroblasts stimulated with IL-1ß increased their generation of the polysaccharide hyaluronan (HA) and increased their expression of the HA synthase enzyme (HAS-2). The aim of this study was to determine the significance of IL-1ß-induced changes in HA and HAS-2 generation. In this study, we found that stimulation of fibroblasts with IL-1ß results in the relocalization of HA associated with the cell to the outer cell membrane, where it forms HAS2- and CD44-dependent cell membrane protrusions. CD44 is concentrated within the membrane protrusions, where it co-localizes with the intracellular adhesion molecule 1. Furthermore, we have identified that these cell protrusions enhance IL-1ß-dependent fibroblast-monocyte binding through MAPK/ERK signaling. Although previous data have indicated the importance of the HA-binding protein TSG-6 in maintaining the transforming growth factor ß1-dependent HA coat, TSG-6 was not essential for the formation of the IL-1ß-dependent HA protrusions, thus identifying it as a key difference between IL-1ß- and transforming growth factor ß1-dependent HA matrices. In summary, these data suggest that IL-1ß-dependent HA generation plays a role in fibroblast immune activation, leading to sequestration of monocytes within inflamed tissue and providing a possible mechanism for perpetual inflammation.
Subject(s)
Cell Surface Extensions/immunology , Fibroblasts/immunology , Hyaluronan Receptors/immunology , Hyaluronic Acid/biosynthesis , Interleukin-1beta/immunology , Monocytes/immunology , Cell Adhesion/immunology , Cell Adhesion Molecules/immunology , Cell Differentiation/immunology , Cell Membrane/immunology , Cells, Cultured , Fibroblasts/physiology , Glucuronosyltransferase/immunology , Humans , Hyaluronan Synthases , Intercellular Adhesion Molecule-1/metabolism , MAP Kinase Signaling System/immunology , Monocytes/physiology , Myofibroblasts/immunology , Transforming Growth Factor beta1/immunologyABSTRACT
Candida albicans strains that are homozygous at the mating type locus can spontaneously and reversibly switch from the normal yeast morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. White-opaque switching also influences the ability of C. albicans to colonize and proliferate in specific host niches and its susceptibility to host defense mechanisms. We used live imaging to observe the interaction of white and opaque cells with host phagocytic cells. For this purpose, we generated derivatives of the switching-competent strain WO-1 that express green fluorescent protein from a white-specific promoter and red fluorescent protein from an opaque-specific promoter or vice versa. When mixed populations of these differentially labeled white and opaque cells were incubated with human polymorphonuclear neutrophils (PMNs) on a glass slide, the neutrophils selectively phagocytosed and killed white cells, despite frequent physical interaction with opaque cells. White cells were attacked only after they started to form a germ tube, indicating that the suppression of filamentation in opaque cells saved them from recognition by the PMNs. In contrast to neutrophils, dendritic cells internalized white as well as opaque cells. However, when embedded in a collagen matrix, the PMNs also phagocytosed both white and opaque cells with similar efficiency. These results suggest that, depending on the environment, white-opaque switching enables C. albicans to escape from specific host defense mechanisms.
Subject(s)
Candida albicans/physiology , Candida albicans/growth & development , Candida albicans/immunology , Cell Surface Extensions/immunology , Cells, Cultured , Collagen/physiology , Dendritic Cells/immunology , Dendritic Cells/microbiology , Gene Expression Regulation, Fungal , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Host-Pathogen Interactions , Humans , Immune Evasion , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Microscopy, Fluorescence , Microscopy, Video , Neutrophils/immunology , Neutrophils/microbiology , Phagocytosis , Red Fluorescent ProteinABSTRACT
Francisella spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. Knowledge is lacking for the virulence factors expressed by Francisella and how these factors are secreted and delivered to host cells. Gram-negative bacteria constitutively release outer membrane vesicles (OMV), which may function in the delivery of virulence factors to host cells. We identified growth conditions under which Francisella novicida produces abundant OMV. Purification of the vesicles revealed the presence of tube-shaped vesicles in addition to typical spherical OMV, and examination of whole bacteria revealed the presence of tubes extending out from the bacterial surface. Recently, both prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes, which appear to function in cell-cell communication and the exchange of molecules. In contrast to these previously characterized structures, the F. novicida tubes are produced in liquid as well as on solid medium and are derived from the OM rather than the cytoplasmic membrane. The production of the OMV and tubes (OMV/T) by F. novicida was coordinately regulated and responsive to both growth medium and growth phase. Proteomic analysis of purified OMV/T identified known Francisella virulence factors among the constituent proteins, suggesting roles for the vesicles in pathogenesis. In support of this, production of OM tubes by F. novicida was stimulated during infection of macrophages and addition of purified OMV/T to macrophages elicited increased release of proinflammatory cytokines. Finally, vaccination with purified OMV/T protected mice from subsequent challenge with highly lethal doses of F. novicida.
Subject(s)
Cell Surface Extensions/metabolism , Francisella/metabolism , Francisella/ultrastructure , Transport Vesicles/metabolism , Transport Vesicles/ultrastructure , Animals , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines/immunology , Cell Communication , Cell Surface Extensions/immunology , Cell Surface Extensions/ultrastructure , Culture Media , Cytokines/biosynthesis , Francisella/immunology , Francisella/pathogenicity , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/prevention & control , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Proteomics , Transport Vesicles/immunology , Vaccination , Virulence Factors/metabolismABSTRACT
Carefully orchestrated intercellular communication is an essential prerequisite for an effective immune response. In recent years tunneling nanotubes (TNT) have emerged as a novel mechanism of cell-cell communication. These long membrane protrusions can establish cytoplasmic continuity between distant cells and enable the exchange of cellular components. In the present study we addressed the question whether these structures can facilitate the intercellular transfer of MHC class I molecules. We found a transmembrane HLA-A2-EGFP but not a soluble HLA-G1s-EGFP fusion protein to be effectively transferred between HeLa cells. Inhibition of actin polymerization significantly reduced the HLA-A2 transfer rate, indicating that transfer is dependent on tunneling nanotubes, whose de novo formation requires actin polymerization. Furthermore, overexpression of the nanotube-inducing protein LST1 promoted transfer of HLA-A2. Moreover, LST1 protein expression is enhanced in antigen presenting cells. Our results indicate that tunneling nanotubes can mediate transfer of MHC class I molecules between distant cells.
Subject(s)
Cell Communication/immunology , Cell Surface Extensions/metabolism , Dendritic Cells/metabolism , HLA-A2 Antigen/metabolism , Actins/antagonists & inhibitors , Actins/metabolism , Antibodies, Monoclonal/pharmacology , Cell Communication/drug effects , Cell Surface Extensions/drug effects , Cell Surface Extensions/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/drug effects , Gene Expression/drug effects , Genes, Reporter , Green Fluorescent Proteins , HLA-A2 Antigen/genetics , HLA-G Antigens/genetics , HLA-G Antigens/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Polymerization/drug effects , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tubulin/metabolismABSTRACT
Myeloid leukocytes form actin-based plasma membrane protrusions, called podosomes, that are implicated in myeloid cell recruitment into tissues and cell migration within the interstitium. In this study, we show that tyrosine kinases of the Abl family are present in podosomes formed by murine and human macrophages. Silencing of Abl expression in bone marrow-derived macrophages and monocyte-derived macrophages by siRNA or Abl enzymatic inhibition with imatinib resulted in the disassembly of macrophage podosomes and the reduction of their capacity to degrade an extracellular matrix and migrate through matrigel matrices and endothelial cell monolayers. Additionally, macrophages deficient in Src-family kinases, which cross-talk with Abl in regulating macrophage migration, also demonstrated podosome disassembly. These findings suggest that podosome disassembly induced by Abl targeting may inhibit podosome-dependent functions such as leukocyte recruitment into inflammatory sites and osteoclast-dependent bone resorption.
Subject(s)
Cell Surface Extensions/immunology , Macrophages/immunology , Oncogene Proteins v-abl/metabolism , Protein-Tyrosine Kinases/metabolism , Actins/metabolism , Animals , Benzamides , Cell Movement/drug effects , Cell Surface Extensions/drug effects , Cells, Cultured , Humans , Imatinib Mesylate , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Oncogene Proteins v-abl/genetics , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering/geneticsABSTRACT
Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.
Subject(s)
Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Surface Extensions/immunology , Endothelial Cells/immunology , Adaptive Immunity , Animals , Antigen Presentation , Antigen-Presenting Cells/cytology , Antigens, Bacterial/immunology , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , CHO Cells , Calcium/immunology , Calcium/metabolism , Calcium Signaling , Cell Communication/immunology , Cell Surface Extensions/ultrastructure , Cricetinae , Endothelial Cells/cytology , Genes, MHC Class II , Humans , Immunologic Memory , Lymphocyte Activation , Receptors, Antigen, T-Cell/biosynthesis , Receptors, Antigen, T-Cell/immunology , Transendothelial and Transepithelial Migration , TransfectionABSTRACT
Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.
Subject(s)
Cell Differentiation/immunology , Cell Movement/immunology , Dendritic Cells/immunology , Microfilament Proteins/physiology , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Movement/genetics , Cell Surface Extensions/immunology , Cell Surface Extensions/pathology , Cell Surface Extensions/ultrastructure , Dendritic Cells/metabolism , Dendritic Cells/ultrastructure , Female , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymph Nodes/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Receptors, OdorantABSTRACT
A critical step of the adaptive response is the detection of foreign peptides on antigen presenting cells by T lymphocytes. It is a major challenge for a T lymphocyte to detect the presence of a few tens of cognate ligands or less on the membrane of a cell exposing millions of protein molecules. Detection is followed by the cell decision to undergo full or partial activation or even to start an inhibitory program. While the measurement of cell proliferation or cytokine synthesis is accepted as a reliable means of monitoring T lymphocyte activation, this requires hours or days to complete, which is a significant drawback to relate decision to particular signaling events or to assess lymphocyte reactivity in patients. Here we show that the contact area formed between T lymphocytes and potentially activating surfaces is exquisitely correlated to the proliferative response measured with the standard CFSE technique. Correlation is even better than the Erk activation that was reported as a digital reporter of cell activation. The simple and accurate method of assessing lymphocyte-to-surface contact extension that we describe might be very useful both to monitor lymphocyte reactivity for clinical purposes and to identify early steps of lymphocyte activation.
Subject(s)
Antibodies, Monoclonal/metabolism , Cell Surface Extensions/pathology , Focal Adhesions/pathology , Lymphocyte Activation , T-Lymphocytes/metabolism , Antibodies, Monoclonal/immunology , CD3 Complex/immunology , Cell Proliferation , Cell Separation , Cell Surface Extensions/immunology , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Flow Cytometry , Fluoresceins/metabolism , Focal Adhesions/immunology , Humans , Microscopy, Interference , Signal Transduction/immunology , Succinimides/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
In mast cells, crosslinking the high-affinity IgE receptor (FcÉRI) results in a dynamic reorganization of the actin cytoskeleton that is associated with membrane ruffling. Although the signaling involved in degranulation has been well described, it is less understood in morphological changes. In this study, we investigated the specific role of conventional protein kinase C (cPKC), a crucial signal for degranulation, in antigen-induced membrane ruffling of mast cells. In RBL-2H3 mast cells, antigen induced a long-lasting membrane ruffling, which was blocked with late-added Gö6976, a specific cPKC inhibitor, indicating that sustained activation of cPKC is required for maintaining the reaction. Immunofluorescence staining of endogenous PKCα/ß and real-time imaging of transfected green fluorescent protein-tagged PKCα/ß demonstrated that in response to antigen both PKCα and PKCßI quickly translocated to the plasma membrane and were colocalized with actin filaments at the ruffling sites. These reactions were blocked by expression of kinase-negative PKCßI, but not kinase-negative PKCα, and by treatment with a specific PKCß inhibitor, LY333531. The adhesion, spreading and membrane ruffling of mouse bone marrow-derived mast cells (BMMCs), which are mostly nonadhesive, were promoted by both antigen and thymeleatoxin. Treatment with Gö6976 abolished all these reactions. Antigen-mediated migration of BMMC was also sensitive to Gö6076 and LY333531. In addition, BMMC adhesion by and migration toward stem cell factor were shown to be dependent on cPKC. Thus, cPKC, at least PKCß subtype, may be critical for the dynamic morphological changes that lead to the migration of mast cells.
Subject(s)
Mast Cells/cytology , Mast Cells/enzymology , Protein Kinase C/metabolism , Actins/metabolism , Animals , Antigens/immunology , Antigens/metabolism , Cell Adhesion/physiology , Cell Degranulation/immunology , Cell Line , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Movement/immunology , Cell Surface Extensions/immunology , Cell Surface Extensions/metabolism , Fibronectins/physiology , Mast Cells/immunology , Mice , Protein Kinase C/immunology , Protein Transport/immunology , Rabbits , Receptors, IgE/immunology , Receptors, IgE/metabolism , Signal Transduction/immunology , Stem Cell Factor/immunology , Stem Cell Factor/metabolismABSTRACT
T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.
Subject(s)
Cell Surface Extensions/metabolism , Immunological Synapses/virology , Nucleocapsid/physiology , T-Lymphocytes/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/virology , Cell Polarity/drug effects , Cell Polarity/immunology , Cell Surface Extensions/immunology , Cells, Cultured , Fluorescent Antibody Technique , HIV-1/metabolism , HIV-1/physiology , Humans , Immunological Synapses/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Models, Biological , Myosin-Light-Chain Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/physiology , T-Lymphocytes/virology , Tissue Distribution , gag Gene Products, Human Immunodeficiency Virus/chemistry , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The Fas/CD95 surface receptor mediates rapid death of various cell types, including autoreactive T cells with the potential for triggering autoimmunity. Here, we present novel aspects of Fas signalling that define a 'social' dimension to receptor-induced apoptosis. Fas stimulation rapidly induces extensive membrane nanotube formation between neighbouring T cells. This is critically dependent on Rho GTPases but not on caspase activation. Bidirectional transfer of membrane and cytosolic elements including active caspases can be observed to occur via these nanotubes. Nanotube formation and intercellular exchanges of death signals are defective in T lymphocytes from patients with autoimmune lymphoproliferative syndrome harbouring mutations in the Fas receptor. We conclude that nanotube-mediated exchanges constitute a novel form of intercellular communication that augments the propagation of death signalling between neighbouring T cells.
Subject(s)
Apoptosis/physiology , Cell Communication/physiology , Cell Surface Extensions/immunology , Cell Surface Extensions/ultrastructure , Nanotubes, Peptide/ultrastructure , Signal Transduction/physiology , T-Lymphocytes/ultrastructure , fas Receptor/metabolism , Autoimmune Lymphoproliferative Syndrome/immunology , Autoimmune Lymphoproliferative Syndrome/pathology , Autoimmune Lymphoproliferative Syndrome/physiopathology , Caspases/metabolism , Cell Line , Cells, Cultured , Extracellular Space/metabolism , Fluorescent Antibody Technique , Humans , Jurkat Cells , Microscopy, Electron, Transmission , Protein Transport/physiology , T-Lymphocytes/immunology , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of antinuclear autoantibodies. Increased apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE, but the underlying mechanisms remain elusive. During apoptosis apoptotic blebs are formed in which autoantigens are clustered. The cellular remnants after blebbing are referred to as apoptotic cell bodies. We undertook this study to compare the effects of apoptotic blebs and apoptotic cell bodies on maturation of dendritic cells (DCs) and their T cell stimulatory capacity in a murine setting. METHODS: The uptake by DCs of apoptotic blebs and apoptotic cell bodies was analyzed by flow cytometry and confocal microscopy. DC maturation and DC-induced T cell activation were determined by measuring expression of costimulatory molecules using flow cytometry and by measuring production of cytokines using enzyme-linked immunosorbent assay. RESULTS: DCs internalized apoptotic blebs more efficiently than apoptotic cell bodies. Incubation of DCs with apoptotic blebs resulted in increased CD40 and CD86 expression and increased interleukin-6 (IL-6) and tumor necrosis factor alpha production, while apoptotic cell bodies had no stimulatory effects. Using chloroquine, apoptotic bleb-induced DC maturation was shown to be independent of Toll-like receptors 3, 7, and 9. Interestingly, in cocultures with allogeneic T cells, bleb-matured DCs induced production of IL-2, interferon-gamma, and, in particular, IL-17, suggesting a Th1/Th17 response. CONCLUSION: Apoptotic blebs, in contrast to apoptotic cell bodies, induce DC maturation, thereby providing DCs with increased Th17 cell stimulatory capacity. These data imply that apoptotic bleb-induced DC maturation represents an important driving force in the autoimmune response in SLE.
Subject(s)
Apoptosis/immunology , Cell Surface Extensions/immunology , Dendritic Cells/immunology , Interleukin-17/immunology , T-Lymphocytes/immunology , 4-Nitroquinoline-1-oxide/toxicity , Animals , Apoptosis/drug effects , Autoantigens/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Differentiation/immunology , Cell Surface Extensions/metabolism , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dendritic Cells/pathology , Disease Models, Animal , Flow Cytometry , Humans , Interleukin-17/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Microscopy, Confocal , Phagocytosis , Quinolones/toxicity , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
Green tea polyphenols have been reported to have anti-inflammatory activities, although the molecular mechanisms responsible for this effect remain unclear. In the present study, we examined the effect of green tea extract and a variety of polyphenolic compounds on spreading of peripheral blood polymorphonuclear leukocytes (PMNs) over fibrinogen-coated surfaces. Green tea extract exerted a biphasic effect on PMN spreading; it induced or suppressed spreading at low and high concentrations, respectively. We also found that pyrogallol-bearing compounds have spreading induction activity. Among the compounds tested, tannic acid (TA) had the strongest activity; the concentrations required for induction of maximal spreading were 2 microM for TA, 200 microM for (-)-epigallocatechin gallate, and 2000 microM for the other active compounds. Furthermore, TA was the only compound showing a biphasic effect similar to that of green tea extract; TA at 20 or 200 microM suppressed spreading. The spreading-stimulatory signal was still latent during PMN exposure to TA at concentrations that inhibited spreading, because the pre-exposed PMNs underwent spreading when plated after removal of free TA by centrifugation. The spreading-inhibitory effect of TA at high concentrations overcame the induction of spreading by other stimuli, including phorbol 12-myristate 13-acetate, hydrogen peroxide, denatured fibrinogen surfaces, and naked plastic surfaces. These results suggest that TA as well as green tea extract is bi-functional, having pro-inflammatory and anti-inflammatory effects at low and high concentrations, respectively. Pharmacological use of TA may thus provide new strategies aimed at regulation of PMN spreading for control of inflammation.
Subject(s)
Camellia sinensis , Neutrophils/drug effects , Plant Extracts/pharmacology , Animals , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Surface Extensions/drug effects , Cell Surface Extensions/immunology , Cells, Cultured , Chemotaxis/drug effects , Chemotaxis/immunology , Fibrinogen/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/pathology , Pyrogallol/pharmacology , Swine , Tannins/pharmacologyABSTRACT
The peritoneal MPhi in the immunological defense removes foreign particles, pathogenic or otherwise, by phagocytosis, and shows movement in search of the target. The macrophage (MPhi) appears in various shapes and sizes, spherical, flattened spindle-shaped, amoeboid, polygonal and with very long extension. On activation, the MPhi shows changes in cell shape, cytokinesis and development of intercellular contacts. A dynamic redistribution of cytoskeleton with cytoplasmic spread and/or extensions occurs with immunomodulators, like Mangiferin (1,3,6,7-tetrahydroxyxanthone-C(2)-beta-D-glucoside). The MPhi isolated from the peritoneal fluid of BALB/c mice pretreated with mangiferin and saline control, on challenge, shows redistribution of cytoplasm with variable morphology. Using the image analyses technique for pattern recognition of individual shapes of MPhi leads to the observation of clustering of cells in the coverslip culture. The stimulation in phagocytosis due to mangiferin, shows cytoplasmic spread, long extensions and intercellular contacts. The individual variations in the cytoplasmic redistribution are due to changes in the balance between the cellular surface area and the long extensions where the shape and nature of the phagocytic particle and the type of MPhi are determinants.