Synchronized turbo apoptosis induced by coldshock

Authors: J H Fransen J W Dieker L B Hilbrands J H Berden J van der Vlag

Publish Date: 2010/10/24

Volume: 16, Issue: 1, Pages: 86-93

PDF Link

Abstract

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus SLE we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells Here we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations In brief granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C After 30–90 min at 37°C more than 80–90 of the cells become early apoptotic Annexin V positive/propidium iodide negative After 24 h of rewarming at 37°C 98 of the cells were late apoptotic secondary necrotic Annexin V positive/propidium iodide positive Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C Internucleosomal chromatin cleavage and caspase activation were other characteristics of this coldshockinduced process of apoptosis Consequently apoptosis could be inhibited by a caspase inhibitor Finally SLEderived antichromatin autoantibodies showed a high affinity for apoptotic blebs generated by coldshock Overall coldshock induced apoptosis is achieved without the addition of toxic compounds or antibodies and quickly leads to synchronized homogeneous apoptotic cell populations which can be applied for various research questions addressing apoptosisApoptosis or programmed cell death is involved in tissue development and maintenance which includes shaping of the immune system and in cellular responses to stress and DNA damage Several disorders are related to an aberrant apoptotic process Roughly too much unwanted apoptosis leads to cell loss disorders like diabetes type 1 while too little apoptosis leads to cell proliferation disorders like cancer 1 2 3 So many research fields address the role of apoptosis In our research we address the role of apoptosis in the development of the antichromatin autoimmune response in systemic lupus erythematosus SLE as will be briefly outlined belowThe hallmark of SLE is the presence of antinuclear antibodies to double stranded DNA nucleosomes histones and other DNA/RNAbinding proteins 4 In patients with SLE apoptotic cells and chromatin can be detected in the circulation and in several tissues which may be explained by an aberrant apoptosis and/or reduced clearance 5 6 7 8 9 During apoptosis autoantigens can be modified by several biochemical processes and they cluster in blebs formed at the apoptotic cell surface Apoptotic blebs containing modified autoantigens can segregate and their contents can be released 10 11 12 This released apoptotic material can be ingested by professional antigen presenting cells the dendritic cells DC which then leads to DC maturation and presentation of modified autoantigens in an immunogenic way to autoreactive T cells 13 14 Activated T cells will stimulate autoreactive B cells producing antinuclear autoantibodies that form immune complexes with released apoptotic chromatin In addition autoreactive B cells can be rescued from deletion by accumulation and continuous presentation of apoptotic material on the surface of follicular dendritic cells in germinal centers as well as by TLR9 mediated activation by DNA and HMGB1 6 15 16 17 Deposition of the formed immune complexes in basement membranes leads to serious inflammation like lupus nephritis So apoptosis can play two key roles in the pathogenesis of SLE First due to an aberrant apoptosis and/or insufficient removal of apoptotic cells the immune system in SLE is activated by released modified apoptotic debris Second aberrant apoptosis may lead to the persistence of autoreactive T and B cells in SLE 6 12 18In our research we address the uptake by antigen presenting cells ie dendritic cells of early and late apoptotic blebs and cell bodies Late and early apoptotic cellderived blebs and cell bodies could differ in the amount and nature of apoptosis induced modifications 13 14 Early apoptotic cells are defined as annexin V AnV positive and propidium iodide PI negative while late apoptotic or secondary necrotic cells are AnV and PI double positive The generation of homogeneous preparations of early and late apoptotic cells is not straightforward Apoptosis can be initiated in several ways for instance by ligands binding to specific death receptors like the TNF receptor family by cellular stress through DNA damage or by deprivation of growth factors or nutrients 19 20 The majority of experimental approaches to induce apoptosis lead to a mixture of cells in different stages of apoptosis Triggering of the Fas–FasL pathway by incubation with antiFas antibodies is most likely one of the most well known methods to obtain a synchronized apoptotic cell populationIn our quest to prepare homogeneous preparations of early and late apoptotic cells we found by serendipity that applying coldshock at 0°C for 2 h followed by a rewarming at 37°C yielded highly homogeneous synchronized apoptotic cell suspensions that served as a source for early and late apoptotic blebs and cell bodies This method can easily be applied to address various research questions related to apoptosisMurine 32D clone 3 cells 32Dcl3 Jurkat cells and WEHI3B cells were cultured in RPMI1640 DM Invitrogen Life Technologies Breda The Netherlands with 10 fetal calf serum FCS Greiner Bio One Alphen aan de Rijn The Netherlands 1 pyruvate 1 glutamax 1 penicillin/streptomycin all from Invitrogen Life Technologies and in the case of 32Dcl3 cells supplemented with 15 WEHI3Bconditioned medium DSMZ Braunschweig Germany 21 Cells were propagated every 2–3 days at 01×106 cells/ml Apoptosis was induced by incubation of 1×106 cells/ml on ice for 2 h unless indicated otherwise followed by rewarming at 37°C in an incubator with 5 CO2 for periods of 30 min up to 24 hApoptosis was determined by staining cells with Annexin VFITC AnV and PI Biovision ITK Diagnostics Uithoorn The Netherlands according to the manufacturer’s protocol and analyzed by flow cytometry FACSCalibur BD Alphen aan de Rijn The Netherlands Polycaspase cleavage was examined by FLICA Fluorochrome Inhibitor of Caspases using the Vybrant FAM polycaspases assay kit Molecular Probes Invitrogen following the instructions of the manufacturer To analyze whether coldshockinduced apoptosis was mediated by caspases cells were prior to coldshock incubated with 100 μM ZVADFMK BD which is a caspase inhibitor and subsequently stained with labeled AnV and PI

Keywords: