This work is funded by R.J. Reynolds Tobacco Company (RJRT) under a collaborative research agreement with Wake Forest University School of Medicine. G.L. Prasad is a full time employee of RJRT.

NOTE: Written Informed consent to do this study was obtained at a local clinical research unit under IRB approval, per Good Clinical Practices. Processing of blood, isolation of PBMCs and other cell culture experiments are performed under sterile conditions, using microbiologically sterile supplies and reagents.
1. WS-CM Preparation
<ol>
	<li>Generate WS-CM as previously described12.
	<ol>
		<li>Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Par...

<ol>
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Res. 6, 74 (2005).</li><li>Vayssier, M., Favatier, F., Pinot, F., Bachelet, M., Polla, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+induces+coordinate+activation+of+HSF+and+inhibition+of+NFkappaB+in+human+monocytes:+effects+on+TNFalpha+release.">Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release.</a> Biochem. Biophys. Res. Commun. 252, 249-256 (1998).</li><li>Witherden, I. R., Vanden Bon, E. J., Goldstraw, P., Ratcliffe, C., Pastorino, U., Tetley, T. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Regulatory+Science:+Research+to+Inform+Regulatory+Action+at+the+Food+and+Drug+Administration's+Center+for+Tobacco+Products.">Tobacco Regulatory Science: Research to Inform Regulatory Action at the Food and Drug Administration's Center for Tobacco Products.</a> Nicotine Tob. Res. , (2014).</li><li>Arimilli, S., Damratoski, B. E., Bombick, B., Borgerding, M. F., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytotoxicity+of+different+tobacco+product+preparations.">Evaluation of cytotoxicity of different tobacco product preparations.</a> Regul. Toxicol. Pharmacol. 64, 350-360 (2012).</li><li>Gao, H., Prasad, G. L., Zacharias, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cell-specific+cytotoxic+responses+of+oral+cavity+cells+to+tobacco+preparations.">Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.</a> Toxicol In Vitro. , (2012).</li><li>Arimilli, S., Damratoski, B. E., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combustible+and+non-combustible+tobacco+product+preparations+differentially+regulate+human+peripheral+blood+mononuclear+cell+functions.">Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.</a> Toxicol. In Vitro. 27, 1992-2004 (2013).</li><li>Arimilli, S., Damratoski, B. E., Chen, P., Jones, B. A., Prasad, G. 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The authors declare that there are no conflicts of interest.

We and others have previously demonstrated that treatment of PBMCs with TPPs suppresses several responses, including expression and secretion of cytokines and functional measures such as target cell killing14. The experimental methods described in the previous work require longer incubation periods and were modest in magnitude14. Given the potential applications of this attractive ex vivo model for basic and applied research, we investigated whether any of the assay parameters in these mult...

A substantial body of knowledge points to the adverse health effects of chronic cigarette smoking, including cardiovascular disease (CVD), chronic obstructive pulmonary disease (COPD) and cancer1,2. Chronic cigarette smoking has been known to cause inflammation and immune suppression, and these alterations are reported to contribute to increased risk of microbial infection and cancer in smokers3. In vitro and ex vivo techniques are useful in elucidating the molecular basis of the pathophysiological effects of cigarette smoke4-9 (Table 1) and are recognized as important tools for guiding the emerging regulation of various tobacco products10,11.
For example, we have demonstrated that combustible tobacco product preparations (TPPs) such as whole smoke-conditioned medium (WS-CM) and total particulate matter (TPM) are far more cytotoxic and damaging to DNA than non-combustible TPPs or nicotine12,13. Consistent with the published work, it was recently reported that combustible TPPs or nicotine12,13. Consistent with the published work, we recently reported that combustible TPPs caused marked immunosuppression. This was evidenced by suppression of Toll- like receptor (TLR)-ligands, stimulated cytokine secretion, and targeted cell (K562) killing by PBMCs in an ex vivo model14. Given the significance of inflammation in cigarette smoke-induced disease processes, further optimization of the assay conditions to evaluate the immune modulatory effects of cigarette smoke is presented in this report.
The ex vivo assays typically measured intracellular and secreted cytokines as well as the cytolytic function of cytotoxic T and NK cells in K562 cell killing assays14. The assays involved pre-incubation with WS-CM and nicotine and subsequent stimulation of PBMCs with TLR agonists over a period of 3 days; the final readouts are performed using enzyme-linked immunosorbent assays (ELISAs) and/or flow cytometry. We utilized bacterial lipopolysaccharide (LPS), which binds to TLR-4 receptors and stimulates PBMCs resulting in the production of intracellular cytokines and secretion of cytokines. In addition to optimization of the various assay steps for evaluating the immunomodulatory effects of TPPs, we also present methods for isolating PBMCs, cell death assays, and IL-8 quantification. These methods may be applied to address other research questions and further refined to evaluate tobacco products in the regulatory context.
Table 1. Published reports of in vitro and ex vivo methods used to study varilus pathophysiological effects of tobacco product preparations. CS, cigarette smoke medium; CSC, cigarette smoke condensate; CSE, cigarette smoke extract; ELISA, enzyme-linked immunosorbent assay; GADPH, glyceraldehyde 3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction; RT, real time quantitative polymerase chain reaction; TS, tobacco smoke.
<table>
	<tbody>
		<tr>
			<td>Author (Year of study)</td>
			<td>Laan et al. (2004)</td>
			<td>Moodie et al. (2004)</td>
			<td>Oltmanns et al. (2005)</td>
			<td>Vayssier (1998)</td>
			<td>Witherden et al. (2004)</td>
			<td>Birrell et al. (2008)</td>
		</tr>
		<tr>
			<td>Cells used</td>
			<td>Human bronchial endothelium cells (BEAS-2B), human neutrophils</td>
			<td>Human alveolar epithelial cells (A549)</td>
			<td>Human airway smooth muscle cells (HASMC)</td>
			<td>Human premonocytic U937 cells, human monocytes</td>
			<td>Alveolar type II epithelial cells (ATII)</td>
			<td>Human monocytic cell line (THP-1), human lung macrophages</td>
		</tr>
		<tr>
			<td>TPP used</td>
			<td>CSE</td>
			<td>CSC</td>
			<td>CSE</td>
			<td>TS</td>
			<td>CSE</td>
			<td>CS</td>
		</tr>
		<tr>
			<td>Method used</td>
			<td>ELISA, qPCR, migration, electromobility shift</td>
			<td>Immunohisto-chemistry, electrophoresis, Arrayscan kit, RT-PCR, ELISA</td>
			<td>ELISA, RT-PCR, qPCR, electrophoresis</td>
			<td>Gel-mobility shift</td>
			<td>Light microscopy, electron microscopy, electrophoresis, ELISA</td>
			<td>qPCR, ELISA, E-toxate kit (Sigma), p65 plate assay (TransAM), electrophoresis, various immunoassay kits</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>IL-8, GM-CF, AP-1, NF-&#954;B, migration</td>
			<td>Histone acetyltransferases, histone deacetylases, NF-&#954;B, IL-8, p-I &#954;B-&#945;, GADPH</td>
			<td>HO-1, GADPH, RANTES, IL-8, eotaxin</td>
			<td>Heat shock/stress proteins (HSP/Hsp70), HF transcription factor, NF-&#954;B, TNF-&#945;</td>
			<td>Surfactant protein (SP-A, SP-C), IL-8, MCP-1, GRO-&#945;, TNF-&#945;, IL-1&#946;, IFN-&#947;</td>
			<td>IL-8, IL-1&#946;, IL-6, TNF-&#945;, MIP1-&#945;, GRO-&#945;, MAPK/JNK/ERK phosphorylation, cJUN:DNA binding, glutathione, p65:DNA binding</td>
		</tr>
		<tr>
			<td>End result</td>
			<td>CSE down-regulates cytokine production via suppression of AP-1 activation.</td>
			<td>H2O2 and CSC enhance acetylation of histone proteins, decrease histone deacetylase activity, differentially regulate proinflammatory cytokine release.</td>
			<td>Cigarette smoke may cause the release of IL-8 from HASMC, enhanced by TNF-&#945;, 20% CSE less IL-8 release, Inhibition of eotaxin and RANTES by cigarette smoke.</td>
			<td>TS activated HF transcription factor, which was associated with Hsp70 overexpression and inhibition of NFkB binding activity and TNF-&#945; release.</td>
			<td>Reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.</td>
			<td>Data provide mechanistic explanation for why smokers have increased respiratory infections. Suppression of the innate response is accompanied by an increase in IL-8.</td>
		</tr>
	</tbody>
</table>

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">12 X 75 tubes</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">2 mL Microtubes</td>
			<td style="width:148px;">Axygen</td>
			<td style="width:119px;">MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">3R4F reference cigarettes&#160;&#160;</td>
			<td style="width:148px;">Univ. of Kentucky, College of Agriculture</td>
			<td style="width:119px;">3R4F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">50 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">500 ml bottle</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">7AAD</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">559925</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well flat bottom plate</td>
			<td>Termo Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">96 well round bottom plates</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture hood</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:119px;">1300 Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Centrifuge</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:119px;">58110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CFSE</td>
			<td style="width:148px;">Molecular Probes Life Technologies</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cluster tubes</td>
			<td style="width:148px;">Corning</td>
			<td>4401</td>
			<td>Harmful if swallowed, carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cytofix/Cytoperm (Permwash)</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555028</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMSO (Dimethyl sulfoxide )</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DPBS</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:119px;">17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FBS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">F2442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCAP Array</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">652099</td>
			<td>Software analyzes CBA data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Filter unit</td>
			<td style="width:148px;">Nalgene</td>
			<td style="width:119px;">156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Canto II</td>
			<td>&#160;8 colors, at Ex 405 and Em785.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Calibur</td>
			<td>&#160;4 colors at Ex 495 and Em 785.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Flow cytometry analysis software</td>
			<td style="width:148px;">Tree Star</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Freezing Container</td>
			<td style="width:148px;">Nalgene</td>
			<td>5100-0001</td>
			<td>Contains DMSO,&#160; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">GogliPlug</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td>Carcinogen, Irritant, Corrosive&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:223px;">H2SO4</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">339741</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human Inflammatory Cytokine Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IFN-&#947; V-500 Antibody</td>
			<td style="width:148px;">BD Horizon</td>
			<td style="width:119px;">561980</td>
			<td>&#160;skin sensitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IL-8 ELISA Kit</td>
			<td style="width:148px;">R and D Systems</td>
			<td style="width:119px;">DY208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isolation Buffer</td>
			<td style="width:148px;">Isolymph, CTL Scientific Corp.</td>
			<td style="width:119px;">1114868</td>
			<td>Flammable liquid, Irritant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Isopropyl alcohol</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">L-Glutamine</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LPS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIP1-&#945; PE Antibody</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">554730</td>
			<td>Acute toxicity, Oral</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Monensin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotine</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3876</td>
			<td>Acute toxicity, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Parafilm</td>
			<td style="width:148px;">Bemis</td>
			<td style="width:119px;">&#8220;M&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>Flammable, Skin irritation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Pen/strep</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Running buffer</td>
			<td style="width:148px;">MACS Running Buffer, Miltenyi Biotech</td>
			<td style="width:119px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Th1/Th2 CBA Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551809</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">TNF-&#945; Alexa Fluor 488 Antibody</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:119px;">502915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Transfer pipette</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tris Base</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

The results were presented as mean &#177; standard error of the mean (four donor samples). The student&#8217;s t-test between treated and untreated control samples was performed using Excel software as well as t-test comparisons for all treatments with their corresponding controls. The statistical significance was indicated by: *, P &#60; 0.05; **, P &#60; 0.005; ***, P &#60; 0.0005.
To measure the effect of exposure to WS-CM and nicotine, PBMCs were treated with different concentrations of WS...

Watch this Scientific Journal Video about Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations at JoVE.com

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

JoVE Journal

Immunology and Infection

11.3K visualizações.  Wake Forest University Health Sciences. Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. 