This work is supported by grants from the Canadian Institutes of Health Research (178918), Fonds de recherche en sant&#233; du Qu&#233;bec &#8211; Vision Health Research Network.

NOTE: Male C57BL/6 mice (5&#8211;7 weeks old) are from Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) and manipulated according to protocols approved by the CHU Sainte-Justine Animal Care Committee. Mouse bronchial tissue explants provide a good source of primary bronchial epithelial cells for investigating the proapoptotic effects of LMPs on epithelial cells. This protocol describes the in vitro generation of LMPs, as well as a method for detecting apoptotic epithelial cells on LM...

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	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. 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MPs are active mediators of intercellular cross talk and their study is promising in many areas of science.11 This study presented a detailed protocol for in vitro large-scale generation of LMPs derived from an apoptotic T cell line. These MPs express a large repertoire of lymphocyte molecules and are biologically implicated in the regulation of cellular and tissue homeostasis. However, LMPs derived from different sources may be biologically different.4,9,12,13
L...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="828">
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			<td style="width:231px;">Whatman International Ltd.</td>
			<td style="width:124px;">1003150</td>
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</table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

LMPs were characterized with annexin V staining10 by fluorescence-activated cell sorting (FACS) analysis and gated using 1 &#181;m beads in which 97% of MPs (&#8804;1 &#181;m) were annexin-V-Cy5 positive (Figure 1A and 1B). Typically, about 2.5 mg of LMPs were obtained following this protocol. Bronchial tissue explants from C57BL/6 mice were subjected to vehicle and LMPs treatment. Histopathological analysis of bronchial sections revealed the effect of LMPs on the structural integrity of the b...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K visualizações.  University of Montréal.  Micropartículas verter à membrana celular (MPs) são vesículas biológicas activas que podem ser isolados e os seus efeitos patofisiológicos investigados em vários modelos. Descrevemos aqui um método para a geração de MPs derivadas de linfócitos T (LMPS) e para demonstrar o seu efeito pró-apoptóticos em células epiteliais das vias respiratórias. 