Name: identification of intracellular signaling events induced in viable cells by interaction with neighboring cells undergoing apoptotic cell death
Uploaded: 2016-12-27T13:00:00
Description: Watch this Scientific Journal Video about Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death at JoVE.com

This work was supported, in whole or in part, by institutional funds from Dr. Jose&#769; A. Arruda and the Section of Nephrology, University of Illinois at Chicago (to J. S. L.); and funding from the Department of Medicine of McGill University and the Research Institute of the McGill University Health Centre (to J. R.)

1. Preparations
Note: In this protocol, two or more cell lines, or primary cell cultures, are independently prepared under specific conditions. These preparations include apoptotic cells (Protocol 1), necrotic cells (Protocol 2), and healthy responder cells (Protocol 3). After independent preparation, apoptotic or necrotic cells are added to responder cells and the resulting signal transduction is monitored (Protocols 4, 5, and 6).
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	<li>Prepare culture media and reagents
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		<li>Pre...

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We provide here a protocol for characterizing the intracellular signaling events induced in viable cells following their exposure to cells undergoing apoptosis, or other forms of cell death. The protocol emphasizes the multiple mechanisms by which exposure to apoptotic cells can modulate intracellular signaling pathways within viable responder cells. These mechanisms include: the interaction (via bridging molecules or surface determinants on the dead cell or its shed vesicles and microvesicles, often termed apoptotic bod...

Apoptosis, or regulated cell death1, contributes in an essential manner to the maintenance and development of tissues. Viewed most simply, apoptosis permits aged, damaged, or excess cells to be eliminated without harm to surrounding tissues2,3. The contribution of apoptosis to tissue homeostasis, however, is considerably more dynamic and varied. Cells dying by apoptosis acquire multiple new activities, both secreted and cell-associated, which enable them to influence the function of adjacent live cells4-10. Earlier studies focused on the ability of apoptotic cells to suppress inflammation11-16, but apoptotic cells also modulate a broad range of cellular functions, including such vital activities as survival4,9,10, proliferation4,9,10, differentiation17, migration18, and growth19. Moreover, these effects are not restricted to professional phagocytes, like macrophages, but extend to virtually all cell types and lineages, including traditionally non-phagocytic cells, such as epithelial and endothelial cells7,9,10,18-21.
The specific response of adjacent live cells following exposure to apoptotic cells depends on multiple factors that relate to both the viable responding cells and the apoptotic cells themselves. For example, although exposure to apoptotic cells inhibits the proliferation of both murine macrophages and kidney proximal tubular epithelial cells (PTECs), these two cell types differ dramatically in their survival response to apoptotic cells4,6,9,10. Apoptotic cells promote the survival of macrophages, but induce the apoptotic death of PTECs4,6,9,10. Notably, the response to apoptotic cells may differ even among responding cells of the same lineage, depending on the responding cell&#39;s organ of origin10 (e.g., kidney PTECs versus mammary epithelial cells) or state of activation22 (e.g., neutrophils). Conversely, apoptotic cells may evoke different responses in the very same cell depending on the nature of the apoptotic stimulus10,23 or the stage of apoptosis10,14.
Given the diverseness and complexity of the response to apoptotic cells, great care must be taken in dissecting the signaling events and pathways responsible for any particular outcome. First, responses requiring direct physical interaction between viable and apoptotic cells must be differentiated from those elicited by soluble mediators released or generated by the apoptotic cell3,6-10. If physical interaction is required, then a further differentiation should be performed. Signaling events may depend on receptor-mediated recognition of the apoptotic cell, independent of subsequent engulfment, or on phagocytic uptake, independent of the specific receptor that binds the apoptotic cell3-7,9,10,19. In the latter instance, the response is not specific to apoptotic cells, and can be triggered by any phagocytic material4,6.
The importance of these distinctions can again be appreciated by contrasting the responses of macrophages and kidney PTECs. For both cell types, exposure to apoptotic cells alters the activity of the pro-survival kinase Akt. Modulation is dependent on physical interaction, since separation of responding and apoptotic cells by a 0.4 &#956;m polycarbonate membrane abolishes the response4,9,10,19. However, the response of PTECs is receptor-mediated and independent of phagocytosis, whereas the response of macrophages is driven by phagocytosis4,9,10,19. This conclusion is reinforced by the fact that exposure to latex beads, a neutral phagocytic stimulus, has no effect on Akt activity in PTECs, but mimics the effect of apoptotic cells in macrophages4,9.
Although less well studied, cells dying by necrosis, or accidental cell death1, also modulate the function of nearby viable cells3-10,19. Like apoptotic cells, necrotic cells exert their effects through a variety of mechanisms, particularly the leakage of intracellular contents through their ruptured cell membrane3,5,6,9,24. Many cells, including PTECs and macrophages, possess distinct non-competing receptors for cells dying by necrosis5,9. Engagement of these receptors induces signaling events that are often opposite to those induced by engagement of the receptors for apoptotic cells4-10,19. For example, in PTECs, necrotic cells increase phosphorylation of Akt, whereas apoptotic cells decrease phosphorylation9,10,19.
Here, we describe a protocol for identifying intracellular signaling events induced in viable kidney PTECs through receptor-mediated recognition of adjacent apoptotic PTECs9,10,19. While the protocol is specific for a conditionally immortalized PTEC cell line known as BU.MPT cells9,10,19,25,26, it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. Importantly, the use of apoptotic cells as a cell stimulus poses certain inherent experimental difficulties not present with soluble ligands. The most important of these is that apoptotic cells must be added as a suspension rather than as a solution. These difficulties, as well as strategies to minimize or circumvent them, are discussed within the protocol. Nearly all of the techniques described in the protocol are straightforward and standard to cell culture. The advantage of this protocol lies in its attention to the multiple mechanisms by which apoptotic cells modulate signal transduction within responding cells. These mechanisms include the binding via surface determinants or bridging molecules to specific receptors on the responding cell, the release of soluble mediators, and/or the engagement of the phagocytic machinery. Necrotic cells are included in all experiments to ensure that results are specific to the mode of cell death, and not a generalized response to dead cells. A careful approach, as recommended in this protocol, is critical to our understanding of the ever expanding influence that dying cells exert on their live neighbors, in both health and disease.

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			<td height="21" style="height:21px;width:461px;">Cell culture materials</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, 1X with [4.5 g/L] glucose, L-glutamine &#38; sodium-pyruvate</td>
			<td>Mediatech (Manassas, VA)</td>
			<td style="width:127px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone Fetal Clone II, fetal bovine serum</td>
			<td style="width:263px;">GE Healthcare (Logan, UT)</td>
			<td style="width:127px;">SH30066.03</td>
			<td>add 10% (v/v)&#160; to the culture medium A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine, 100X</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">10378-016</td>
			<td>add 100 units/mL to the culture media A and B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-Interferon, mouse recombinant, from Escherichia coli</td>
			<td style="width:263px;">Calbiochem, Millipore (Billerica, MA)</td>
			<td style="width:127px;">407303</td>
			<td>add 10 units/mL to the culture medium A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Falcon polystyrene tissue-culture treated dishes, non-pyrogenic</td>
			<td style="width:263px;">Corning (Durham, NC)</td>
			<td style="width:127px;">353003</td>
			<td>sterile, 100 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Falcon polystyrene centrifuge tubes, conical bottom</td>
			<td style="width:263px;">Corning (Durham, NC)</td>
			<td style="width:127px;">352095</td>
			<td>sterile, 15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Trypsin-EDTA, 0.05%, Gibco</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td style="width:127px;">25300062</td>
			<td>sterile, 100 mL</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals and inhibitors</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staurosporine, from Streptomyces sp.</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">S4400</td>
			<td>use at [1 &#956;g/mL] for 3 h to induce apoptosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Epidermal growth factor (EGF), [1 mg/ml]</td>
			<td style="width:263px;">Millipore (Billerica, MA)</td>
			<td style="width:127px;">EA140</td>
			<td>use at 50 nM to stimulate responder cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">UltraPure, 0.5 M EDTA, pH 8.0</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">15575-038</td>
			<td>use 5 mM to detach adherent cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Trypan blue solution, 0.4%</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">91691049</td>
			<td>for dead cells staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Paraformaldehyde Solution, 4% in DPBS</td>
			<td style="width:263px;">Affymetrix (Santa Clara, CA)</td>
			<td>19943</td>
			<td>use 0.4% (v/v) in 1&#215; DPBS for cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dulbecco&#39;s phosphate-buffered saline (DPBS), 1&#215;, Gibco</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td>14040133</td>
			<td>stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Dulbecco&#39;s phosphate-buffered saline (DPBS), Ca+2- and Mg+2-free, 1&#215;, Gibco</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">14190367</td>
			<td>stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Cytochalasin D, 4 mM in dimethyl sulfoxide (DMSO), from Zygosporium mansonii</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">C8273</td>
			<td>use at [4 &#956;g/mL] to inhibit phagocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">D2650</td>
			<td>solvent for cytochalasin D</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell lysis buffer ingredients</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Tris-buffered saline (TBS), 10&#215;, pH 7.4</td>
			<td style="width:263px;">BioRad (Hercules, CA)</td>
			<td style="width:127px;">1706435</td>
			<td>for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium pyrophosphate</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">221368</td>
			<td>10 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium deoxycholate</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">D6750</td>
			<td>0.5% w/v for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">L3771&#160;</td>
			<td>0.1% w/v for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">G5516</td>
			<td>10% for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium fluoride</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">450022</td>
			<td>25 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Complete mini EDTA-free protease inhibitor cocktail tablet</td>
			<td style="width:263px;">Roche Diagnostics (Indianapolis, IN)</td>
			<td style="width:127px;">4693159001</td>
			<td>for cell lysis buffer, 1 tablet per 10 mL of buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Triton X-100</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">T8787</td>
			<td>10% for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dithiothreitol (DTT)</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">11DTT00005</td>
			<td>1 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">4800263</td>
			<td>1 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium orthovandate</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td>2159664</td>
			<td>10 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sorvall T6000B centrifuge</td>
			<td style="width:263px;">Du Pont (Wilmington, DE)</td>
			<td style="width:127px;">T6000B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Fisher Tissuemat, water bath</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sonic Dismembrator (sonicator), model 100</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miscellaneous</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Cell counting chamber, Neubauer</td>
			<td style="width:263px;">Hawksley (Lancing, Sussex, UK)</td>
			<td style="width:127px;">AC2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Annexin V-FITC Apoptosis Detection Kit II</td>
			<td style="width:263px;">Millipore (Billerica, MA)</td>
			<td style="width:127px;">CBA059</td>
			<td>for PI and Annexin V staining to determine the stage of apoptosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Eppendorf microcntrifuge tubes, 1.5 ml</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Transwell permeable support, 0.4 &#956;m polycarbonate membrane</td>
			<td style="width:263px;">Corning (Corning, NY)</td>
			<td style="width:127px;">3413</td>
			<td>sterile, polystyrene, tissue culture treated</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibodies</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Phospho-glycogen synthase kinase-3&#945;/&#946; (GSK-3&#945;/&#946;) (Ser21/9), polyclonal antibody</td>
			<td style="width:263px;">Cell Signaling Technology</td>
			<td style="width:127px;">9331</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antobody ( (14C10) )</td>
			<td style="width:263px;">Cell Signaling Technology</td>
			<td style="width:127px;">2118</td>
			<td>rabbit monoclonal antibody</td>
		</tr>
	</tbody>
</table>

identification of intracellular signaling events induced in viable cells by interaction with neighboring cells undergoing apoptotic cell death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

In Figure 1, we provide a schematic depicting the timing and critical steps involved in the preparation of apoptotic cells (Protocol 1) and necrotic cells (Protocol 2) and the stimulation of responder cells with suspensions of apoptotic and necrotic cells (Protocol 4).
In Figure 2, we provide representative results showing the effect of apoptotic cells on phosphorylation of the two isoforms of t...

Watch this Scientific Journal Video about Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death at JoVE.com

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

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