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Michael L Dustin

Publications and source records attributed to Michael L Dustin.

At least 37 records · Page 2Linked to original sources
PubMed2005

Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo.

The maturation status of dendritic cells (DCs) determines whether they prime or tolerize T cells. We targeted ovalbumin peptide exclusively to DCs in situ using an antibody to DEC-205 and studied the interaction of DCs with naive CD4(+) T cells in tolerizing or priming conditions. We used two-photon microscopy to simultaneously track antigen-specific OT-II T cells, nonspecific T cells and DCs in lymph nodes of living mice. In both tolerance and immunity, OT-II cells arrested on DCs near high endothelial venules beginning shortly after extravasation and regained their baseline speed by 18 h. Thus, early antigen-dependent T cell arrest on DCs is a shared feature of tolerance and priming associated with activation and proliferation.

Animals↗
PubMed2005

ATP mediates rapid microglial response to local brain injury in vivo.

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

Adenosine Triphosphate↗
PubMed2005

Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes.

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL.

Cell Adhesion↗
PubMed2005

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10-20 microm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

Animals↗
PubMed2005

A model for CD2/CD58-mediated adhesion strengthening.

Stable cell adhesion is vital for structural integrity and functional efficacy. Yet how low affinity adhesion molecules such as CD2 and CD58 can produce stable cell adhesion is still not completely understood. In this paper, we present a theoretical model that simulates the accumulation of CD2 and CD58 in the contact area of a Jurkat T lymphoblast and a CD58-containing substrate. The cell is assumed to have a spherical shape initially and it is allowed to spread gradually on a circular substrate. Mobile CD2 and CD58 can diffuse freely on both the cell and substrate. Their binding in the contact area is controlled by first-order kinetics. The contact area grows linearly with the total number of CD2/CD58 bonds. Cellular deformation and cytoskeleton involvement were not considered. This time-dependent moving-boundary problem was solved with the Crank-Nicolson finite difference scheme and the variable space grid method. Our simulated results are in reasonable agreement with the experimental observations. The role of diffusion becomes more and more prominent during the contact area increase, which is not sensitive to the kinetic rate constants tested in this study. However, it is very sensitive to the dissociation equilibrium constant and the concentrations of CD2 and CD58.

Adhesiveness↗
PubMed2005

ICAM-1 co-stimulates target cells to facilitate antigen presentation.

Adhesion molecules are known to mediate cell-cell interactions, particularly those between T cells and antigen-presenting or target cells. Recent studies identified ICAM-1 as a co-stimulatory ligand that binds to lymphocyte function associated antigen-1 (LFA-1), thereby promoting the activation of T cells. As ICAM-1 is expressed on virtually any cell, it becomes a crucial molecule for the activation of CD8(+) T cells in the absence of co-stimulation provided by CD80 and CD86 molecules. In addition, ICAM-1 might function as cell-surface receptor, capable of initiating intracellular signaling. ICAM-1 is associated with other cell molecules, including MHC-I proteins, and our recent data show that productive engagement of ICAM-1 on target cells leads to recruitment of the MHC-I proteins to the contact area and enhances presentation of cognate peptide MHC-I complexes to cytotoxic T cells.

Antigen Presentation↗
PubMed2005

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling.

In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells.

Calcium Signaling↗
PubMed2004

Visualizing dendritic cell networks in vivo.

In the steady state, dendritic cells (DCs) in the lymph node induce T cell tolerance to self antigens. Innate signals trigger the maturation of tissue DCs, which migrate into lymph nodes and activate T cells. To examine DCs in vivo, we produced transgenic mice whose DCs expressed enhanced yellow fluorescent protein. Two-photon microscopy of lymph nodes in live mice showed that most of the steady-state DCs were enmeshed in an extensive network and remained in place while actively probing adjacent T cells with their processes. Mature DCs were more motile than steady-state DCs and were rapidly dispersed and integrated into the sessile network, facilitating their interaction with migrating T cells.

Animals↗
PubMed2004

T cell receptor antagonism interferes with MHC clustering and integrin patterning during immunological synapse formation.

T cell activation by nonself peptide-major histocompatibility complex (MHC) antigenic complexes can be blocked by particular sequence variants in a process termed T cell receptor antagonism. The inhibition mechanism is not understood, although such variants are encountered in viral infections and may aid immune evasion. Here, we study the effect of antagonist peptides on immunological synapse formation by T cells. This cellular communication process features early integrin engagement and T cell motility arrest, referred to as the "stop signal." We find that synapses formed on membranes presenting antagonist-agonist complexes display reduced MHC density, which leads to reduced T cell proliferation that is not overcome by the costimulatory ligands CD48 and B7-1. Most T cells fail to arrest and crawl slowly with a dense ICAM-1 crescent at the leading edge. Similar aberrant patterns of LFA-1/ICAM-1 engagement in live T-B couples correlate with reduced calcium flux and IL-2 secretion. Hence, antagonist peptides selectively disable MHC clustering and the stop signal, whereas LFA-1 valency up-regulation occurs normally.

Animals↗
PubMed2004

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Animals↗
PubMed2004

Stop and go traffic to tune T cell responses.

Adaptive immune responses are initiated by interactions of T cells with antigen-presenting cells, but the basic nature of these interactions during an immune response in vivo has been a matter of speculation. While some in vitro systems provide evidence for stable interactions, referred to as immunological synapses, compelling evidence supports T cell activation through serial transient interactions. Deep tissue intravital and organ culture microscopy studies suggest that both modes of interaction are employed, but new issues have emerged. This review will discuss in vitro results that framed the hypotheses that are currently being tested in vivo. I present a model in which TCR stop signals compete with chemokine-mediated go signals to adjust the duration of immunological synapse formation and tune the immune response between tolerance and full activation.

Animals↗
PubMed2004

What is the importance of the immunological synapse?

The immunological synapse (IS) has proved to be a stimulating concept, particularly in provoking discussion on the similarity of intercellular communication controlling disparate biological processes. Recent studies have clarified some of the underlying molecular mechanisms and functions of the IS. For both T cells and natural killer (NK) cells, assembly of the IS can be described in stages with distinct cytoskeletal requirements. Functions of the IS vary with circumstance and include directing secretion and integrating positive and negative signals to determine the extent of response.

Animals↗
PubMed2004

LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation.

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.

Animals↗
PubMed2004

Membranes as messengers in T cell adhesion signaling.

Talin and RapL are components of molecular pathways that regulate the avidity of the integrin lymphocyte function-associated antigen 1 (LFA-1) for its ligand, intercellular adhesion molecule 1. In this review, we discuss recent advances in our understanding of LFA-1 affinity regulation and signaling and discuss a scenario for how Talin and Rap1 might act in synergy to achieve regulation of LFA-1 that is tailored to the specific functional requirements of different situations. Speedy delivery of signals may be crucial, and membrane trafficking from endosomes and the Golgi apparatus seem to be essential in delivering the messages from spatially segregated surface receptors.

Animals↗
PubMed2004

Cytotoxic T lymphocytes form an antigen-independent ring junction.

Immunological synapses are organized cell-cell junctions between T lymphocytes and APCs composed of an adhesion ring, the peripheral supramolecular activation cluster (pSMAC), and a central T cell receptor cluster, the central supramolecular activation cluster (cSMAC). In CD8(+) cytotoxic T lymphocytes, the immunological synapse is thought to facilitate specific killing by confining cytotoxic agents to the synaptic cleft. We have investigated the interaction of human CTLs and helper T cells with supported planar bilayers containing ICAM-1. This artificial substrate provides identical ligands to CD4(+) and CD8(+) T cells, allowing a quantitative comparison. We found that cytotoxic T lymphocytes form a ring junction similar to a pSMAC in response to high surface densities of ICAM-1 in the planar bilayer. MICA, a ligand for NKG2D, facilitated the ring junction formation at lower surface densities of ICAM-1. ICAM-1 and MICA are upregulated in tissues by inflammation- and stress-associated signaling, respectively. Activated CD8(+) T cells formed fivefold more ring junctions than did activated CD4(+) T cells. The ring junction contained lymphocyte function associated antigen-1 and talin, but did not trigger polarization and granule translocation to the interface. This result has specific implications for the mechanism of effective CTL hunting for antigen in tissues. Abnormalities in this process may alter CTL reactivity.

Antigen-Presenting Cells↗
PubMed2003

The immunological synapse balances T cell receptor signaling and degradation.

The immunological synapse is a specialized cell-cell junction between T cell and antigen-presenting cell surfaces. It is characterized by a central cluster of antigen receptors, a ring of integrin family adhesion molecules, and temporal stability over hours. The role of this specific organization in signaling for T cell activation has been controversial. We use in vitro and in silico experiments to determine that the immunological synapse acts as a type of adaptive controller that both boosts T cell receptor triggering and attenuates strong signals.

Adaptor Proteins, Signal Transducing↗
PubMed2003

In vivo imaging approaches in animal models of rheumatoid arthritis.

The interaction of activated leukocytes with the rheumatoid synovial environment is a key process in arthritis. Understanding this process will play an important role in designing effective treatments. In vivo imaging approaches combined with molecular genetics in animal models provide important tools to address these issues. The present review will focus on approaches to in vivo imaging, with particular attention to approaches that are proving useful for, or have promise for, research on animal models of rheumatoid arthritis. These approaches will probably shed light on the specific local mechanisms involved in chronic inflammation and provide real time monitoring approaches to follow cellular and molecular events related to disease development.

Animals↗