Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Summary

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.

Abstract

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-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α 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.

Introduction

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 cancer^1,2. 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 smoke^4-9 (Table 1) and are recognized as important tools for guiding the e....

Protocol

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

1. Generate WS-CM as previously described¹².
   1. Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red using the following smoking regimen: 35-60-2, puff volume in ml, puff interval in sec, and ....

Results

The results were presented as mean ± standard error of the mean (four donor samples). The student’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 < 0.05; **, P < 0.005; ***, P < 0.0005.

To measure the effect of exposure to WS-CM and nicotine, PBMCs were treated with different concentrations of WS-CM and nicoti.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 x 75 mm tubes	BD Falcon	352058
15 ml conical tubes	Corning	430790
2 ml microtubes	Axygen	MCT-150-C-S
3R4F reference cigarettes	Univ. of Kentucky, College of Agriculture	3R4F
50 ml conical tubes	Corning	430828
500 ml bottle	Corning	430282
7AAD	BD Pharmingen	559925
96-well flat bottom plate	Termo Nunc	439454
96-well round bottom plates	BD Falcon	353077
Cell culture hood	Thermo Scientific	1300 Series A2
Centrifuge	Eppendorf	58110R
CFSE	Molecular Probes Life Technologies	C34554
Cluster tubes	Corning	4401	Harmful if swallowed, carcinogen
Cytofix/Cytoperm (Permwash)	BD Biosciences	555028	Flammable
DMSO (dimethyl sulfoxide )	Sigma-Aldrich	D8418
DPBS	Lonza	17-512F
FBS	Sigma-Aldrich	F2442
FCAP Array	BD Biosciences	652099	Software analyzes CBA data
Filter unit	Nalgene	156-4020
Flow cytometer	BD Biosciences	FACS Canto II	8 colors, at Ex 405 and Em 785.
Flow cytometer	BD Biosciences	FACS Calibur	4 colors at Ex 495 and Em 785.
Flow cytometry analysis software	Tree Star	FlowJo
Freezing container	Nalgene	5100-0001	Contains DMSO, irritant
GogliPlug	BD Biosciences	555029	Carcinogen, irritant, corrosive
H2SO4	Sigma-Aldrich	339741
Human Inflammatory Cytokine Kit	BD Biosciences	551811
IFN-γ V-500 Antibody	BD Horizon	561980	skin sensitizer
IL-8 ELISA Kit	R and D Systems	DY208
Isolation buffer	Isolymph, CTL Scientific Corp.	1114868	Flammable liquid, irritant
Isopropyl alcohol	Sigma-Aldrich	W292907
L-Glutamine	Gibco Life Technologies	25030-081
LPS	Sigma-Aldrich	L2630
MIP1-α PE Antibody	BD Pharmingen	554730	Acute toxicity, oral
Monensin	Sigma-Aldrich	M5273
NaCl	Sigma-Aldrich	S7653
Nicotine	Sigma-Aldrich	N3876	Acute toxicity, environmental hazard
Parafilm	Bemis	“M”
Paraformaldehyde	Sigma-Aldrich	P6148	Flammable, skin irritation
Pen/strep	Gibco Life Technologies	15140-122
RPMI 1640	Gibco Life Technologies	11875-093
Running buffer	MACS Running Buffer, Miltenyi Biotech	130-091-221
Th1/Th2 CBA Kit	BD Biosciences	551809
TNF-α Alexa Fluor 488 Antibody	BioLegend	502915
Transfer pipette	Fisher Scientific	13-711-20
Tris Base	Sigma-Aldrich	T1503