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 emerging regulation of various tobacco products^10,11.

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 nicotine^12,13. Consistent with the published work, it was recently reported that combustible TPPs or nicotine^12,13. 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.

Author (Year of study)	Laan et al. (2004)	Moodie et al. (2004)	Oltmanns et al. (2005)	Vayssier (1998)	Witherden et al. (2004)	Birrell et al. (2008)
Cells used	Human bronchial endothelium cells (BEAS-2B), human neutrophils	Human alveolar epithelial cells (A549)	Human airway smooth muscle cells (HASMC)	Human premonocytic U937 cells, human monocytes	Alveolar type II epithelial cells (ATII)	Human monocytic cell line (THP-1), human lung macrophages
TPP used	CSE	CSC	CSE	TS	CSE	CS
Method used	ELISA, qPCR, migration, electromobility shift	Immunohisto-chemistry, electrophoresis, Arrayscan kit, RT-PCR, ELISA	ELISA, RT-PCR, qPCR, electrophoresis	Gel-mobility shift	Light microscopy, electron microscopy, electrophoresis, ELISA	qPCR, ELISA, E-toxate kit (Sigma), p65 plate assay (TransAM), electrophoresis, various immunoassay kits
Measure	IL-8, GM-CF, AP-1, NF-κB, migration	Histone acetyltransferases, histone deacetylases, NF-κB, IL-8, p-I κB-α, GADPH	HO-1, GADPH, RANTES, IL-8, eotaxin	Heat shock/stress proteins (HSP/Hsp70), HF transcription factor, NF-κB, TNF-α	Surfactant protein (SP-A, SP-C), IL-8, MCP-1, GRO-α, TNF-α, IL-1β, IFN-γ	IL-8, IL-1β, IL-6, TNF-α, MIP1-α, GRO-α, MAPK/JNK/ERK phosphorylation, cJUN:DNA binding, glutathione, p65:DNA binding
End result	CSE down-regulates cytokine production via suppression of AP-1 activation.	H2O2 and CSC enhance acetylation of histone proteins, decrease histone deacetylase activity, differentially regulate proinflammatory cytokine release.	Cigarette smoke may cause the release of IL-8 from HASMC, enhanced by TNF-α, 20% CSE less IL-8 release, Inhibition of eotaxin and RANTES by cigarette smoke.	TS activated HF transcription factor, which was associated with Hsp70 overexpression and inhibition of NFkB binding activity and TNF-α release.	Reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.	Data provide mechanistic explanation for why smokers have increased respiratory infections. Suppression of the innate response is accompanied by an increase in IL-8.

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 puff duration in sec, respectively. Each preparation generates a 20 ml sample.
2. Label tube(s) with date, time completed, and cigarette name and number. Store the 500 µl aliquots at -80 °C immediately after smoking is completed.
3. Analyze aliquots of frozen WS-CM to determine the levels of nicotine, tobacco specific nitrosamines, and polycyclic aromatic hydrocarbons as previously described¹².

2. Isolation of PBMCs

1. Collect fresh blood from healthy donors (who are non-consumers of tobacco products) Isolate PBMCs from fresh blood as described below under a separate approval from Wake Forest Baptist Health IRB¹⁵.
   1. Prior to the arrival of the blood bag, have a 500 ml bottle, scissors, isolation buffer, and Dulbecco’s phosphate buffered saline (DPBS) (RT) ready in a biosafety level 2 (BSL-2) cell culture hood. Isolation buffer must be protected from light.
   2. Hold the blood bag upside down and cut the tube just below where it has been clamped leaving at least 3 cm of tubing.
   3. Remove the cap from the 500 ml bottle and hold the tube above the bottle opening. Pick up the blood bag and invert it to allow the blood to flow freely from the bag into the bottle until the bag is empty. Allow the blood to flow onto the inside wall of the bottle versus straight down as to avoid creating bubbles.
   4. Pour isolation buffer into the bottle at a 1:5 ratio of isolation buffer to blood. Cap the bottle tightly and gently invert it, end-over-end, 10 times. Leave the bottle in the cell culture hood to incubate with lights off, for 1 hr at RT.
   5. A light, straw-colored layer will build up above the blood. Remove this layer using a 25 ml serological pipette into 50 ml conical tubes. The collected amount may vary from 50 - 300 ml, depending on the subject who donated blood.
   6. Centrifuge the tubes at 200 x g for 10 min at RT.
   7. Aspirate the translucent supernatant, leaving the dark blood-colored pellet. The pellet will be loose but viscous.
   8. Pipette 3 ml of isolation buffer into 15 ml conical tubes.
   9. To the resuspend pellet, add 20 ml of DPBS for every 50 ml of straw-colored liquid collected in step 2.1.5. Vortex to mix thoroughly, and consolidate the resuspended liquid from multiple tubes. This liquid contains suspended blood cells.
   10. With a transfer pipet, transfer 5 ml of the suspended blood cells onto 3 ml of isolation buffer in step 2.1.8. Tilt the 15 ml conical tube that contains the cell suspension slowly and gently to create two separate layers. Centrifuge the tubes at 400 x g for 40 min at RT with minimal acceleration and without brake.
   11. Use a transfer pipet to remove the resulting cloudy middle layer (buffy coat) containing PBMCs into a 50 ml conical tube. Avoid drawing other clear layers below it. Transfer no more than 25 ml into each 50 ml conical tube.
   12. Add 25 ml of cold running buffer (or more to fill the entire remaining volume of the 50 ml conical tube) to wash the cells. Centrifuge the cells at 400 x g for 10 min at 4 °C.
   13. Resuspend the pellet with 10 ml running buffer. This contains PBMCs. Count the cells and use immediately or place on ice for freezing.

3. Freezing and Thawing the PBMCs

1. Centrifuge the PBMCs that were collected in step 2.1.13 for 10 min at 400 x g.
2. Fill the freezing container with isopropyl alcohol per the manufacturer’s instructions. CAUTION: Isopropyl alcohol is flammable and acutely toxic.
3. Resuspend the pellet with RPMI 1640 medium (4 °C) containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). This is RPMI 1640 freezing medium. Resuspend the pellet with an amount of freezing medium that will result in a suspension having about 5 x 10⁷ cells/ml. The number of cells available for freezing will vary.
4. Dispense 1 ml aliquots of cell suspension into 2 ml cryotubes and place the cryotubes in the freezing container. Place the freezing container with cryotubes in a freezer at -80 °C to store O/N and then remove the cryotubes and transfer to store in a cryogenic freezer between -150 °C to -190 °C.
5. Remove the cryotube from cryogenic storage and thaw it rapidly with gentle agitation in a water bath at 37 °C.
6. Immediately transfer the thawed PBMCs in the cryotube into a 15 ml conical tube with 10 ml RPMI 1640 complete medium (4 °C) containing 10% FBS, 1% Pen/Strep and 1% L-glutamine. The contents of the thawed cryotube should be transferred as soon as possible to obtain maximal cell viability¹⁵.
7. Centrifuge the PBMCs at 400 x g for 10 min.
8. Resuspend the pellet with 5 ml RPMI 1640 complete medium and count the cells.
9. Measure cell viability by established methods such as trypan blue exclusion method. Generally cell viability with this method is about 90 - 95%. The PBMCs are now ready to use in experiments.

4. Cell Death Determination

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be changed accordingly.

1. Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well, as indicated below.
2. Dilute WS-CM to the following concentrations: 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 µg/mL of equi-nicotine units (based on the nicotine content in WS-CM)¹².
3. Dilute nicotine in RPMI medium to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 µg/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.
4. Add 100 µl of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 10⁶ cells/well. The total volume of cells plus WS-CM or nicotine will be 200 µl/well.
5. For the purpose of this study, prepare two sets of plates as above. Cover the plates and incubate one plate for 24 hr and one plate for 3 hr at 37 °C and 5% CO₂. Adjust time points as necessary.
6. Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 µl of ice cold running buffer, and repeat the washing step one more time.
7. Add 95 µl of running buffer followed by 5 µl of 7-aminoactinomycin D (7AAD) to each well for a total volume of 100 µl/well. Incubate the plate in the dark at RT for 15 min.
8. Add 100 µl of running buffer to cluster tubes. Transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.
9. Determine the percentage of 7AAD-positive cells using flow cytometry analysis software.

5. EC₅₀ Determination

1. The EC₅₀ values of WS-CM and nicotine are determined by 7AAD-positive staining of PBMCs.
2. The EC₅₀ value is defined as the concentration at which 50% of the cells were no longer viable in a 24 hr assay, and the values are expressed as μg of equi-nicotine units/ml.
3. For the purposes of this study, the EC₅₀ values were determined to be 1.56 μg/ml and 1,650 µg/ml for WS-CM and nicotine, respectively.

6. Secreted Cytokines

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.

1. Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well at the concentration of 0.3, 1.56, 3, and 5 µg/ml of equi-nicotine units.
2. Dilute nicotine to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 µg/mL. CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 10⁶ cells/well. The total volume of cells plus WS-CM or nicotine will be 200 µl/well.
4. Cover the plate and incubate for 3 h at 37 °C and 5% CO₂.
5. Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally suspending cells with 200 µl of ice cold running buffer and repeating the washing step one more time.
6. Add 200 µl of RPMI complete medium, and repeat the washing step one more time.
7. Add 200 µl of 10 µg/ml LPS medium to each well.
8. Cover the plate and incubate for 4 hr, 24 hr, 48 hr, or 72 hr at 37 °C and 5% CO₂. Adjust incubation times as necessary.
9. Centrifuge the plate at 300 × g for 3 min.
10. Take 175 µl of supernatant from each well and store in a freezer at -80 °C to perform the assays in steps 7 and 8.

7. Cytometric Bead Array Assay

1. Thaw the cell supernatants prepared from step 6.10 and use in the CBA assay. Perform the cytometric bead array (CBA) assay as per the manufacturer’s instructions.

8. IL-8 ELISA

1. Thaw the cell supernatants from step 6.10 and use in the IL-8 ELISA. Perform the ELISA assay as per the manufacturer’s instructions.

9. Intracellular Staining and Flow Cytometry

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.

1. Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well at the concentration of 0.3, 1.56, 3, and 5 µg/ml of equi-nicotine units.
2. In the same plate, dilute nicotine to the following concentrations: 2, 10, 50, 100, 500, 2000, and 4000 µg/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs suspended in RPMI complete medium at a concentration of 1 × 10⁶ cells/well. The total volume of cells plus WS-CM or nicotine will be 200 µl/well.
4. Cover the plate and incubate for 3 hr at 37 °C and 5% CO₂.
5. Wash the cells at RT by centrifuging at 300 × g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 µl of ice cold running buffer, and repeat the washing step one more time.
6. Add 200 µl of RPMI complete medium to the plate, and repeat the wash step one more time.
7. Prepare the working concentrations of 2 µl/ml GolgiPlug and 10 µg/ml LPS using RPMI complete medium and add 200 µl to each well.
8. Incubate the plate for 6 hr at 37 °C and 5% CO₂.
9. At the end of step 9.8, wash the cells with running buffer (4 °C) and spinning at 300 x g for 3 min at 4 °C.
10. Add 100 µl of Cytofix to each well and incubate for 20 min at 4 °C.
11. Wash the cells 3 times as described in step 9.9 with 1x Permwash (4 °C) at 300 x g for 3 min at 4 °C.
12. Add 45 µL of 1x Cytoperm to each well followed by 5 µl of each of one of the following antibodies to each well: TNF-α-Alexa Fluor 488, IFN-γ V500, MIP-1α PE. Incubate at 4 °C for 30 min.
13. Wash the cells two times as described in step 9.9 with 1x Permwash (4 °C) and one time with running buffer (4 °C) at 300 x g for 3 min at 4 °C.
14. Resuspend the cells with 200 µl of 2% paraformaldehyde (4 °C). Transfer the cells into 12 × 75 mm tubes, and analyze the samples on flow cytometer. CAUTION: Paraformaldehyde is corrosive, acutely toxic and a health hazard.

10. K562 Killing Assay

NOTE: K562 cells should be grown in culture at 37 °C and 5% CO₂ with RPMI complete medium until they reach 80% confluence before the assay.

1. Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 µl of DMSO to the vial.
2. Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well to achieve the desired equi-nicotine units or nicotine concentrations for each well. CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs into RPMI complete medium at a concentration of 1.5 x 10⁶ cells/well. The total volume of cells with WS-CM or nicotine will be 200 µl/well.
4. Cover the plate and incubate for 3 hr at 37 °C and 5% CO₂.
5. Wash the K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.
6. Resuspend the cells with 10 ml of DPBS and count the K562 cells.
7. Prepare CFSE working solution by adding 1 µl of CFSE stock solution to 1 ml DPBS.
8. Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 - 2 x 10⁷ cells. Vortex and incubate precisely for 2 min at RT.
9. Immediately add 200 μl of FBS. Vortex and incubate precisely for 2 min at RT.
10. Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.
11. Remove the RPMI supernatant and break the pellet and resuspend the cells with 10 ml of RPMI complete medium and count the CFSE-labeled K562 cells.
12. Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 µl of RPMI complete medium and repeat washing step one more time.
13. Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 10⁶ PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 °C and 5% CO₂.
14. Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.
15. Add 200 µl of running buffer and repeat the washing step described in step 10.14 one more time.
16. Add 95 µl of running buffer followed by 5 µl of 7AAD to each well for a total volume of 100 µl/well. Incubate the plate in the dark at RT for 15 min.
17. Add 100 µl of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.
18. Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.

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