Simon V. Hunt, MA DPhil.
(1) library project: the use of cell-division history to measure cell proliferation
1. Measurement of intracellular calcium transients during B and T cell activation
2. Development of a cell population array imager
3. Effects of dihydropyridines on lymphocyte calcium transients
4. Effects of insect extracts on mammalian lymphocyte proliferation
1. The first three projects are interlinked. When the antigen receptors on the plasma membrane of freshly-isolated normal B lymphocytes are polyclonally triggered by an agonist that mimics antigen, such as anti-IgM or anti-IgD, calcium channels in the membrane open promptly. Within the next minute or so the chemical and electrical potentials across the membrane cause calcium ions (Ca++) to rush in. The consequent intracellular rise and decay in calcium provides one of the signals that alert the cell to alter its gene activity, eventually either to become an antibody-manufacturing cell or, sometimes, to be rendered impotent (“tolerised”). This is a particular example of the general phenomenon by which calcium ions can provide amplitude- or frequency-modulated information to communicate to the cell nucleus (Berridge, Bootman et al. 1998) . One of our interests has been to manipulate intracellular Ca++ stores by depletion with the sesquiterpenoid lactone thapsigargin and by deprivation of extracellular calcium We then observe the effects on the calcium entry pathway.
2. Much of the research on calcium transients in lymphocytes has used cultured cell lines. Our focus is instead on lymphocytes in as near-physiological state as we can find them, i.e. not under the insistent selection pressure of continuous hyper-efficient cell division in artificial medium. We are particularly interested to characterise the variety of differences of form of the calcium signals when comparing one freshly-isolated (“ex vivo”) lymphocyte population with another. Partly this is because there are very interesting features that demonstrably distinguish an activating signal from a tolerising one (Carruthers, Arrol et al. 2000) . A severe bottleneck in recording transients kinetically has been the limitations on numbers of cells that can be analysed. As few as 20-50 cells are all that can be analysed at one time by video-imaging in a fluorescence microscope. So we are developing with Wellcome grant funding an array imager that will measure the kinetics of calcium transients in 20000 or more lymphocytes simultaneously.and partly because our instinctive approach is that of the natural historian attempting to relate form to function. We believe that there may be classes of functional response that correspond with lymphocyte subset. This has for instance been suggested for T lymphocytes taken from patients with rheumatoid arthritis compared with normals
3. We have found (Akha, Willmott et al. 1996) and others have confirmed (Briede, Daija et al. 1999) that the family of dihydropyridine drugs exemplified by nicardipine (at around 10 micromolar) can at least partially prevent the entry of calcium ions (reviewed by Grafton and Thwaite, 2001). This is very surprising since dihydropyridines specifically block the so-called L-type channels that are found in electrically-excitable cells (muscle, neurones, etc.) but B cells, unlike electrically excitable cells, do not react to depolarisation of their membrane potential by opening their calcium channels. Our recent graduate student, Dr Anna Obolensky, has measured the IC50s of a range of dihydropyridines on rat T and B lymphocytes and on their immature precursors (papers in preparation). We’d like to understand the explanation of this paradox. Molecular studies of a lymphocyte calcium channel that I began in the lab of Prof Jefferies at the University of British Columbia, Vancouver, have subsequently revealed a Cav1.4 1 (CACNA1F; see OMIM entry) variant with key regions of sequence truncated (Kotturi et al, 2003). The variation could explain the lack of voltage-sensitivity and the quite high IC50 for dihydropyridines. This work might conceivably have possible practical implications because of the very widespread use of dihydropyridines in treating hypertension and angina, although there have been no reports, after years of extended use, of diminished antibody formation in patients taking these drugs.
4. In conjunction with Dr Jeyaraney Kathirithamby of the Department of Zoology we are pursuing a chance observation on the inhibitory effects of certain insect extracts on mammalian lymphocyte proliferation in vitro. We aim to biochemically characterise in outline the agent responsible for this effect. A number of project students have been engaged on this work, most recently John Openshaw and Keith Farrell-Dillon.
We use Fluo-3 and Fluo-4 as Ca-sensitive
fluorescent dyes excitable at the 488 nm wavelength of standard benchtop
flow cytometers to load lymphocytes. The
Lymphocyte proliferation assays
We routinely set up 96-well cultures of lymphocytes stimulated by standard mitogens (ConA, LPS, anti-Ig etc) for observation of the effects of drugs and toxins as inhibitors. Many of our assays are done with the fluorescence-halving technique using carboxy-fluorescein succinimidyl ester diacetate (CFSE-DA), devised by Bruce Lyons and co-workers (Lyons, Hasbold et al 2001)
We frequently employ conventional ELISA assays and a variety of cell subset separation techniques.
We have in the past used video imaging microscopy of single cells loaded with calcium-sensitive dyes (Fura-2), in collaboration with the group of Prof Antony Galione in the Pharmacology Department.
Technical assistant: Wendy Brownsill
Post-doctoral Research Fellow: Dr Oleg Salata
Most recent project student: Keith Farrell-Dillon 3rd year medic: was awarded a Wellcome Trust Summer Vacation Studentship 2005
Dr Steve Young
(Immunity and Infection,
Dr Stephen Payne
(Dept of Engineering,
Prof Wilf Jefferies (
Kathirithamby (Dept Zoology,
Project student: Keith Farrell-Dillon (Balliol) 3rd year medic 2004-5.
Summer student, from Univ of Wurzburg: Rebekka
Krumbach . Now at Lab
of Molecular Biology,
Lecture Notes (restricted access: these need your Weblearn username and password)
Year 1: Blood cells and haemopoiesis
Year 2: Principles
Akha, A. A. S., N. J. Willmott, et al. (1996). "Anti-Ig-Induced Calcium Influx in Rat B-Lymphocytes Mediated By cGMP Through a Dihydropyridine-Sensitive Channel." Journal of Biological Chemistry 271(13): 7297-7300. In contrast to excitable tissues where calcium channels are well characterized, the nature of the B lymphocyte calcium channel is unresolved. Here, we demonstrate by single cell analysis of freshly isolated rat B cells that the anti-immunoglobulin (Ig)-induced calcium influx takes place through a channel which shares pharmacologic and serologic properties with the L-type calcium channel found in excitable tissues, It is sensitive to the dihydropyridines nicardipine and Bay K 8644, to calciseptine, and to an anti-peptide antibody raised against the alpha(1) subunit of the L-type calcium channel, but is voltage-insensitive. Anti-alpha(1) and anti-alpha(2) antibodies stain B but not T lymphocytes, Application of a cGMP agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and examining the effect of a guanylyl cyclase inhibitor on the anti-Ig response show that cGMP mediates the influx, This possibly involves a cGMP-dependent protein kinase, The anti-Ig- induced response is not abolished by prior treatment of B cells with a high dose of thapsigargin. These findings undermine the widely held belief of a categorical divide between excitable and non-excitable tissue calcium channels, demonstrate the limitations of the capacitative calcium influx theory, and point to a distinction between the calcium response mechanisms utilized by B and T lymphocytes. http://www.jbc.org/cgi/content/full/271/13/7297
Barritt, G. J. (1999). "Receptor-activated Ca2+ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2+ signalling requirements." Biochemical Journal 337(Pt2): 153-169. Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage- operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Nat, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals.
Berridge, M. J., M. D. Bootman, et al. (1998). "Calcium--a life and death signal [news]." Nature 395(6703): 645-648. http://dx.doi.org/10.1038/27094
Briede, J., D. Daija, et al. (1999). "Effects of some 1,4-dihydropyridine Ca antagonists on the blast transformation of rat spleen lymphocytes." Cell Biochemistry and Function 17(2): 97-105. Ca antagonists of different classes (verapamil, nifedipine, nicardipine, diltiazem) in a concentration of 10(-5) M and higher are known to suppress Ca2+ transport into the lymphocyte cytosol? changing a normal response of lymphocytes to mitogens and antigens and so inhibiting their proliferation, as well as IL-2-induced cell proliferation, and their receptor expression on the surface of lymphocytes without cell cytotoxicity. In the present work we studied the effect of some 1,4-dihydropyridines (DHP) such as nimodipine, nicardipine, nifedipine, niludipine, cerebrocrast, etaftoron, as well as metabolites of cerebrocrast: compounds 7 and 8, (four of the last were synthesized in the Latvian Institute of Organic Synthesis) on rat spleen isolated lymphocyte activation and proliferation in vitro following stimulation with the mitogens concanavalin A (Con A) and recombinant interleukin-2 (IL-2), insulin and insulin antibodies. Based on the experimental results we conclude that in low concentrations (10(-7) to 10(-9) M) the tested 1,4-DHP Ca antagonists stimulated the process of rat spleen lymphocyte proliferation and DNA synthesis, especially cerebrocrast. It is proposed that these Ca antagonists, as well as causing a concentration decrease of Ca2+, also activated phosphodiesterase, which in its turn, suppressed cAMP accumulation in the lymphocytes and eventually increased Ca2+ ion transport in the cells. Cerebrocrast among all the studied DHP Ca antagonists was the most potent in studies of activation of the lymphocytes in the presence of Con A, IL-2 and insulin, which indicates the number of suppressor and helper lymphocytes and formation of insulin and interleukin receptors on their membrane surface. The increase in the lymphocyte suppressive activity produced by this compound effect can prevent diabetes mellitus types I and II at the stages of pre-diabetes, early and distant diabetes, from hyperexpression of insulin and its receptor antibodies. Copyright (C) 1999 John Wiley & Sons, Ltd.
Carruthers, D. M., H. P. Arrol, et al. (2000). "Dysregulated intracellular Ca2+ stores and Ca2+ signaling in synovial fluid T lymphocytes from patients with chronic inflammatory arthritis." Arthritis Rheum 43(6): 1257-1265. OBJECTIVE: Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals. METHODS: Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion- imaging system, and the spatial distribution of Ca2+ within intracellular organelles. RESULTS: There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P 0.05). Non-ER Ca2+ stores were not similarly affected. CONCLUSION: We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity. http://dx.doi.org/10.1002/1529-0131(200006)43:6<1257::AID-ANR8>3.0.CO;2-Q
Grafton, G. and L. Thwaite (2001). "Calcium channels in lymphocytes." Immunology 104(2): 119-126. http://dx.doi.org/10.1046/j.0019-2805.2001.01321.x
Healy, J. I. and C. C. Goodnow (1998). "Positive versus negative signaling by lymphocyte antigen receptors." Annu Rev Immunol 16: 645-670. Antigen receptors on lymphocytes play a central role in immune regulation by transmitting signals that positively or negatively regulate lymphocyte survival, migration, growth, and differentiation. This review focuses on how opposing positive or negative cellular responses are brought about by antigen receptor signaling. Four types of extracellular inputs shape the response to antigen: (a) the concentration of antigen; (b) the avidity with which antigen is bound; (c) the timing and duration of antigen encounter; and (d) the association of antigen with costimuli from pathogens, the innate immune system, or other lymphocytes. Intracellular signaling by antigen receptors is not an all-or-none event, and these external variables alter both the quantity and quality of signaling. Recent findings in B lymphocytes have clearly illustrated that these external inputs affect the magnitude and duration of the intracellular calcium response, which in turn contributes to differential triggering of the transcriptional regulators NF kappa B, JNK, NFAT, and ERK. The regulation of calcium responses involves a network of tyrosine kinases (e.g. lyn, syk), tyrosine or lipid phosphatases (CD45, SHP-1, SHIP), and accessory molecules (CD21/CD19, CD22, FcR gamma 2b). Understanding the biochemistry and logic behind these integrative processes will allow development of more selective and efficient pharmaceuticals that suppress, modify, or augment immune responses in autoimmunity, transplantation, allergy, vaccines, and cancer.
Lyons, A.B., J. Hasbold, and P.D. Hodgkin, (2001) "Flow cytometric analysis of cell division history using dilution of carboxyfluorescein diacetate succinimidyl ester, a stably integrated fluorescent probe", in Methods in Cell Biology, 63: 375-398
Putney, J. W. and C. M. P. Ribeiro (2000). "Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores." Cellular and Molecular Life Sciences 57(8-9): 1272-1286. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11028918&dopt=Abstract
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