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Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.
Copyright © 2014 Elsevier Inc. All rights reserved.
The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.
Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Copyright Â© 2011 Elsevier B.V. All rights reserved.
We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endothelial solute barrier, and that this effect was reversed by restoring 'normal' (1.2 mM) calcium (1). This effect was shown to be dependent on cadherins, however the molecular mechanisms through which barrier was altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low calcium exposure. Endothelial permeability was significantly increased by exposure to low (0.12 mM) calcium; this effect was attenuated by pre-treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 x 10(-7) M) for 30 min. Cell border retraction and gap formation produced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of endothelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediated endocytosis was also prevented by the actin filament toxin, cytochalasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calcium mediated loss of endothelial barrier function is mediated in part by a PKC dependent endocytosis of endothelial cadherins, which may involve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-actin mediated endocytosis of cadherin junctional elements.
Immunization of C57BL/6 (B6) mice with heat-killed Sendai virus generates a Sendai virus-specific CD8+ T cell response. This suggests that APC have the capacity to take up and present exogenous (nonreplicative) Sendai virus Ag on MHC class I molecules. Little is known about the intracellular requirements for processing of this form of Ag and its presentation on MHC class I. Therefore, we have studied the processing and presentation of heat-killed Sendai virus Ag on MHC class I molecules in splenic APC. Heat-killed Sendai virus Ags were efficiently processed by normal B6 as well as by TAP-1(-/-) splenic APC. Presentation was MHC class I restricted, since no presentation was seen by APC from TAP-1/beta2m-/- mice that lack expression of MHC class I. Presentation occurred even in the presence of brefeldin A, but was blocked by cytochalasin D as well as chloroquine. Finally, B6 as well as TAP-1(-/-) splenic APC, loaded with heat-killed Sendai virus Ag in vitro, primed naive CD8+ T cells in vivo. These studies suggest the existence of a TAP-independent pathway for Ag presentation on MHC class I in normal splenic APC, bearing many similarities with the MHC class II pathway for Ag presentation. The present results are discussed in relation to the events underlying the processing and presentation of exogenous Ag on MHC class I, the molecular basis for CD8+ T cell priming during viral infections, and prospects for vaccine development.
The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.
Depolarization of leech neurons growing on extracellular matrix extract (ECM) leads to cessation of neurite outgrowth, rounding up of the peripheral regions of the growth cone, loss of filopodia, and neurite retraction. These responses depend on the influx of calcium (Neely, 1993). The aim of the present experiments was to analyze how the cytoskeleton becomes reorganized as growth cones change their morphology. Immunocytochemistry revealed a loss of microfilaments in the tips of neurites growing on ECM after depolarization. Leech neurons cultured on a different substrate, the plant lectin concanavalin A (ConA), continue to grow during and after depolarization (Grumbacher-Reinert and Nicholls, 1992; Neely, 1993). As expected, we did not observe any change in the distribution of microfilaments after depolarization on ConA. Since there is evidence that this lack of response is due to a reduced calcium influx during depolarization of neurons on ConA (Ross et al., 1988), the effect of the calcium ionophore A23187 on the outgrowth of these cells was analyzed. In the absence of depolarization, this ionophore caused cessation of growth cone motility and a loss of microfilaments, while microtubules were not affected. Cytochalasin D, a microfilament-disrupting agent, induced changes in growth cone morphology and neurite retraction similar to those observed after depolarization and calcium influx. Application of phalloidin, a drug that stabilizes microfilaments, inhibited depolarization-induced retraction of neurites on ECM. By contrast, stabilization of microtubules with taxol did not prevent depolarization from inducing changes in growth cone morphology and neurite growth. These experiments show that changes in growth cone morphology and motility of leech neurons induced by depolarization and calcium influx are accompanied by a dramatic change in the organization of microfilaments, but not microtubules.
Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F-actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low-dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F-actin was measured by FACS analysis of NBDphallacidin-stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 +/- 0.04, 1.60 +/- 0.21, 1.10 +/- 0.18, and 1.05 +/- 0.14) associated with four distinct shapes (round-smooth, round-ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (greater than 10-7M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5-20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP (less than 10-9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI less than 1.35-fold basal results in blebbed shape; polymerization greater than 1.35-fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F-actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for greater than 5 sec; polar shape with polymerization greater than or equal to 30 sec to RFI greater than 1.35-fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.