Toxicology in Vitro 27 (2013) 19922004

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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Combustible and non-combustible tobacco product preparations

differentially regulate human peripheral blood mononuclear cell
functions
Subhashini Arimilli a,, Brad E. Damratoski a, G.L. Prasad b
a
b

Department of Microbiology & Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, United States
R&D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102, United States

a r t i c l e

i n f o

Article history:
Received 12 April 2013
Accepted 29 June 2013
Available online 11 July 2013
Keywords:
Tobacco product preparations (TPPs)
EC50
T cells
NK cells
PBMCs
Poly I:C
LPS
Cytotoxicity assay

a b s t r a c t
Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in
protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST)
products affects immune responses. In this study, we assessed the effects of two preparations of cigarette
smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated
human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested
included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product
preparation in complete articial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:
polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of
intracellular IFN-c and TNF-a was evident in NK cells and T cells treated with WS-CM and TPM.
Consistently, attenuation of ligand-induced secretion of cytokines (IL-1b, IL-10, IL-12 and TNF-a) from
PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the
expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human
PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle
confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations.
Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented
herein suggests that the function of NK cells and T cells is inuenced by exposure to TPPs (based on
equi-nicotine units) in the following order: WS-CM > TPM > ST/CAS. These ndings are consistent with
the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that
may contribute to increased microbial infections and cancer incidence among smokers.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Chronic cigarette smoking is known to affect immune responses
and compromise host defense against microbial infections and tumor surveillance (Holt and Keast, 1977). The mechanisms of
immunomodulatory effects of cigarette smoke on innate and adaptive immunity are active areas of investigation and subjects of
several recent reviews (Goncalves et al., 2011; Lee et al., 2012;
Yao and Rahman, 2011). Several clinical studies show that chronic
smoking induces inammation as seen by increases in white blood

Corresponding author. Address: Department of Microbiology & Immunology,

Wake Forest Baptist Health, Wake Forest Biotech Place, Room 2N-052, 575
Patterson Avenue, Winston Salem, NC 27101, United States. Tel.: +1 336 713
1390; fax: +1 336 716 9928.
E-mail address: sarimill@wakehealth.edu (S. Arimilli).
0887-2333/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tiv.2013.06.015

cells (Frost-Pineda et al., 2011) and the distribution and function of

leukocyte subsets are altered in smokers (Hoser et al., 2003; RoosEngstrand et al., 2010; Rumora et al., 2008). Similarly, in vitro studies show that exposure to cigarette smoke (or its components) results in altered immune responses as measured by decreased
interferon-gamma (IFN-c) and tumor necrosis factor-alpha (TNFa) secretion (Ouyang et al., 2000). Another characteristic response
to exposure to cigarette smoke (or its constituent phases) is increased secretion of interleukin-8 (IL-8) in several cell types
(example, human aortic and bronchial epithelial cells) (Nordskog
et al., 2005; Parsanejad et al., 2008).
Cigarette smoking adversely affects the functions of components of innate immunity, including natural killer cells (NKs) and
Toll-like receptors (TLRs). NK cells are a vital part of the innate
immune system and play an important role against microbial
infections and tumor surveillance through secretion of an array

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

of cytokines including IFN-c and TNF-a and by cytolysis of infected

or neoplastic cells (Bancroft, 1993; Biron, 1997; Biron et al., 1999;
Cerwenka and Lanier, 2001; Mian et al., 2008; Nadigel et al., 2011).
NK cell functions have also been shown to be signicantly reduced
by cigarette smoke (Mehta et al., 2008; Mian et al., 2008). TLRs are
involved in innate immunity by recognizing molecules that are
broadly shared by pathogens, and they activate a cascade of signaling pathways, triggering NF-jB and type-1 interferon production
(Reimer et al., 2008).
T cells are involved in the initiation and regulation of innate and
adaptive immune responses. T cell subsets play specic roles in
eliciting immune response, and smoking has been reported to decrease CD4/CD8 cell ratios (Runo et al., 2007). Furthermore, exposure to cigarette smoke alters T cell function including cytokine
secretion, anergy and proliferation (Chang et al., 1990; Lambert
et al., 2005; Petro and Zhang, 1997; Sopori and Kozak, 1998).
Cigarette smoke extracts inhibit the secretion of cytokines such
as IL-1b, IL-2, IFN-c and TNF-a by PBMCs (Ouyang et al., 2000).
These proinammatory cytokines are capable of driving NK cell
and cytotoxic T cell responses that are critical to tumor suppression (Ouyang et al., 2000; Yoneda et al., 1993). Given the importance of these cell types in the immune response, it has been
suggested that T cell and NK cell anergy resulting from smoking
could signicantly impair the ability to ght viral and bacterial
infections (Mehta et al., 2008; Phipps et al., 2010) as well as tumor
surveillance (Hogan et al., 2011b).
While cigarette smoking represents the most common form of
tobacco consumption, non-combustible tobacco such as, moist
snuff, snus, and other smoke-free products, exist in the marketplace. Relative to cigarettes, health risks associated with the use
of smokeless tobacco (ST) products have shown to be lower
(Hatsukami et al., 2002; Zeller et al., 2009). While the effects of cigarette smoking on immune function has been subject of active research, relatively less is known of how the use of ST alters immune
response. For example, some in vitro studies suggest ST elicits
immunostimulatory responses as observed by altered cytokine
secretion (Goud et al., 1993; Johnson et al., 1994, 1996; Petro
et al., 1999; Petro and Zhang, 1997). Therefore, we have initiated
investigation into the relative effects of combustible and non-combustible tobacco products using HL60 cells, PBMCs, and oral cavity
cells as representative in vitro models for systemic and local
(mucosal) exposure, respectively (Arimilli et al., 2012a; Gao
et al., 2013).
Cigarette smoke contains a particulate phase and a gasvapor
phase. The particulate phase, dissolved in DMSO (or alternate solvent) is known as total particulate matter (TPM) and is commonly
used in cell culture studies. A different method of exposing cells to
cigarette smoke involves the use of smoke-conditioned medium
generated by passing cigarette smoke through it; this preparation
is referred to as cigarette smoke extract (Sopori, 2002), wholesmoke conditioned medium (WS-CM) (Arimilli et al., 2012a) or
aqueous extract. Each of these TPPs is chemically distinct and
therefore could elicit different responses. Smokeless tobacco extracts may be generated in several different ways: example, direct
extraction in cell culture medium (Mitchell et al., 2010; Petro,
2003) or extraction in complete articial saliva (CAS) (Arimilli
et al., 2012a; Gao et al., 2013).
We have prepared WS-CM and TPM from 3R4F cigarettes and
extracts from 2S3 reference moist snuff ST in CAS, respectively.
Since these TPPs are chemically distinct, we used the nicotine concentration of the TPPs as a common measure to determine the
exposure of different TPPs. The biological effects due to TPP exposure were compared based on the nicotine content of the TPPs used
in various treatments in in vitro and ex vivo studies, and we termed
it equi-nicotine unit paradigm (Arimilli et al., 2012a; Gao et al.,
2013).

1993

The purpose of this study was to determine how the combustible and non-combustible TPPs modulate select immune responses.
We used PBMCs collected from non-smoking donors, exposed
them to TPPs and determined their responses upon stimulation
with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). Poly I:C, a synthetic double-stranded RNA analog, and
lipopolysaccharide (LPS), a major component of gram-negative
bacteria cell wall, have been used extensively to study the various
pathways of innate immune activation associated with specic
TLRs (Reimer et al., 2008). Poly I:C and LPS bind to TLRs and activate NF-jB, TNF-a and other signaling molecules. The ex vivo culture model has been used to investigate the mechanisms of
immunosuppression in smokers which are linked to the increased
susceptibility of smokers to infections and to tumors (Chen et al.,
2007; Lambert et al., 2005; Lee et al., 2012; Ouyang et al., 2000).
In this manuscript, we investigated the functional responses of
PBMCs, NK cells and T cells pre-treated acutely with cigarette
smoke-derived (combustible) and smokeless (non-combustible)
TPPs followed by stimulation with poly I:C and LPS. We also comparatively assessed the effects of nicotine pre-treatment on the
responsiveness of PBMCs to TLR ligand stimulation.

2. Materials and methods

2.1. Tobacco product preparations and EC50 values
The TPPs were prepared as described previously (Arimilli et al.,
2012a). Briey, TPM was prepared by smoking 3R4F reference cigarettes using the standard ISO method (Johnson et al., 2009b)
(35 mL, 60 s, 2 s; puff volume, frequency and duration, respectively) and dissolving the particulate phase, trapped on the Cambridge lter pad, in DMSO. Smokeless tobacco extract was
prepared by extracting 2S3 reference smokeless tobacco (ST)
(North Carolina State University Tobacco Services Analytical Laboratory) for 2 h in complete articial saliva (CAS) using a published
method (Chou and Hee, 1994). The CAS consists of mucin, salts
(potassium chloride, sodium chloride, calcium chloride dehydrate,
di-potasium hydrogen phosphate and magnesium chloride hexahydrate), urea, glucose and enzymes (alpha-amylase, lysozyme
and acid phosphatase).
We presented the above information at the 64th Tobacco Science
Research Conference held in Hilton Head, SC in 20101. WS-CM was
prepared by passing smoke from four 3R4F cigarettes through 20 mL
of RPMI 1640 medium (Invitrogen, Grand Island, NY) without phenol
red. Nicotine free base (SigmaAldrich, Milwaukee, WI) was used as a
reference. Aliquots of frozen TPPs were analyzed for nicotine, tobacco
specic nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons
(PAHs) at Labstat International (Kitchener, Ontario, Canada) using
published methods (Rickert et al., 2009; Wu et al., 2008).
We tested TPPs and nicotine at different doses based on the
equi-nicotine unit paradigm. The EC50 values of different TPPs were
determined by 7-aminoactinomycin D (7AAD) positive staining of
PBMCs. The EC50 is dened as the concentration at which 50% of
the cells were no longer viable in a 24 h assay and the values are
expressed as lg of equi-nicotine units/mL (Arimilli et al., 2012a).
The EC50 values of combustible preparations TPM and WS-CM were
determined to be 2.58 lg/mL and 1.56 lg/mL of equi-nicotine units,
respectively (Arimilli et al., 2012a). As discussed in a previous
publication Arimilli et al. (2012a), EC50 values for non-combustible
ST/CAS could not be determined due to its low cytotoxicity, which
required addition of substantial (>30%) volumes of ST/CAS into cell
1
Standardization of the Preparation of Smokeless Tobacco Extracts for Assessment
of Biological Effects. Kathy Fowler, Jo Ann Hill, Betsy Bombick and G.L. Prasad.
Research and Development, R.J. Reynolds Tobacco Company, Winston-Salem, NC USA.

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S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

culture medium. Separately, CAS at higher volumes altered basal

and TLR ligand-induced immune response which interfered with
the assessment of the effects of treatment at higher doses of ST/
CAS (data not shown). Hence, we present results with ST/CAS at
2 lg/mL of equi-nicotine units, which is comparable to the dose used
for combustible TPPs. The nicotine EC50 value was determined to be
1650 lg/mL (Arimilli et al., 2012a).
A range of lower doses of TPPs and nicotine were also used in
some of the experiments. The numbers in the parentheses in each
gure indicate the dose of lg/mL nicotine units used in those
experiments. Equivalent volumes of media, DMSO and CAS were
used as controls for WS-CM, TPM and ST/CAS, respectively.

2.2. Isolation of PBMCs

Fresh blood was collected from healthy non-smoking donors
after written informed consent (who were non-consumers of tobacco products) at a local Clinical Contract Research Organization
(Piedmont Medical Group, Winston-Salem, NC) under IRB approval. PBMCs were isolated from fresh blood as described earlier
(Arimilli et al., 2012b) under Wake Forest Baptist Health IRB approval. Briey, PBMCs were isolated by standard density gradient
centrifugation by using Isolymph (CTL Scientic Supply Corp., Deer
Park, NY). Isolated PBMCs were cryopreserved for further use.

2.5. Cytotoxicity assay

K562 cells were purchased from ATCC (Manassas VA, USA) and
grown in RPMI complete media. One vial of cryopreserved PBMCs
was thawed and washed, and a live cell count was taken. Different
concentrations of TPPs were prepared in 100 lL media. 1.5 million
PBMCs in a 50 lL volume were added to each well and incubated
for 1.5 h at 37 C in 96-well round bottom plates. K562 target cells
were labeled with carboxy uorescein succinimidyl ester (CFSE)
and added at a density of 100,000 cells/well (target:effector ratio
is 1:15). Cell co-cultures were incubated at 37 C for an additional
4 h. Immediately after the incubation period, cells were stained
with 7AAD (which labels the dead cells) and the killing of CFSE-labeled K562 target cells was evaluated after 7AAD staining by ow
cytometric analysis (BD Biosciences, San Jose, CA). Flow data were
analyzed using Flow Jo software (Tree Star, Ashland, OR).
2.6. Statistical comparisons
The results were presented as the mean the standard error of
the mean (four donor samples). The t-test between treatment and
untreated control samples was performed using Sigma Plot (version 9) for all treatments with their corresponding controls. The
statistical signicance was indicated by: , P < 0.05; , P < 0.005;

, P < 0.0005.
3. Results

2.3. Cell staining and ow cytometry

PBMCs were treated with indicated concentrations (nicotine
units) of WS-CM, TPM, ST/CAS and neat nicotine for 3 h in a 24well plate at 3 million cells/well in 2 mL RPMI complete media.
Dosing was based on an equi-nicotine exposure paradigm. Cells
were washed after 3 h treatment and re-plated in a 48-well plate
at 1 million cells/well/mL with and without the presence of
10 lg/mL poly I:C or LPS. After a 67 h incubation, supernatants
were taken for secreted cytokine analysis. Cells were plated onto
a 96-well plate at 106/mL and further incubated for 4 h with
200 lL of media with Golgiplug (1 lg/mL) (BD Biosciences, San
Jose, CA) to measure intracellular cytokines (Arimilli et al., 2006;
Mian et al., 2008). Cells were then harvested, washed and surface
stained for CD56-PE and CD2-FITC, CD69-APC monoclonal antibodies (mAB) for 30 min at 4 C.
After washing, cells were xed and permeabilized with BD Biosciences Cytox/Cytoperm buffer for 20 min on ice in the dark.
Cells were then stained with anti-IFN-c-PE, anti-TNF-a-APC and
anti-perforin-FITC mAB and incubated for an additional 30 min
on ice. After washing the cells, they were re-suspended in a total
volume of 200 lL of 2% paraformaldehyde for FACS analysis. Forward scatter, side scatter, uorescence intensity and percent positivity were measured after acquiring 100,000 cells per sample by
ow cytometry (BD Biosciences, San Jose, CA), and the data were
analyzed using Cell Quest (BD Biosciences, San Jose, CA) and Flow
Jo (Tree Star, Ashland, OR) software.

2.4. Cytometric bead array assay

PBMC culture supernatants were harvested after 67 h of poly I:C
or LPS stimulation from the above experiments. A Cytometric Bead
Array (CBA) for human cytokine secretion was used as described
previously (Arimilli et al., 2007). Briey, we measured IL-1b, IL-6,
IL-8, IL-10, IL-12, and TNF-a (BD Biosciences, San Jose, CA) by ow
cytometry according to the manufacturers instructions.

3.1. Suppression of TNF-a+ and IFN-c+ T cell populations in PBMCs

treated with TPPs
We sought to evaluate intracellular cytokine levels in T cells
after treating with combustible and non-combustible TPPs. In the
rst set of experiments, we measured the induction of intracellular
TNF-a and IFN-c by ow cytometry after PBMCs were treated with
TPPs and stimulated with either poly I:C or LPS. Stimulation with
TLR ligands resulted in robust and statistically signicant increases
in TNF-a+ and IFN-c+ cells compared to unstimulated controls
(basal).
First, we assessed whether the pretreatment of PBMCs with
TPPs compromised their ability to produce TNF-a+ cells when stimulated with poly I:C and LPS (Fig. 1). Fig. 1A presents representative
ow cytometric data of T cells positive for TNF-a after treatment
with the TPPs followed by no stimulation (top panel), poly I:C stimulation (middle panel) and LPS stimulation (bottom panel). TPPs by
themselves induced basal number of TNF-a+ T cells. A dose-dependent suppression of TNF-a+ T cells in stimulated PBMCs by WS-CM
is evident (Fig. 1B, top panel). Although a 26% reduction from control with WS-CM at 0.5 lg/mL was detected, a statistically signicant reduction in number of TNF-a+ T cells (72%) was observed at
1.56 lg/mL of equi-nicotine units of WS-CM. Treatment with TPM
did not result in statistically signicant differences at the doses
tested. Treatment with ST/CAS at 2 lg/mL of equi-nicotine units
did not alter the number of TNF-a producing T cells.
T cells treated with nicotine (101650 lg/mL) did not yield statistically signicant differences in their TNF-a+ T cell numbers to
poly I:C and LPS stimulation. Exposure to 1650 lg/mL of nicotine
resulted in higher basal levels of TNF-a+ T cells, which were unaltered by stimulation with the TLR ligands.
We next evaluated changes in the number of IFN-c+ T cells
(Fig. 2) with TPP treatment followed by poly I:C and LPS stimulation. A set of representative ow cytometric images is included
in Fig. 2A and summary data from four donors is presented in
Fig. 2B. The TPPs induced a minimal number of IFN-c+ T cells under
basal (non-stimulated) conditions. Control and DMSO treated cells

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

1995

Fig. 1. Reduction of intracellular TNF-a+ T cells by different TPPs. PBMCs were exposed to varying concentrations of WS-CM, TPM, ST/CAS, nicotine and their corresponding
vehicle controls for 3 h with no stimulation (top panel) or stimulated with poly I:C (middle panel) and LPS (bottom panel) for 67 h. The vehicle controls for WS-CM is medium,
for TPM it is DMSO and for ST/CAS it is CAS; the volumes of vehicle control correspond to the volumes used for the TPPs. Intracellular TNF-a+ T cells were quantied by ow
cytometry. (A) Representative ow cytometric data after acquiring 100,000 cells per sample. (B) Combined data from four independent experiments using four different
donors PBMCs and indicated concentrations of TPPs and nicotine. The statistical signicance was indicated by: , P < 0.005; , P < 0.0005.

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S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

Fig. 2. Suppression of intracellular IFN-c+ T cells by different TPPs. PBMCs were exposed to TPPs as indicated and stimulated with poly I:C and LPS as described in the caption
to Fig. 1. Intracellular IFN-c+ T cells were quantied by ow cytometry. (A) Representative ow cytometric data. (B) Combined data from four independent experiments using
four different donors PBMCs and indicated concentrations of TPPs and nicotine. The statistical signicance was indicated by: , P < 0.05; , P < 0.005.

showed an increase in the number of IFN-c+ T cells after poly I:C

and LPS induction. Treatment with WS-CM resulted in near complete suppression of IFN-c+ T cells induced by poly I:C and LPS,
while incubation with TPM resulted in statistically signicant dose
dependent reduction of IFN-c+ T cells.

In terms of equi-nicotine units, the combustible TPPs, WS-CM

and TPM, were far more potent suppressors of IFN-c+ T cells
(Fig. 2B, top panel) than ST/CAS or nicotine (Fig. 2B, bottom panel).
Treatment with combustible TPPs resulted in statistically
signicant suppression of IFN-c+ T cells at or below 2.58 lg/mL

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

of equi-nicotine units. Measurable suppression of the cytokine-positive cells was observed only at the highest concentrations of nicotine, although these changes were not statistically signicant.
Thus, a dose-dependent suppression of IFN-c+ T cells in response to TPM treatment was observed, whereas WS-CM was more
potent in suppressing TNF-a+ and IFN-c+ T cells at both doses
tested. The combustible TPPs exerted more pronounced effects relative to ST/CAS and nicotine. Furthermore, IFN-c+ T cells appear to
be compromised more severely by combustible TPPs and nicotine
(1650 lg/mL) than TNF-a+ T cells in the stimulated PBMCs.
3.2. Suppression of TNF-a+ NK cell populations in PBMCs treated with
TPPs
Because of the importance of NK cells in innate immunity, we
next tested the effect of TPPs on the induction of TNF-a+ NK cells
in poly I:C-and LPS-stimulated PBMCs. Fig. 3A shows the ow
cytometry data of differential suppression of TNF-a in NK cells
by various TPPs. Consistent with the data obtained with T cells,
NK cells treated with WS-CM showed the lowest number of TNFa+ NK cells after stimulation with poly I:C (Fig. 3A, middle panel)
and LPS (Fig. 3A, lower panel) when compared to the control cells.
Fig. 3B summarizes four experiments and demonstrates that the
inhibition of TNF-a+ NK cells are more pronounced with combustible TPPs than ST/CAS at equi-nicotine units (WS-CM > TPM > ST/
CAS). PBMCs treated with WS-CM showed 90% reduction and
TPM showed 64% reduction of intracellular TNF-a+ NK cells, which
were statistically signicant, upon stimulation with TLR ligands.
However, pre-treatment with ST/CAS (2) did not result in statistically signicant changes relative to vehicle control. While pretreatment with lower doses (10 and 500 lg/mL) of nicotine did
not induce statistically signicant changes in TNF-a+ NK cells, at
a higher dose (1650 lg/mL) produced a statistically signicant
reduction after LPS stimulation (Fig. 3B).
3.3. Effect of TPPs on CD69 expression in NK cells
Since the induction of TNF-a+ NK cells is reduced by TPPs in the
stimulated NK cells, we further investigated whether TPPs interfered with NK cell differentiation and function. CD69 is an early
inducible cell surface glycoprotein expressed during lymphoid
activation and is involved in NK cell function (Borrego et al.,
1999). PBMCs were treated with TPPs, and CD69 expression on
NK cell population was analyzed by ow cytometry after poly I:C
and LPS stimulation (Fig. 4). Pre-treatment of PBMCs with the TPPs
resulted in increased CD69 expression upon stimulation with poly
I:C and LPS. It is interesting to note that the WS-CM (1.56) treatment increased CD69 expressing NK cells under basal conditions
by 5-fold, whereas poly I:C or LPS did not further enhance the
CD69 expression. Exposure to nicotine (1650 lg/mL) did not increase the basal levels of the induction of CD69 relative to the untreated cells.
3.4. Inhibition of secreted cytokines in TPP-treated PBMCs
Next we assessed the effect of TPPs on the secreted cytokines.
PBMCs were treated with different TPPs and stimulated with either
poly I:C (Fig. 5) or LPS (Fig. 6). Cell culture supernatants were used
to measure the levels of a panel of cytokines. Among the TPPs
tested, WS-CM exerted statistically signicant and profound inhibitory effect (which was statistically signicant, P < 0.0005) on the
secretion of the cytokines. Levels of IL-1b, IL-6, IL-10 and TNF-a
were profoundly reduced in WS-CM-treated PBMCs stimulated
with poly I:C or LPS. While IL-8 and IL-12 secretion was reduced
by 94% and 68% with poly I:C, respectively, they were reduced by
91% and 67% with LPS, respectively.

1997

Treatment with TPM also reduced the secretion of several cytokines upon stimulation with the TLR ligands. Statistically signicant reductions were observed in IL-10 (>60%), IL-12 (>68%) and
TNF-a (>80%) secretion with both TLR ligands (Figs. 5 and 6).
TPM also suppressed IL-1b and IL-6 levels in LPS-stimulated cells
(Fig. 6). Pre-treatment with ST/CAS (2) resulted in statistically signicant reductions in IL-6 levels with poly I:C, but not with LPS
stimulation (Figs. 5 and 6).
3.5. Suppression of PBMC cell target killing ability
As the intracellular and secreted cytokines are suppressed by
pre-treatment with TPPs to varying degrees, we tested whether
the functional properties, particularly target cell killing by NK cells
and CD8+ T cells in the PBMC pool was compromised from the
exposure to TPPs. These two cell types are known as cytolytic effector cells that confer innate protection against viral and bacterial
infections and are involved in anti-tumor surveillance. We utilized
the K562 cell line as a target in the cytolytic assay and a set of representative ow cytometric images of cytolysis is shown in Fig. 7A.
The cytolytic data on multiple donor PMBCs is presented in Fig. 7B
and shows a statistically signicant decrease of K562 cell killing
ability with WS-CM (65%) treatment. Treatment with ST/CAS (2)
or nicotine (1650) did not reduce the target cell killing ability
(Fig. 7B).
3.6. Effect of TPP exposure on perforin levels in total PBMCs and NK
cells
We next investigated whether the altered cytolytic activity of
PBMCs and NK cells is due to differences in the endogenous perforin levels. Perforin, a cytoplasmic granular protein, is known to
mediate target cell killing. We therefore determined perforin levels
in TPP-treated PBMCs (Fig. 8, upper panel) and NK cells (Fig. 8, lower panel).
WS-CM (1.56)-treated PBMCs and NK cells showed a statistically signicant reduction (70% and 78%, respectively) in perforin
levels. Treatment with TPM, ST/CAS and nicotine did not result in
statistically signicant changes in perforin levels in PBMC populations or NK cells.
4. Discussion
The purpose of this work is to assess how the exposure of combustible and non-combustible TPPs inuences immune responses;
particularly induction, secretion of cytokines and cytolytic activity
by PBMCs. Cigarette smoking has been known to elicit inammation and alter a wide range of innate and adaptive immune responses (Goncalves et al., 2011). Such altered immune responses
have been linked to compromised host defense against microbial
infections and tumor surveillance (Cerwenka and Lanier, 2001).
The key nding of our study is that the exposure to combustible
TPPs (WS-CM and TPM), on an equi-nicotine unit basis, caused potent immunosuppression relative to ST and nicotine, as measured
by NK cell and T cell functions.
Due to their critical role in modulating immune functions in response to microbial pathogens and in tumor surveillance, NK cells
and T cells have been studied for possible adverse effects of smoking. Our ndings are in general agreement with the previously
published work which showed that the responsiveness of T cells
and NK cells to poly I:C, a TLR3 ligand, is signicantly attenuated
by WS-CM (Mian et al., 2008, 2009a,b).
That chronic cigarette smoking causes increased inammation
and yet results in compromised immune responses has been
widely known (Lee et al., 2012; US Department of Health and Hu-

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S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

Fig. 3. Suppression of intracellular TNF-a+ NK cells with different TPPs. PBMCs were exposed to different concentrations of WS-CM, TPM, ST/CAS and their corresponding
vehicle controls as described in the caption to Fig. 1. Intracellular TNF-a+ NK cells were quantied by ow cytometry. (A) Representative ow cytometric data. (B) Combined
data from four independent experiments using four different donors PBMCs and indicated concentrations of TPPs. The statistical signicance was indicated by: , P < 0.05; ,
P < 0.005; , P < 0.0005.

man Services, 2010). A number of different experimental models,

similar to that described herein, have been utilized to dissect the
mechanisms. However, the effects of ST on immune responses
are incompletely understood, and the results appear to be

dependent on the cell types and other experimental conditions.

For example, ST exposure was shown to cause immunostimulatory
and proinammatory effects (Goud et al., 1993; Petro, 2003), as
well as immunosuppression (Fine et al., 2002; Johnson et al.,

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

1999

Fig. 4. Stimulation of CD69 expression in NK cells with different TPPs. PBMCs were exposed to different concentrations of WS-CM, TPM, ST/CAS, nicotine and their
corresponding vehicle controls for 3 h and stimulated with poly I:C, LPS or no stimulation for 67 h. CD69 expressing NK cells were quantied by ow cytometry (data were
combined from four independent experiments using PBMCs from ve different donors). The statistical signicance was indicated by: , P < 0.0005.

Fig. 5. Attenuation of cytokine secretion by TPPs following poly I:C stimulation. PBMCs were exposed to different concentrations of WS-CM, TPM, ST/CAS and their
corresponding vehicle controls for 3 h and stimulated with poly I:C for 67 h. Levels of cytokines in the culture supernatants were determined using a cytometric bead array
and ow cytometer. The statistical signicance was indicated by: , P < 0.05; , P < 0.005; , P < 0.0005.

2009a). Some of the widely characterized consequences of exposure to cigarette smoke (or its constituent phases) are cytotoxicity
and inammation, as measured by increased secretion of cytokines
such as IL-8. Recently we reported that exposure to combustible

TPPs results in cytotoxicity relative to ST/CAS and increased secretion of IL-8 in PBMCs and other cultured cells (Arimilli et al.,
2012a). Further, we showed that T-helper (CD4+) cells appear to
be more sensitive to the exposure to TPPs compared to other

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S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

Fig. 6. Attenuation of cytokine secretion by TPPs following LPS stimulation. PBMCs were exposed to TPPs, stimulated with LPS for 67 h, and the secreted cytokines were
measured as described in the caption of Fig. 5. The statistical signicance was indicated by: , P < 0.05; , P < 0.005; , P < 0.0005.

leukocyte subsets tested in that study. Although we exposed

PBMCs in this study at the EC50 equi-nicotine units of WS-CM
and TPM, the pre-treatment was for only 3 h and no cytotoxicity
was evident either at the end of pre-treatment or stimulation with
TLR ligands for 67 h (data not shown).
One of the strengths of this study is that, for the rst time, the
effects of exposure to combustible and non-combustible TPPs are
comparatively evaluated in a single system. We have utilized a
well characterized TLR stimulated PBMC system to assess the cellular and cytokine secretion in response to TPP exposure. Further,
we have reported the biological responses in equi-nicotine units
for comparison of the effects of TPPs.
The cytokines secreted by NK cells and T cells play a crucial role
in modulating immune responses and subsequent cell killing
(Mian et al., 2008). Our nding that WS-CM exposure markedly
suppresses T cell and NK cell responsiveness is in agreement with
previous work (Mian et al., 2008, 2009a,b). Further, we demonstrate that the WS-CM also suppresses the secretion of several
cytokines, which is consistent with published data (Ouyang et al.,
2000). For example, IFN-c is key in both innate and adaptive immune responses and is secreted by NK cells, cytotoxic T cells and
T helper cells to induce multiple cellular effects such as inhibition
of cell proliferation, immunoregulation and apoptotic activity
(Schoenborn and Wilson, 2007; Schroder et al., 2004).
Pretreatment of PBMCs with commercial cigarette smoke extracts have previously been shown to suppress the production of
IL-1b, IL-2, IFN-c and TNF-a by greater than 90% without signicant loss of cell viability (Lambert et al., 2005; Ouyang et al.,
2000). Further, smoke extracts from 1R3 reference cigarettes have
been shown to impair NK cell functions (Mian et al., 2008). Our
data with WS-CM (which other authors referred to as smoke extracts) are in agreement with these ndings and also show the relative effects of different TPPs tested herein. Acrolein, catechol and
hydroquinone have been shown to contribute to the observed

impairment of the immune responses (Lambert et al., 2005; Ouyang et al., 2000).
Similarly, TNF-a is important in suppression of tumorigenesis
and infection as well as inducing immune cell activation and proliferation. TNF-a is critical for activation of the NF-jB pathway
(Van Antwerp et al., 1996) and thus near complete abolition of
secretion by WS-CM and markedly diminished secretion of TNFa by TPM, could signicantly contribute to immunosuppression
(Figs. 1, 2, 5 and 6). Consistent with our results, cigarette smoke extract was shown to signicantly reduce the production of TNF-a
and IFN-c levels of invariant NK cells stimulated with a-galactosylceramide (Hogan et al., 2011a).
Besides TNF-a, secretion of IL-1b, IL-6, IL-8, IL-10 in poly I:C
(Fig. 5) and LPS-stimulated PBMCs (Fig. 6) was essentially
blocked by treatment with WS-CM, whereas secretion of IL-12
was markedly reduced. Treatment with TPM, however, resulted
in a signicant reduction in IL-10, IL-12 and TNF-a, with no
reductions in the secretion of the other cytokines. There is
overall directional concordance between the levels of the
intracellular cytokine and the secreted cytokine (Figs. 5 and 6)
in response to WS-CM. Thus, a qualitatively important difference
between the effects of WS-CM and TPM (and ST) on IL-1b,
zIL-6 and IL-10 cytokine secretion in stimulated PBMCs was
evident.
To investigate the effect of TPPs on early anti-inammatory responses we assessed the expression of CD69 after treatment with
TPPs. CD69 is a pleiotropic immune regulator, involved in the activation and differentiation of a wide variety of hematopoietic cells,
including NK cells (Ziegler et al., 1994), and the expression of macrophage CD69 is known to increase upon cigarette smoke exposure
(Tsuyusaki et al., 2011). Data presented in Fig. 4 show that CD69
expression increases markedly in WS-CM-treated NK cells without
poly I:C or LPS stimulation, and stimulation with poly I:C or LPS did
not further enhance CD69 in cells treated with WS-CM. TPM and

S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

2001

Fig. 7. Reduction of PBMCs cytolytic ability with different TPPs. PBMCs were treated with varying concentrations of TPPs and nicotine for 1.5 h. CFSE-labeled K562 cells were
then added as target cells and incubated for additional 4 h. Cells are stained with 7AAD and ow cytometry was used to gauge killing ability. (A) The representative ow
cytometry results with percent killing shown in the gated boxes. (B) Combined data from four independent experiments using PBMCs from four different donors. The
statistical signicance was indicated by: , P < 0.05.

ST/CAS (2)-treated cells, although exhibiting higher basal levels of

CD69, responded to the ligand stimulation.
Cigarette smoke contains many different classes of chemicals.
The identity of the chemicals in cigarette smoke which are responsible for immune suppression is not completely established. For

example, a, b unsaturated aldehydes, such as acrolein and crotonaldehyde have been shown to cause cytokine suppression, but not
the saturated aldehydes (acetaldehyde, propionaldehyde and
butyraldehyde) (Lambert et al., 2005, 2007). Our ongoing work
shows that the WS-CM used in the current study contains both

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S. Arimilli et al. / Toxicology in Vitro 27 (2013) 19922004

Fig. 8. Inhibition of perforin levels in PBMCs and NK cells with different TPPs. Cells
were exposed to varying concentrations of WS-CM, TPM, ST/CAS, nicotine and their
corresponding vehicle controls. Upper panel shows percent perforin-positive
PBMCs from four different donors PBMCs. Lower panel shows percent perforin
positive NK cells in the PBMC population. The statistical signicance was indicated
by: , P < 0.05; , P < 0.0005.

the a, b unsaturated aldehydes and the saturated aldehydes. It is

possible that the cytokine suppression observed in this study also
may be mediated by the a, b unsaturated aldehydes. However,
additional work remains to be done in determining the components of the WS-CM that are responsible for suppression of the target cell killing.
NK cells and CD8+ T cells play critical roles in tumor surveillance and defending against microbial infections through cytolysis of target cells. Several investigators have assessed NK cell
function in response to cigarette smoke exposure and reported
a decrease in target cell killing by the exposed cells (Ferson
et al., 1979; Hogan et al., 2011b; Mehta et al., 2008; Mian
et al., 2008). Consistent with those ndings, we report here that
exposure to WS-CM signicantly inhibits target cell killing, with
WS-CM being the most potent of the TPPs tested in this study
(Fig. 7). One possible mechanism through which WS-CM interferes with the NK cell and CD8+ cell function is likely through
its ability to block the expression of perforin (Fig. 8). Perforin
is largely responsible for mediating the cytolysis of targeted cells
(Smyth et al., 1999). Although TPM exposure did not interfere
with the induction of perforin (Fig. 8), a near 50% decrease in
cell killing was observed, suggesting the involvement of additional proteases in NK cell-mediated cytolysis. Previous studies
have suggested that anti-tumor cell activity of NK cells is reduced in smokers compared to non-smokers (Ferson et al.,
1979). Thus TPPs, particularly WS-CM, diminishes perforin basal

and stimulated expression in NK cells, which in turn, may compromise their cytolytic activity.
One of the confounding factors of this study relates to the cell
type-specic interference of CAS, the vehicle/solvent used for the
exposure of ST. As observed in our recent work, CAS, by itself, appears to induce IL-8 secretion at higher doses (425 lg/mL) in a 24 h
exposure (Arimilli et al., 2012a). The vehicle effects were more pronounced at a higher volume of CAS (115 lL) needed to treat cells at
425 lg/mL equi-nicotine dose (data not shown). Consistent with
these ndings, we have recently reported that a-amylase in CAS
elicits inammatory responses, including the expression of TNF-a
in dermal broblasts (Malpass et al., 2013). Collectively, these ndings suggest a need to carefully evaluate the effects of higher doses
of ST/CAS on PBMCs and other cell types. It should be noted that
the concentrations of TPPs employed in this work are expected
to exceed those typically observed in chronic tobacco usage (Benowitz et al., 2009).
In contrast to the effects of the combustible TPPs and ST/CAS,
the effects of nicotine were signicantly less remarkable on stimulated PBMCs. Generally, any nicotine effects were evident only at
the highest dose (EC50 1650 lg/mL) tested.
In summary we have examined the role of NK cells and T cells in
the innate and adaptive immune responses and how in vitro treatment with TPPs can impact the immune system. In this manuscript
we have used both combustible and non-combustible tobacco
product preparations to treat PBMCs and then stimulated with
poly I:C and LPS. We showed a reduction of multiple cytokines
and the cytolytic ability of PBMCs which are important for fully
functional immune responses. We have demonstrated that human
NK and T cell function was reduced with TPPs in an order of WSCM > TPM > ST/CAS. Our ndings indicate that combustible TPPs,
most notably WS-CM, attenuate cellular functions by suppressing
cytokine production and cytolytic ability to a greater extent at lower nicotine equivalent units than smokeless TPPs. These ndings
may provide a mechanistic explanation why smokers are more
prone to viral and bacterial infections, and cancers. Investigating
the molecular mechanisms underlining these effects and effects
on other cytokines regulating PBMC functions will provide further
insight into the potential impact of cigarette smoke on the immune
system.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgments
We sincerely thank Dr. Peter Chen for his statistical analysis of
the work. We also thank Dr. Evan Gregg for his help in manuscript
preparation. 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.
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