Characterisation of lymphocyte responses to Ca 2+ in Scott syndrome

© 2004 Schattauer GmbH, Stuttgart

Lettersto Letter tothe theEditor Editor Characterisation of lymphocyte responses to Ca2+ in Scott syndrome

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Correspondence to: Dr. James Elliott Membrane Transport Biology Group MRC Clinical Sciences Centre Faculty of Medicine Imperial College Hammersmith Hospital Campus Du Cane Rd London W12 0NN, UK Tel.: +44 20 8383 8269, Fax: +44 20 8383 8337 E-mail: [email protected] Received July 1, 2003 Accepted after resubmission August 18, 2003

Thromb Haemost 2004; 91: 412 – 5

Figure 1: Responses of Scott and control lymphocytes to calcium ionophore A23187. A) Scott and control EBV transformed B cell lines were equilibrated with annexin VFITC and PI (Becton Dickinson, CA). The cells were analysed by flow cytometric analysis. Baseline fluorescence was established and then cells were stimulated with A23187. Plots depict the change in fluorescence of cell bound Annexin VFITC which is a surrogate marker of PS exposure.To further demonstrate an increase in PS exposure EBV transformed B cells were washed and resuspended with 2 µM human prothrombin, 5 pM human factor Xa and 25 pM human factor V in a volume of 200 µl. Aliquots were withdrawn at various time points before and following the addition of A23187 and assayed for thrombin activity using the chromogenic substrate S-2238. B) Freshly isolated lymphocytes obtained by venepuncture from the Scott patient and controls were washed and resuspended in phenol red-free DMEM (Sigma). Donors of control blood were healthy female volunteers aged between 24 and 60. Blood was collected from the Scott patient on three separate visits to Hammersmith Hospital. Lymphocytes were stained with antiCD4APC and equilibrated with annexin VFITC and PI prior to FACS analysis. After establishing baseline fluorescence, cells were stimulated with A23187. Density plots of annexin VFITC fluorescence in PI– cells staining positively for CD4 are shown and the number of CD4+ cells and their annexin VFITC fluorescence is shown towards the end of the experiment (R1). C) In the same experiment as represented in B, but without restricting analysis to CD4+ lymphocytes, the appearance of small, (forward light scatter low) cells (marked as gate R2) is shown following stimulation of control lymphocytes with A23187. Cells within R2 were annexin VFITCnegative (not shown). In contrast Scott cells stimulated with A23187 showed no appearance of small cells.The relative sizes of control and Scott PI– cells towards the end of the experiment (within gate R3 and similarly gated control cells). Results are representative of three separate experiments. D) Ca2+-uptake by Scott and control lymphocytes was measured by flow cytometric analysis. Lymphocytes labelled with antiCD4APC antibody were incubated with the Ca2+-indicator Fluo-4 AM, after baseline fluorescence was established; Ca2+ uptake was induced with A23187. Increased intracellular Ca2+ in the CD4+ population is indicated by raised Fluo-4 fluorescence (mean + S.E. fluorescence units.Two representative experiments are shown. E) Cell shrinkage in Scott and control lymphocytes was analysed by flow cytometry. Lymphocytes were stained with anti-CD4PE, anti-CD4CYCHROME antibodies in order to distinguish between Scott and control cells. Samples were mixed, baseline flow cytometric parameters established, and stimulated with A23187. Shrinkage of cells positively stained with CD4 antibodies was measured by reduction in forward light scatter.The results of two independent experiments are shown.The data are representative of three comparable experiments performed on separate occasions using three different control subjects.

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Letter to the Editor

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Letter to the Editor

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James I. Elliott, Andrew D. Mumford, Christiane Albrecht, Peter W. Collins, John C. Giddings, Christopher F. Higgins, Edward G. D.Tuddenham, John H. McVey

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UK jìååáñ fÌ e~êãëã~ jI dáÇÇáåÖë gÌ Éí ~äKK píçêÉJãÉÇá~íÉÇ Å~äÅáìã Éåíêó áå íÜÉ êÉÖìä~íáçå çÑ éÜçëéÜ~íáÇóäëÉêáåÉ ÉñéçëìêÉ áå ÄäççÇ ÅÉääë Ñêçã pÅçíí é~íáÉåíëK qÜêçãÄ e~Éãçëí OMMPX UVW SUTJVRK VK m~êêó aeI dáÇÇáåÖë gÌ _äççã îK c~ãáäá~ä Ü~Éãçëí~íáÅ ÇÉÑÉÅí ~ëëçÅá~íÉÇ ïáíÜ êÉÇìÅÉÇ éêçíÜêçãÄáå ÅçåëìãéíáçåK _ê g e~Éã~íçä NVUMX QQW POPJPQK NMK hçàáã~ eI kÉïíçåJk~ëÜ aI tÉáëë egI Éí ~äKK mêçÇìÅíáçå ~åÇ ÅÜ~ê~ÅíÉêáò~íáçå çÑ íê~åëJ ÑçêãÉÇ _JäóãéÜçÅóíÉë ÉñéêÉëëáåÖ íÜÉ ãÉãJ

Äê~åÉ ÇÉÑÉÅí çÑ pÅçíí ëóåÇêçãÉK g `äáå fåîÉëí NVVQX VQW OOPTJQQK NNK bääáçíí gfI eáÖÖáåë `cK fh`~N ~Åíáîáíó áë êÉJ èìáêÉÇ Ñçê ÅÉää ëÜêáåâ~ÖÉI éÜçëéÜ~íáÇóäëÉêáåÉ íê~åëäçÅ~íáçå ~åÇ ÇÉ~íÜ áå q äóãéÜçÅóíÉ ~éçéJ íçëáëK bj_l oÉéçêíë OMMOX QW NUVJVQK NOK páãë mgI táÉÇãÉê qI bëãçå `qI Éí ~äKK ^ëëÉãJ Ääó çÑ íÜÉ éä~íÉäÉí éêçíÜêçãÄáå~ëÉ ÅçãéäÉñ áë äáåâÉÇ íç îÉëáÅìä~íáçå çÑ íÜÉ éä~íÉäÉí éä~ëã~ ãÉãÄê~åÉK píìÇáÉë áå pÅçíí ëóåÇêçãÉW ~å áëçJ ä~íÉÇ ÇÉÑÉÅí áå éä~íÉäÉí éêçÅç~Öìä~åí ~ÅíáîáíóK g _áçä `ÜÉã NVUVX OSQW NTMQVJRTK

Erratum

få íÜÉ ~êíáÅäÉ Äó h~åÉáÇÉêI Éí ~äK ÉåíáíäÉÇ póåÇÉÅ~åJQJ ÇÉéÉåÇÉåí ëáÖå~äáåÖ áå íÜÉ áåÜáÄáíáçå çÑ ÉåÇçíçñáåJáåÇìÅÉÇ ÉåÇçíÜÉäá~ä ~ÇÜÉêÉåÅÉ çÑ åÉìíêçéÜáäë Äó ~åíáíÜêçãÄáåÒ éìÄäáëÜÉÇ áå qÜêçãÄçëáë ~åÇ e~Éãçëí~ëáë aÉÅÉãÄÉê OMMP EqÜêçãÄ e~Éãçëí OMMPX VMW NNRMJTF íÜÉ ÑçääçïáåÖ Éêêçê ï~ë ã~ÇÉW

Materials and methods

qÜÉ éêÉé~ê~íáçå çÑ ~åíáíÜêçãÄáå ìëÉÇ Ñçê íÜÉ ëíìÇó ï~ë îáêìëJ áå~Åíáî~íÉÇ Üìã~å éä~ëã~íáÅ ~åíáíÜêçãÄáå fff ÅçåÅÉåíê~íÉ E^îÉåíáë _ÉÜêáåÖI j~êÄìêÖI dÉêã~åóFK

415
