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Biomedical subjects

Michael D Cahalan

Publications and source records attributed to Michael D Cahalan.

31 records · Page 2Linked to original sources
PubMed2003

Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells.

FM1-43, a fluorescent styryl dye that penetrates into and stains membranes, was used to investigate kinetics of constitutive endocytosis and to visualize the fate of endocytic organelles in resting and activated human T lymphocytes. The rate of dye accumulation was strongly temperature dependent and approximately 10-fold higher in activated than in resting T cells. Elevation of cytosolic free Ca2+ concentration with thapsigargin or ionomycin further accelerated the rate of FM1-43 accumulation associated with cytosolic actin polymerization. Direct modulation of actin polymerization affected membrane trafficking. Actin condensation beneath the plasma membrane with calyculin A abolished FM1-43 internalization, whereas actin depolymerization with cytochalasin D had no effect. Photoconversion of DAB by FM1-43 revealed altered endocytic compartment targeting associated with T cell activation. Internalized cargo was carried to lysosome-like compartments in resting T cells and to multivesicular bodies (MVB) in activated T cells. Externalization of exosomes from MVB occurred commonly in activated but not in resting T cells. T cell exosomes contained raft-associated CD3 proteins, GM1 glycosphingolipids, and phosphatidylserine at the outer membrane leaflet. The present study demonstrates the utility of FM1-43 as a marker of membrane trafficking in T cells and reveals possible mechanisms of its modulation during T cell activation.

Actins↗
PubMed2003

Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naive CD4(+) T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

Adoptive Transfer↗
PubMed2003

A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.

Amino Acids↗
PubMed2003

MIC channels are inhibited by internal divalent cations but not ATP.

TRPM7 channels are nonselective cation channels that possess a functional alpha-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg(2+)-nucleotide-inhibited metal) or MIC (for Mg(2+)-inhibited cation). Here, we report that internal Mg(2+) rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg(2+) chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg(2+) rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl(2) and MgATP solutions contain similar amounts of free Mg(2+), explaining the fact that numeric values of Mg(2+) and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg(2+) is not unique in its inhibitory action, as Ba(2+), Sr(2+), Zn(2+), and Mn(2+) can substitute for Mg(2+), causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations.

Adenosine Triphosphate↗
PubMed2003

Polyvalent cations as permeant probes of MIC and TRPM7 pores.

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Animals↗
PubMed2003

Real-time imaging of lymphocytes in vivo.

New preparations, fluorescent probes and imaging techniques are providing the means to observe the behavior of cells in the tissue environment of lymphoid organs. In particular, when combined with two-photon laser microscopy, intravital imaging of surgically exposed lymph nodes provides a unique view of lymphocyte migration and antigen presentation as it occurs within the living animal. The view is emerging that lymphocytes migrate randomly within lymphoid organs, and that lymphocyte contact with antigen-presenting cells may be a stochastic process rather than one guided by chemokine gradients.

Animals↗
PubMed2003

A stochastic view of lymphocyte motility and trafficking within the lymph node.

Two-photon microscopy is providing literal insight into the cellular dynamics of lymphoid organs and, guided by analysis of three-dimensional images, into mechanisms that underlie cell migration and antigen recognition in vivo. This review describes lymphocyte motility and antigen recognition in the native tissue environment and compares these results with a much more extensive literature on lymphocyte motility, signaling, and chemotaxis in vitro. We discuss the in vitro literature on dynamic aspects of lymphocyte motility, chemotaxis, and the response to antigen and present the view that random migration of lymphocytes may drive a stochastic mechanism of antigen recognition in lymphoid organs, rather than being guided by chemotaxis.

Animals↗
PubMed2002

Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.

Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to "swarms" and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.

Adoptive Transfer↗
PubMed2002

Two-photon tissue imaging: seeing the immune system in a fresh light.

Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Two-photon microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.

Animals↗
PubMed2002

Distinct properties of CRAC and MIC channels in RBL cells.

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

Animals↗
PubMed2001

Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel.

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.

Amino Acid Sequence↗