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H A Wilson

Publications and source records attributed to H A Wilson.

11 recordsLinked to original sources
PubMed1987

Crosslinkage of B lymphocyte surface immunoglobulin by anti-Ig or antigen induces prolonged oscillation of intracellular ionized calcium.

Our results indicate that B lymphocytes stimulated with anti-Ig or antigen exhibit repetitive [Ca2+]i transients which persist for hours. The magnitude of these transients favors an important and ongoing role for [Ca2+]i elevation in antigen driven B cell activation. Repetitive Ca2+ transients may prove to be a prevalent mechanism of Ca2+ signaling. In preliminary experiments (with L. E. Samelson and R. D. Klausner), we have observed Ca2+ transients in cloned T cells stimulated with antigen. Woods et al. have described repetitive free Ca2+ transients in hepatocytes stimulated with extracellular ligands promoting glycogenolysis, and suggest that the intervals of base-line [Ca2+]i levels explain the absence of mitochondrial overload in chronically stimulated cells. These considerations apply equally to B lymphocytes and recommend caution in delineating the range of Ca2+-mediated functions by prolonged coculture of cells with Ca2+ ionophores. Our experiments were done in a simple recording chamber with one cell type. No cell interactions were observed. Given the variety of indicator dyes now available, the technical approach we present, augmented by a more sophisticated recording chamber, is a potentially powerful tool for examining the intrinsic, and T- or accessory cell-dependent, physiology of B cell differentiation.

Animals↗
PubMed1987

The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor.

We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.

Animals↗
PubMed1987

Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti-IgM and anti-IgD.

The accurate measurement of ionized intracellular calcium [Ca++]i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo-1, is described. We have developed a dependable in situ calibration technique that indicates a resting [Ca++]i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of [Ca++]i transients does not occur. The [Ca++]i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti-immunoglobulin is heterogeneous. With maximal stimulus, the peak [Ca++]i response occurs in 10 to 20 seconds with most cells reaching levels greater than/1 microM. Mean [Ca++]i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti-delta is a more potent stimulus of increased [Ca++]i than anti-mu in terms of both [Ca++]i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the [Ca++]i response.

Animals↗
PubMed1985

Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components.

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.

B-Lymphocytes↗
PubMed1985

Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements.

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (psi) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of psi than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte psi was calculated to be -70 mV. T and B lymphocytes maintain a constant psi as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (EK) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+-sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4-aminopyridine (4-AP, 10 mM) were observed to return to resting psi by A23187-induced elevation of [Ca2+]i. Cells depolarized by quinine (100 microM) were unaffected by A23187. The Ca2+-activated channel does not contribute to resting psi in T cells since it may be selectively blocked by quinine (20 microM) or modulated by calmodulin antagonists (5 microM trifluperazine) without affecting resting psi.

4-Aminopyridine↗
PubMed1979

Association of IgG anti-brain antibodies with central nervous system dysfunction in systemic lupus erythematosus.

Sera from 20 patients with systemic lupus erythematosus (SLE) and active central nervous system (CNS) dysfunction were examined by indirect immunofluorescence for antibodies to neuronal membrane determinants. Warm-reactive IgG antibodies were demonstrable in 82% (9/11) of patients with clinical evidence for seizures or diffuse CNS disease, but these antibodies generally were absent in non-CNS SLE sera or when focal neurologic deficit or psychosis was the primary CNS manifestation. Cold-reactive antibodies of the IgM class were equally prevalent in patients with or without CNS disease and appeared to be more directly correlated with extra-CNS systemic illness. Absorption experiments with lymphocytes, brain homogenate, and various other tissues suggested a predominant brain-specificity for IgG antibodies and partial lymphocyte cross-reactivity for IgM antibodies. Interpretations of this special association between IgG anti-brain antibodies and diffuse CNS dysfunction in SLE are discussed.

Autoantibodies↗
PubMed1972

Inhibition of catecholamine uptake in the isolated rat heart by haloalkylamines related to phenoxybenzamine.

1. Twenty-one haloalkylamine derivatives were tested as inhibitors of both the neuronal uptake of (3)H-noradrenaline (NA) by the Uptake(1) mechanism and the extraneuronal uptake of (3)H-NA by the Uptake(2) mechanism in the isolated rat heart.2. At a concentration of 50 muM most of the compounds tested caused a significant inhibition of both uptake processes, although there were wide differences in the relative effects on Uptake(1) and Uptake(2). Some tentative structure activity relationships for uptake inhibition were formulated from these results.3. Phenoxybenzamine was confirmed to be a potent inhibitor of both the Uptake(2) and Uptake(1) mechanisms, with IC50 values for these two systems of 2.8 muM and 0.9 muM respectively.4. The substances N-(9-fluorenyl)-N-methyl-beta-chloroethylamine (SKF 550), N-(3,4-dimethoxyphenylisopropyl)-N-benzyl-beta-chloroethylamin (SKF 625A) and N-(4-methoxyphenoxyisopropyl)-N-benzyl-beta-chloroethylamine (SKF 784A) were significantly more potent than phenoxybenzamine as Uptake(2) inhibitors, and were all less potent than phenoxybenzamine as Uptake(1) inhibitors. The compound SKF 550 is the most potent and selective inhibitor of Uptake(2) so far described. It has an IC50 for Uptake(2) of 0.08 muM, and an IC50 for Uptake(1) of approximately 40.0 muM.5. Comparison of the present results with the known activities of these blocking agents suggests that no correlation exists between adrenoceptor blocking activity and ability of the substances to act as inhibitors of Uptake(2) or Uptake(1).

Animals↗