ABSTRACT
Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1ß is particularly important. IL-1ß is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1ß activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1ß processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe, we also demonstrated that the pro-IL-1ß processing occurs all over the cytoplasm.
Subject(s)
Energy Transfer/immunology , Inflammasomes/genetics , Inflammasomes/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Luminescent Measurements/methods , Macrophages/immunology , Animals , Bacterial Proteins/genetics , Cell Culture Techniques , Cell Line, Transformed , Cell Line, Tumor , HEK293 Cells , Humans , Luciferases, Renilla/genetics , Luciferases, Renilla/metabolism , Luminescent Proteins/genetics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/immunologyABSTRACT
Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by approximately 14 to 15 nm and/or energies of approximately 1.5 kcal mol(-1) relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.
Subject(s)
Energy Transfer/immunology , Fluoresceins/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Immunoglobulin Fab Fragments/immunology , Animals , Antibody Affinity , Antigen-Antibody Complex , Fluorescein-5-isothiocyanate/chemistry , Ligands , Rabbits , Spectrometry, FluorescenceABSTRACT
Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3epsilon is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.
Subject(s)
CD3 Complex , CD8 Antigens/metabolism , Histocompatibility Antigens Class I/physiology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Animals , Biomarkers/analysis , Cell Membrane/immunology , Cell Membrane/metabolism , Energy Transfer/immunology , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/immunology , Peptide Fragments/metabolism , Phosphatidylethanolamines/metabolism , Phycocyanin/metabolism , Spectrometry, Fluorescence , T-Lymphocytes/immunologyABSTRACT
Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.
Subject(s)
Acetylcysteine/analogs & derivatives , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , HLA-A2 Antigen/metabolism , Peptide Fragments/immunology , Peptide Fragments/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/metabolism , Acetylcysteine/pharmacology , Amino Acid Sequence , Antigen Presentation/drug effects , Antigen Presentation/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cell Line, Transformed , Cysteine Proteinase Inhibitors/pharmacology , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/genetics , Energy Transfer/drug effects , Energy Transfer/genetics , Energy Transfer/immunology , Gene Products, tax/immunology , Gene Products, tax/metabolism , Green Fluorescent Proteins , HLA-A2 Antigen/genetics , HeLa Cells , Human T-lymphotropic virus 1/immunology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/immunology , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/genetics , Protein Transport/drug effects , Protein Transport/genetics , Protein Transport/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Spectrometry, FluorescenceABSTRACT
Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.
Subject(s)
CD8 Antigens/chemistry , Energy Transfer/immunology , HLA Antigens/chemistry , Histocompatibility Antigens Class I/chemistry , Models, Molecular , Receptors, Antigen, T-Cell/chemistry , Antigen-Presenting Cells/chemistry , Antigen-Presenting Cells/immunology , B-Lymphocytes/chemistry , B-Lymphocytes/immunology , Cell Line, Transformed , Cell Membrane/chemistry , Cell Membrane/immunology , Computer Simulation , Crystallography, X-Ray/methods , Epitopes, B-Lymphocyte/chemistry , HLA-A2 Antigen/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Peptide Mapping , Spectrometry, Fluorescence/methods , beta 2-Microglobulin/chemistryABSTRACT
Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca(2+) concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca(2+) mobilization, whereas mAbs against the beta(2) integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca(2+) signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.
Subject(s)
L-Selectin/physiology , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Cell Surface/physiology , Signal Transduction/immunology , Urokinase-Type Plasminogen Activator/metabolism , Carbohydrate Metabolism , Cell Adhesion/immunology , Energy Transfer/immunology , Glycosylation , Humans , L-Selectin/immunology , L-Selectin/metabolism , Ligands , Neutrophil Activation/immunology , Neutrophils/enzymology , Protein Binding/immunology , Protein Structure, Tertiary , Receptors, Urokinase Plasminogen Activator , Spectrometry, FluorescenceABSTRACT
Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3.