Nicotinic Attenuation of Central Nervous
System Inflammation and Autoimmunity
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J Immunol 2009; 182:1730-1739; ;
doi: 10.4049/jimmunol.182.3.1730
http://www.jimmunol.org/content/182/3/1730
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References
Fu-Dong Shi, Wen-Hua Piao, Yen-Ping Kuo, Denise I.
Campagnolo, Timothy L. Vollmer and Ronald J. Lukas
The Journal of Immunology
Nicotinic Attenuation of Central Nervous System Inflammation
and Autoimmunity1
Fu-Dong Shi,2* Wen-Hua Piao,3,4* Yen-Ping Kuo,5† Denise I. Campagnolo,*
Timothy L. Vollmer,6* and Ronald J. Lukas2†
A
multitude of studies suggest that nicotine, a psychoactive component of tobacco products that acts as does the
natural neurotransmitter, acetylcholine, on nicotinic acetylcholine receptors (nAChRs)7 found in many organ systems, has profound immunological effects (1, 2). During ontogeny, nicotine exposure can modulate T (3) and B cell development and activation (4).
Nicotine exposure also suppresses the T cell response and alters the
differentiation, phenotype, and functions of APCs including dendritic
cells (5, 6) and macrophages (7). Findings have been mixed about
effects of nicotine exposure on immune responses in vivo. Exposure
to nicotine and/or related agents appears to dampen inflammatory responses and reduce mortality in a mouse model of sepsis (8) and to
protect against induction of type 1 diabetes in mice (9). In addition,
cigarette smoke inhalation produces sustained suppression of humoral
autoimmunity in a murine model of systemic lupus erythematosus
*Department of Neurology and †Department of Neurobiology, Barrow Neurological
Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ 85013
Received for publication August 19, 2008. Accepted for publication November
18, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants from Barrow Neurological Foundation
and the National Institutes of Health (R01 AI052463). The contents of this report are
solely the responsibility of the authors and do not necessarily represent the views of
the aforementioned awarding agencies.
2
Address correspondence and reprint requests to Dr. Fu-Dong Shi or Dr. Ronald J.
Lukas, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, 350
West Thomas Road, Phoenix, AZ 85013. E-mail addresses: [email protected]
or [email protected]
3
W.-H.P. and F.-D.S. contributed equally to this work.
4
Current address: Department of Clinical Laboratory, Ningxia People’s Hospital,
Yinchuan, Ningxia Province, People’s Republic of China.
5
Current address: School of Osteopathic Medicine in Arizona, A. T. Still University,
5850 East Still Circle, Mesa, AZ 80206.
6
Current address: Department of Neurology, University of Colorado School of Medicine, Denver, CO 80045.
7
Abbreviations used in this paper: nAChR, nicotinic acetylcholine receptor; MS,
multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin
oligodendrocyte glycoprotein; PLP, proteolipid protein.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org
(10). Moreover, several epidemiological studies reveal a strong inverse correlation between smoking and human autoimmune responses
manifest as systemic lupus erythematosus and ulcerative colitis (10,
11). By contrast, other studies suggest that smoking behavior in humans might exacerbate multiple sclerosis (MS) and Crohn’s disease
(12–14). These apparently conflicting observations suggest that the
impact of nicotine on immune responses in vivo is complex, being
potentially influenced by drug dosage and duration of exposure, by
the specific organ systems involved in the immune response, by the
stage and type of disease, and by the level of involvement of autoimmune and inflammatory mechanisms.
Inflammatory and immune responses within the CNS are capable
of shaping the clinical outcome of brain diseases including stroke,
trauma, Alzheimer’s disease, Parkinson’s disease, epilepsy, encephalomyelitis, and MS (15). Compared with other organ systems, the
CNS has several unique properties with respect to immune responses.
First, the spectrum of APCs differs from that in the periphery; in the
CNS, resident microglia and astrocytes are active participants (16,
17). Second, peripheral immune cells migrating into the CNS are
reactivated upon encountering myelin and other Ags, enhancing their
capacity to recognize a wide spectrum of ambient Ags through “determinant spreading” (18). Third, the nature and magnitude of immune responses within the CNS are likely influenced by signals intrinsic to this unique microenvironment, including local cytokine
signaling, but also including presumably higher levels and more concentrated signaling by chemical messengers such as acetylcholine.
More work is needed to elucidate roles of nAChRs and nicotinic cholinergic signaling on immune responses in the periphery, but this need
for further investigation may be even more acute for studies of immune and inflammatory responses in the CNS where additional cell
types express nAChRs, especially given implication of nAChRs and
nicotinic signaling in stroke, Alzheimer’s disease, Parkinson’s disease, epilepsy, and perhaps other brain diseases (19, 20).
To ascertain influences of nicotine exposure on a CNS autoimmune response, we used the murine experimental autoimmune encephalomyelitis (EAE) model of MS. Immunization of C57BL/6
(B6) mice with myelin oligodendrocyte glycoprotein (MOG) peptide activates T cells in the periphery and subsequently generates
pronounced cellular infiltration and demyelination in the CNS and
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The expression of nicotinic acetylcholine receptors by neurons, microglia, and astrocytes suggests possibly diverse mechanisms by
which natural nicotinic cholinergic signaling and exposure to nicotine could modulate immune responses within the CNS. In this
study, we show that nicotine exposure significantly delays and attenuates inflammatory and autoimmune responses to myelin Ags
in the mouse experimental autoimmune encephalomyelitis model. In the periphery, nicotine exposure inhibits the proliferation of
autoreactive T cells and alters the cytokine profile of helper T cells. In the CNS, nicotine exposure selectively reduces numbers of
CD11cⴙ dendritic and CD11bⴙ infiltrating monocytes and resident microglial cells and down-regulates the expression of MHC
class II, CD80, and CD86 molecules on these cells. The results underscore roles of nicotinic acetylcholine receptors and nicotinic
cholinergic signaling in inflammatory and immune responses and suggest novel therapeutic options for the treatment of inflammatory and autoimmune disorders, including those that affect the CNS. The Journal of Immunology, 2009, 182: 1730 –1739.
The Journal of Immunology
a monophasic neurological deficiency that resembles a form of MS
in humans, acute disseminated encephalomyelitis (21). We found
that nicotine exposure delays and dramatically attenuates CNS inflammation and autoimmune responsiveness to myelin Ags, suggesting several novel mechanisms of neuroimmune interaction.
Materials and Methods
Mice
B6 (H-2b) mice purchased from Taconic Farms were housed in pathogenfree animal facilities. Female mice used were 7– 8 wk of age at the experiment’s inception. Experiments were conducted in accordance with institutional guidelines.
Ags
The murine MOG35–55 peptide (M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-YR-N-G-K) and proteolipid protein (PLP)139 –151 peptide (H-S-L-G-K-W-LG-H-P-D-K-F) were synthesized by BioSynthesis.
Induction of acute EAE and adoptive transfer of EAE
Nicotine treatment
Nicotine bitartrate was purchased from Sigma-Aldrich. A 100 mg/ml solution in PBS or a solution of PBS alone was freshly prepared 24 h before
pump implantation and loaded into Alzet osmotic minipumps (model
1007D, Durect Corporation). The pumps were implanted s.c. on the right
side of the back of the mouse and continuously delivered either PBS or
nicotine salt at 12 ␮l/day for 7 days, and then the pumps were removed.
This equated to delivery of 0.39 mg of nicotine-free base per mouse per
day. For an ⬃30 g mouse, which is at the upper end of weight for animals
used in the study, this equates to ⬃13 mg of nicotine-free base/kg/day or
⬃0.54 mg of nicotine free base/kg/h. Plasma nicotine levels in mice are
⬃100 –200 ng/ml (⬃0.6 –1.2 mM) after infusion of ⬃2– 4 mg/kg/h of drug
and ⬃45 ng/ml (⬃280 nM) after infusion at ⬃0.5 mg/kg/h (23). For comparison, human smokers have peak plasma nicotine levels of 10 –50 ng/ml
(⬃60 –310 nM; Ref. 23). Thus, nicotine levels in plasma (extrapolated to
be ⬃49 ng/ml or ⬃300 nM) of mice used in the studies are comparable to
those in the plasma of human smokers. Some control mice received PBS
via direct injections rather than through minipumps, but either delivery
method produced similar results.
To evaluate the effects of nicotine pretreatment on EAE-associated autoimmune responses, mice received nicotine or PBS daily for 7 days starting on the day of or 7 days before MOG immunization. To analyze effects
of nicotine exposure on an activated autoimmune response, mice received
nicotine or PBS daily for 7 days starting on day 7, which is the first day of
manifestation of disease signs, after EAE induction. For the adoptive EAE
transfer study, lymphocytes used for secondary injection were isolated
from mice treated for 7 days with nicotine or PBS initiated at the time of
MOG immunization.
Preparation of tissues and histological staining
Mice were anesthetized with pentobarbital on the 25th day after immunization and perfused by intracardiac puncture with 50 ml of cold PBS.
Spinal cords were removed and fixed in 10% formalin/PBS. Paraffin-embedded, longitudinal sections running from the cervical enlargement of the
cord were prepared and stained for H&E, myelin (luxol fast blue), and
axons (Biechowsky silver). Manual tracing was used to define the degree
of inflammation, demyelination, and axonal damage across the entire spinal
cord section for each mouse. Pathological changes in each spinal cord were
scored as follows: 0, no changes; 1, focal area involvement; 2, ⬍5% of
total myelin area involved; 3, 5–10% of total myelin area involved; 4,
10 –20% involved area; 5, ⬎20% of total myelin area involved (22).
T cell proliferation assays
Mononuclear cells were isolated from the spleens of EAE mice at day 11
post immunization that were treated with nicotine or PBS. Cells were suspended in culture medium containing DMEM (Life Technologies) supplemented with 1% (v/v) MEM (Life Technologies), 2 mM glutamine (Flow
Laboratory), 50 IU/ml penicillin, 50 mg/ml streptomycin, and with 10%
(v/v) FCS (all from Life Technologies). Four ⫻ 105 cells in 200 ␮l of
culture medium were placed in each well of 96-well, round-bottom microtiter plates (Nunc). Ten microliters of immunizing Ag MOG35–55 peptide
(10 ␮g/ml), control myelin Ag PLP139-151 peptide (10 ␮g/ml), or Con A (5
␮g/ml) (Sigma-Aldrich) were then added (triplicates per condition). After
3 days of incubation, the cells were pulsed for 18 h with 10-␮l aliquots
containing 1 ␮Ci of [methyl-3H]thymidine (specific activity of 42 Ci/
mmol; MP Biomedicals) per well. Cells were harvested onto glass fiber
filters and thymidine incorporation proportional to the degree of cell proliferation was then measured. The results are expressed as cpm.
Single cell suspensions (4 ⫻ 107 cells) were prepared and labeled with 0.5
␮M CFSE at 37°C for 10 min. Subsequently, when cells were cultured, levels
of CFSE staining declined with each cell division, allowing for cell proliferation to be monitored. Cells with or without CFSE were incubated at 37°C for
3 days in round-bottom plates (2 ⫻ 106 cells/well) with or without Ags (MOG
10 ␮g/ml). After harvesting, cells were stained for surface markers with fluorochrome-conjugated mAbs including anti-CD3-PE/Cy5 (17A2), anti-CD4allophycocyanin/Cy7 (GK1.5), and anti-CD8␣-PE/Cy7 (53-6.7) (BD Biosciences). Isotype-matched negative mAbs were used as controls.
Cell viability and apoptosis assay
Cell viability was assessed by trypan blue dye exclusion. For detection of
cell apoptosis, spleen mononuclear cell suspensions were collected from
PBS- or nicotine-treated mice on day 11 (peak stage). Single cell suspensions were washed in PBS and resuspended in binding buffer containing
annexin V-FITC to monitor apoptosis- or necrosis-associated plasma membrane alterations and propidium iodide to monitor cell death-associated
DNA exposure (both from BD Biosciences) for 20 min at room temperature. The samples were analyzed on a FACSAria using Diva software (BD
Biosciences).
Spleen and CNS cell isolation and flow cytometric analysis
Spleen mononuclear cell suspensions were collected from PBS- or nicotine-treated mice on day 11 (i.e., at the peak of the EAE response). Single
cell suspensions were prepared and stained for one or more of the following Ags (targeted by the indicated Ab fluorescently tagged with either
FITC, PE, allophycocyanin, PE-Cy5, or PE-Cy7): CD25 (PC61.5), CD3
(17A2), CD4 (GK1.5), CD8 (53– 6.7), NK1.1 (PK136), CD11b (M1/70),
CD11c (HL3), CD19 (1D3), CD80 (16-10A1), CD86 (GL1), and MHC
class II (M5/114.15). Intracellular Foxp3 (FJK-16s) staining was performed according to the manufacturer’s protocol (eBioscience). Appropriate isotype controls were always included. All samples were analyzed on
a FACSAria using Diva. The absolute number of a particular cell subset
was calculated based on the percentage of cells stained for the appropriate
markers determined by FACSAria flow cytometry and the number of
mononuclear cells per mouse spleen defined based on hemocytometer
counts.
For CNS cell isolates, at day 11 after EAE induction, mice were sacrificed
and perfused with PBS delivered transcardially to eliminate contaminating
blood cells in the CNS. CNS mononuclear cells were then isolated from the
brains and spinal cords of five to six mice based on their characteristic sedimentation features on Percoll density gradients (30⬃70%) and stained for cell
surface markers as for splenocytes. Abs were directly labeled and analyzed as
done for splenocytes. Absolute numbers and percentages of particular CNS
mononuclear cell subsets were determined as described above and in Ref. 22.
Cytokine quantification
Single cell suspensions were incubated at 37°C for 3 days in round-bottom
plates (2 ⫻ 106 cells/well) with or without Ags (MOG 10 ␮g/ml, PLP 10
␮g/ml, or Con A 2.5 ␮g/ml) and then stimulated with PMA (20 ng/ml)/
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To induce acute EAE, B6 mice were injected s.c. in the hind flank with 200
␮g of MOG35–55 peptide in CFA (Difco) containing 500 ␮g of nonviable,
desiccated Mycobacterium tuberculosis. On the day of and 2 days after
immunization, the mice also were inoculated with 200 ng of pertussis toxin
(List Biologic) i.p.
For adoptive transfer of EAE (passively induced EAE), lymph node
cells were extracted from primary inoculants described above on day 8
after immunization, and cells were cultured at a density of 2 ⫻ 106/ml in
Click’s Eagle’s Ham’s amino acids medium supplemented with 15% FCS,
20 ng/ml rIL-12, and 50 ␮g/ml MOG35–55 peptide. After 4 days of culture,
cells were harvested, and 3 ⫻ 107 viable cells were injected i.p. into each
recipient mouse irradiated at 350 rad 1 h earlier (22). For both actively and
passively induced EAE, the mice were monitored daily for symptoms
scored on an arbitrary scale of 0 –5 with 0.5 increments: 0, no symptoms;
1, flaccid tail; 2, hind limb weakness or abnormal gait; 3, complete hind
limb paralysis; 4, complete hind limb paralysis with forelimb weakness or
paralysis; 5, moribund or deceased. Values presented indicate averages of
disease symptom scores on a given day, the absolute maximum score seen
for all animals tested, the mean score for all animals on each day and from
the time of initial presentation of symptoms until termination of the experiment, or the terminal (day of the end of the experiment) disease symptom score.
1731
1732
NICOTINE IN CNS INFLAMMATION
ionomycin (1 ␮g/ml)/brefeldin A (5 ␮g/ml) for 5 h at 37°C. After harvesting, cells were stained for surface markers with fluorochrome-conjugated
mAbs targeting CD3, CD4, and/or CD8 as described above and/or for
intracellular cytokines using anti-IFN-␥, anti-IL-4, anti-IL-10, or antiIL-17 mAbs conjugated with Alexa 647 after fixation and permeabilization
using Cytofix/Cytoperm kit (BD Biosciences). All samples were analyzed
on a FACSAria using Diva. For assessment of effects of treatments on
cytokine induction, supernatants were collected 3 days after in vitro boosting. IFN-␥, IL-10, IL-2, and TGF-␤ were measured by optEIA kits (BD
Pharmingen and eBioscience).
Quantification of MOG-reactive Abs
MOG-reactive Abs were quantified using ELISA. In brief, microtiter plates
(Corning Glass Works) were coated with 100 ␮l/well of murine MOG35–55
(10 ␮g/ml) overnight and blocked with 10% FBS (4°C). Mouse serum
samples obtained on day 11 (peak responses after EAE induction) were
then added to the plates and incubated overnight at 4°C. Plates were then
incubated for 2 h with biotinylated rabbit anti-mouse IgG, IgG1, IgG2a,
IgA, IgG3, or IgG2b (Invitrogen) followed by addition of alkaline phosphatase-conjugated ABC reagent (Dakopatts; R&D Systems). Immune
complexes were visualized colorimetrically after exposure to p-nitrophenyl-phosphate and quantified based on assessments of OD at 450 nm.
Differences between groups were evaluated by performing ANOVA. Fisher’s exact test and Mann-Whitney’s U test were applied to analyze disease
incidence and severity, respectively, of symptomatic manifestations.
Results
Nicotinic attenuation of actively induced, and adoptively
transferred, EAE
To determine how nicotine might influence the course of EAE, B6
mice were infused using an implantable minipump with PBS or
with nicotine at a dose of ⬃13 mg/kg/day for 7 days. The choice
on dosage and route of administration was based on recently published guidelines for testing the effects of nicotine in vivo (23) and
to create in mice plasma nicotine levels comparable to those found
in human cigarette smokers (⬃300 nM). Nicotine delivery under
these conditions had no observable effects on animal behavior.
A single injection of MOG35–55 peptide in CFA augmented with
pertussis toxin induced moderate to severe EAE in untreated animals or in animals receiving control infusions of PBS only (maximum disease symptom score 4.25 ⫾ 0.80, mean disease symptom
score over days 7–26 of 2.99 ⫾ 0.67; Fig. 1). The average time
after immunization to initial presentation of disease symptoms was
7.6 ⫾ 0.53 days in these control animals (Fig. 1A). Disease symptoms subsequently increased, with paralysis being evident at days
9 to 14 after immunization, before control animals showed a slow
and incomplete recovery stabilizing ⬃25–26 days after immunization (i.e., at termination of the experiment; terminal disease
symptom score of ⬃2.75 ⫾ 0.25; Fig. 1). By contrast, mice receiving nicotine 7 days before or on the day of immunization had
a delayed onset of EAE, with disease symptoms first becoming
evident 11 days after immunization and peaking 16 –18 days after
immunization (Fig. 1A). Moreover, severity of EAE was attenuated in these nicotine-treated animals, reaching mean maximum
disease symptom scores of 3.17 ⫾ 0.78 for the pretreatment group
(Fig. 1A) and 3.13 ⫾ 0.25 for the nicotine cotreatment group (Fig.
1A). Differences in times to initial presentation, times to peak
symptoms, and maximum or mean disease severities between controls receiving PBS and nicotine pre- or cotreated animals all were
statistically significant ( p ⬍ 0.01). Also striking was the ability of
nicotine pre- or cotreatment to accelerate the rate of recovery and
to increase the extent of recovery from EAE (Fig. 1A). Disease
symptom scores fell to ⬍1.0 by the experiments’ termination
(⬃25–26 days after immunization; disease symptom scores were
0.85 ⫾ 0.25 for nicotine cotreatment or 0.83 ⫾ 0.26 for nicotine
pretreatment; p ⬍ 0.01 compared with PBS controls). Thus, nic-
FIGURE 1. Nicotinic prevents EAE and ameliorates CNS inflammation, demyelination, and axonal damage in B6 mice. PBS (control; ⽧) or
nicotine at a dose of ⬃13 mg/kg (F) were administered daily for a total of
7 days for treatment that started 7 days before (pretreatment; day ⫺7;
green), on the day of (cotreatment; day 0; red), or 7 days after (delayed
treatment; day ⫹ 7, blue) immunization with myelin Ags (A). In the adoptive transfer study (B), T cells from immunized animals that were exposed
to PBS only (control; ⽧) or nicotine (red F) on the day of immunization
were introduced into naive animals, and evolution of EAE in recipients was
monitored. Mice were observed daily and scored for disease symptoms.
Data for each time point represent the average disease symptom score ⫾
SEM (see Materials and Methods) at the given time (days) for 10 –12 (A)
or 5 (B) mice in each group. At day 25 post immunization, mice were
sacrificed, and spinal cord samples were fixed in formalin and then processed as longitudinal sections for H&E (left), Luxol Fast Blue (LFB;
middle), or Biechowsky silver (right) staining. C–E, control group. F–H,
nicotine-treated group. Original photos were taken at ⫻200. I, Semiquantitative analyses of inflammation, demyelination, and axonal damage in
tissues from control (solid bars) or nicotine-treated (cross-hatched bars)
animals. Histopathological indices were scored as described in Materials
and Methods. n ⫽ 3 for all experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 based
in the Mann-Whitney U test.
otine exposure preceding or simultaneous with disease induction
significantly delayed EAE onset, attenuated EAE severity, and
promoted disease recovery.
If nicotine treatment began 7 days after immunization, i.e.,
when disease symptoms (flaccid tail, hind limb weakness, etc.)
typically are first evident and when myelin-reactive T cells have
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Statistical analysis
The Journal of Immunology
1733
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already become activated, there was no striking difference in the
time to peak effect (Fig. 1A). However, the maximum and mean
severities of EAE were attenuated (3.97 ⫾ 0.26 and 2.99 ⫾
0.67, respectively, for controls; 3.17 ⫾ 0.24 and 1.89 ⫾ 0.29,
respectively, for delayed nicotine treatment; p ⬍ 0.05), and
disease severity at termination of the experiment was even more
reduced (2.75 ⫾ 0.25 for controls and 0.49 ⫾ 0.01 for delayed
nicotine treatment; p ⬍ 0.01; Fig. 1A). Therefore, attenuation of
disease severity and acceleration of recovery also were evident
even if nicotine exposure was delayed until the day when disease symptoms first became evident and even when myelinreactive T cells were activated.
EAE is a T cell mediated disease that can be produced in
recipients of encephalitogenic T cells (24). Thus, we performed
adoptive transfer experiments in which T cells from immunized
animals that were exposed to PBS only or nicotine starting at
the time of immunization was introduced into naive animals,
and evolution of EAE in recipients was monitored. Robust development of EAE was evident in control animals. The maximum and mean disease severity scores in these animals were
5.0 ⫾ 0.25 and 4.22 ⫾ 0.36, respectively (Fig. 1B). By contrast,
disease severity scores were 2.5 ⫾ 0.82, p ⬍ 0.001 (maximum)
and 1.0 ⫾ 0.15, p ⬍ 0.001 (mean) for recipients from nicotinetreated animals (Fig. 1B). Moreover, whereas there was no recovery at all over the course of the study in recipients of encephalitogenic T cells, recipients of cells from animals
immunized and treated with nicotine showed nearly complete
resolution of symptoms by the end of the experiment (Fig. 1B).
These results showing protection against adoptive transfer of
EAE suggest that nicotine exposure may modulate (directly or
indirectly) T cell properties/functions.
Nicotine exposure ameliorates CNS inflammation, demyelination,
and axonal damage
Pronounced cellular infiltration, demyelination, and axonal damage
are pathological hallmarks of EAE and MS. To evaluate whether nicotine can alter these pathological changes, we conducted histological
examinations of spinal cords isolated 25 days after immunization
from control mice immunized to produce EAE and from mice immunized but also exposed to nicotine for 7 days starting on the day of
immunization.
Examination of white matter in longitudinal sections of these tissues from control mice stained with H&E revealed marked multifocal
and lymphohistiocytic inflammation that was both perivascular and
diffuse (Fig. 1C). Myelin loss as revealed by luxol fast blue staining
was widespread, especially around inflamed areas (Fig. 1D). In sharp
contrast, most sections from nicotine-treated mice had few infiltrating
cells, and myelin sheets were largely preserved (Fig. 1, F and G).
Furthermore, axonal damage revealed by silver staining was clearly
present in the submeningeal areas of PBS control mice, whereas axons from nicotine-treated mice were scarcely affected and then only in
regions immediately surrounding foci of inflammation (Fig. 1, E and
H). Quantitative analysis of histological indices showed that inflammation, demyelination, and axonal degeneration were significantly
greater in PBS-treated compared with nicotine-treated mice (Fig. 1I;
p ⬍ 0.01). Therefore, nicotine exposure protected the CNS against
inflammation, demyelination, and axonal damage that are pathological hallmarks of EAE.
Nicotine treatment alters the peripheral lymphocyte
subpopulation during EAE
Nicotine exposure can modify T and B cell development and survival,
although a consensus has yet to develop regarding mechanisms involved and how differences observed may relate to the degree of cell
FIGURE 2. Nicotine alters the peripheral lymphocyte subpopulation
during EAE. On the day of immunization to induce acute EAE, mice were
treated with PBS (control) or with nicotine at a dose of ⬃13 mg/kg daily
for a total of 7 days. Mice were sacrificed on day 11 after immunization,
and splenic mononuclear cells were isolated as described in Materials and
Methods. Dot plots illustrating flow cytometry results generated after gating on lymphocytes (by forward vs side scatter) are shown for T and B
cells. A–C, Representative dot plot results for CD4⫹, CD8⫹, NK, NKT
cells, and CD3⫺CD19⫹ cells. D, Absolute numbers of CD4⫹, CD8⫹,
CD3⫹, and CD3⫺CD19⫹ cells from control (solid bars) or nicotine-treated
(cross-hatched bars) animals (n ⫽ 5 mice each group).
maturity during drug exposure (25). To define effects of nicotine exposure on lymphocyte status during an autoimmune response to
MOG, we quantified various lymphocyte subpopulations based on
1734
cellular phenotype from mice immunized with MOG under control
conditions or in concert with nicotine treatment commenced on the
day of MOG immunization (day 0). Results obtained for animals
FIGURE 4. Nicotine reduces the
expression of MHC class II, B7-1,
and B7-2 on APCs in the periphery.
Mice were immunized to induce acute
EAE and treated with PBS (control;
solid bars) or with nicotine (crosshatched bars) at a dose of ⬃13 mg/kg
daily for a total of 7 days. Mice were
sacrificed on day 11 after immunization, and splenic mononuclear cells
were isolated as described in Materials and Methods. MHC II, B7-1
(CD80) or B7-2 (CD86) expression
was analyzed with gating on macrophages (CD11b⫹, A) or on dendritic
cells (CD11c⫹, B) in the periphery
(left columns are representative histogram plots; right columns are absolute numbers; n ⫽ 5 mice each group;
ⴱ, p ⬍ 0.05 vs PBS; ⴱⴱ, p ⬍ 0.01 vs
PBS).
treated with nicotine 7 days before of after immunization were similar
and so are not detailed in this study.
Compared with control EAE mice, nicotine-treated mice had various degrees of reductions in the percentages and numbers of CD3⫹,
CD4⫹, or CD8⫹ T cells or CD19⫹CD3⫺ B cells among splenocytes
sampled on day 11 post immunization (Fig. 2, A–D; p ⬍ 0.05). Concurrently, there were no significant effects of nicotine treatment on
numbers or proportions of NK cells (CD3⫺NK1.1⫹), NKT cells
(CD3⫹NK1.1⫺), or CD4⫹CD25⫹ regulatory T cells (Fig. 2B and
3A). Interestingly, the expression of Foxp3 was clearly augmented in
the nicotine-treated mice (Fig. 3, B and C).
Although the numbers and percentages of peripheral CD11b⫹
mononuclear/microglial (Fig. 4A) and CD11c⫹ dendritic (Fig. 4B)
cells were not dramatically altered by nicotine treatment, reductions in
the expression of MHC class II, CD80, and CD86 were notable on
these cells (Fig. 4). Similar data were also obtained from assessments
of cells derived from lymph nodes or peripheral blood and at several
other time points during the course of EAE (data not shown).
Nicotine exposure inhibits autoreactive T cell expansion
To address how nicotine affected expansion of MOG35–55-specific T
cell responses in nicotine-treated EAE mice, splenic mononuclear
cells were isolated at peak of the automimmune response (11 days
post immunization) from animals immunized on the same day as initiation of control or nicotine treatment. T cell proliferation in response
to myelin Ags was quantified by measuring [3H]lthymidine incorporation and based on CFSE assays. Compared with baseline proliferation responses in tissue culture medium, there were slightly but not
significantly higher responses in both control and nicotine-treated
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FIGURE 3. Nicotine augments the expression of FoxP3 on regulatory T
cells. Mice were immunized to induce acute EAE and treated with PBS (control) or with nicotine at a dose of ⬃13 mg/kg daily for a total of 7 days. Mice
were sacrificed on day 11 after immunization, and splenic mononuclear cells
were isolated as described in Materials and Methods. A, Representative dot
plots showing flow cytometry results and percentages of CD4⫹CD25⫹ T cells
gated on lymphocytes (n ⫽ 5). B, The expression of Foxp3 in relation to
CD25⫹ cells was determined by gating on CD4⫹ cells (n ⫽ 5). C, The average
percentage of CD4⫹CD25⫹ T cells and Treg⫹/FoxP3⫹CD4⫹ cells in PBStreated (control; solid bars) or nicotine-treated (cross-hatched bars) mice.
NICOTINE IN CNS INFLAMMATION
The Journal of Immunology
1735
Table I. Cytokines secretion was measured by ELISA at the different
time pointsa
Time Points of Measurement
Day 11
Cytokine
Control
Nicotine
IFN-␥ (ng/ml)
IL-10 (pg/ml)
TGF-␤ (pg/ml)
IL-2 (pg/ml)
IL-4 (pg/ml)
IL-17 (pg/ml)
166 ⫾ 32
113 ⫾ 36
128 ⫾ 41
432 ⫾ 36
UTb
171 ⫾ 77
Day 26
Control
Nicotine
64 ⫾ 3.0ⴱⴱ 96 ⫾ 35 34 ⫾ 12ⴱ
164 ⫾ 30ⴱ
96 ⫾ 35 134 ⫾ 32ⴱ
264 ⫾ 52ⴱ
96 ⫾ 35 164 ⫾ 42ⴱ
294 ⫾ 72ⴱ
206 ⫾ 69 104 ⫾ 48ⴱ
UTb
UTb
UTb
131 ⫾ 62
106 ⫾ 39 84 ⫾ 38
a
Mice were immunized to induce acute EAE and treated with PBS or with nicotine at a dose of ⬃13 mg/kg daily for a total of 7 days. Mice were sacrificed at the
indicated days after immunization, and splenic mononuclear cells were isolated and
cultured in the presence of MOG (see Materials and Methods). Supernatants were
harvested after 36 h, and IFN-␥, IL-17, IL-2, IL-10, or TGF-␤1 secretion was determined by ELISA. Data are means ⫾ SD of values for nine mice per group; ⴱ, p ⬍ 0.05
vs. PBS; ⴱⴱ, p ⬍ 0.01 vs. PBS.
b
UT, Undetectable; ND, not done.
Exposure to nicotine alters Th cell cytokine profile
FIGURE 5. Nicotine inhibits peripheral autoreactive T cell responses.
Mice were immunized to induce acute EAE and treated with PBS (control;
solid bars) or with nicotine (cross-hatched bars) at a dose of ⬃13 mg/kg daily
for a total of 7 days. Mice were sacrificed on day 11 after immunization, and
splenic mononuclear cells were isolated as described in Materials and Methods. A, The proliferation of Ag-specific splenic cells from control (solid bars)
or with nicotine-treated (cross-hatched bars) mice was measured as [3H]thymidine incorporation by these cells cultured in the absence of Ag (tissue culture medium; TCM) or with PLP or MOG. Results are expressed as mean
cpm ⫾ SD. B, Representative dot plots of flow cytometry results showing
proliferation responses assessed based on CFSE staining (arrow points in direction of increased cell division) of CD3⫹, CD4⫹, or CD8⫹ T cells. C, Representative dot plots of flow cytometry results showing assessments of splenic
cell apoptosis and death based on annexin V and PI double staining. Results
are from one of three independent experiments (n ⫽ 5 mice each group; ⴱ, p ⬍
0.05 vs PBS; ⴱⴱ, p ⬍ 0.01 vs PBS).
groups to PLP, but responses to MOG in nicotine-treated animals
were no higher and were significantly ( p ⬍ 0.05) lower than responses of cells from control animals to MOG challenge (Fig. 5A).
Similarly, there were fewer CD3⫹, CD4⫹, and CD8⫹ T cells with
lower levels of CFSE staining, indicative of a reduced T cell proliferative response to MOG in nicotine-treated mice (Fig. 5B). By con-
To further characterize effects of nicotine exposure on autoreactive T
cells, splenocytes isolated at the peak of the EAE response (11 days
after immunization) from animals subjected to control PBS or nicotine treatment starting on the day of immunization were then subjected to 3 days of cell culture alone or in the presence of Ags (MOG
10 ␮g/ml, PLP 10 ␮g/ml, or Con A 2.5 ␮g/ml) and then stimulated
with PMA (20 ng/ml)/ionomycin (1 ␮g/ml)/brefeldin A (5 ␮g/ml) for
5 h. After harvesting, cells were stained for cell surface markers
and/or fixed and permeabilized to allow staining for intracellular cytokines, all of which were quantified using flow cytometry, and cell
culture supernatants were collected and assayed for cytokine release
by enzyme immunoassays.
Secreted IFN-␥ by splenocytes isolated from nicotine-treated animals was reduced (⬃64 ⫾ 3.0 ng/ml compared with ⬃166 ⫾ 32
ng/ml in controls; p ⬍ 0.01 at day 11; ⬃96 ⫾ 35 ng/ml compared
Table II. Cytokines expression was detected by FACS at the different
time pointsa
Time Points of Measurement
Day 11
Cytokine (%)
⫹
⫹
IFN-␥ /CD8
IL-10⫹/CD4⫹
TGF-␤⫹/CD4⫹
IL-2⫹/CD4⫹
IL-4⫹/CD4⫹
IL-17⫹/CD4⫹
Day 26
Control
Nicotine
Control
Nicotine
5.4 ⫾ 1.2
1.0 ⫾ 0.4
NDb
NDb
UTb
2.4 ⫾ 0.8
2.3 ⫾ 0.5ⴱ
1.7 ⫾ 0.6
NDb
NDb
UTb
1.7 ⫾ 0.7
1.4 ⫾ 0.5
0.7 ⫾ 0.3
NDb
NDb
UTb
0.9 ⫾ 0.4
0.9 ⫾ 0.2ⴱ
1.1 ⫾ 0.4
NDb
NDb
UTb
1.0 ⫾ 0.4
a
Mice were immunized to induce acute EAE and treated with PBS or with nicotine at a dose of ⬃13 mg/kg daily for a total of 7 days. Mice were sacrificed at the
indicated days after immunization, and splenic mononuclear cells were isolated and
cultured in the presence of MOG (see Materials and Methods). Cells were collected
after 36 h, and IFN-␥, IL-17, or IL-10 expression was determined by FACS. Data are
means ⫾ SD of values for nine mice per group; ⴱ, p ⬍ 0.05 vs. PBS; ⴱⴱ, p ⬍ 0.01
vs. PBS.
b
UT, Undetectable; ND, not done.
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trast, annexin V and propidium iodide double staining for cells undergoing apoptosis or necrosis revealed no increase in cell death upon
nicotine exposure, at least at the current dose (Fig. 5C). Thus, the
reduced numbers of several lymphocyte subpopulations recorded in
Fig. 3 were likely the result of a selective decline in proliferation
related to nicotine exposure rather than an increase in apoptosis.
1736
NICOTINE IN CNS INFLAMMATION
FIGURE 6. Nicotine alters autoantibody isotypes. Mice were immunized to induce acute EAE and treated with PBS (control; solid bars) or
with nicotine (cross-hatched bars) at a dose of ⬃13 mg/kg daily for a total
of 7 days. Sera were collected on day 11 after immunization, and titers of
MOG-specific IgG (1/500), IgG1 (1/500), IgG2b (1/500), IgG3 (1/100),
IgA (1/100), and IgG2a (1/2) were determined by ELISA. Data are
means ⫾ SD for three mice per group. Similar results were obtained in two
independent experiments. (ⴱ, p ⬍ 0.05 vs PBS; ⴱⴱ, p ⬍ 0.01 vs PBS).
Treatment with nicotine alters autoantibody isotypes
Autoantigen responses are influenced by helper T cell levels
and actions. For example, in mice, IFN-␥ secreted from Th1
cells is believed to drive the IgG2b response, whereas the IgG1
response is driven by Th2 cells principally involved in Abmediated immunity and help direct toward B cells. We used
ELISAs to measure MOG-specific Abs of specific isotypes
FIGURE 8. Nicotine inhibits activation-induced MHC class II expression and suppresses costimulatory molecules in APCs. Mononuclear cells
were isolated from the CNS of mice 11 days after immunization to induce
acute EAE and treatment with PBS (control; solid bars) or with nicotine
(cross-hatched bars) at a dose of ⬃13 mg/kg daily for a total of 7 days. A,
A representative dot plot illustrates results of staining for CD11b⫹ macrophage/microglia (CD11b⫹CD45⫹) populations on mononuclear cells. B,
Absolute numbers of macrophage/microglial cell (CD11b⫹CD45⫹) are
shown. C and E, Representative histograms and D and F, plots of absolute
numbers of MHC II, B7-1 (CD80) or B7-2 (CD86) expression on gated,
live macrophages (CD11b⫹) or dendritic cells (CD11c⫹) in the CNS
(ⴱ, p ⬍ 0.05 vs PBS; ⴱⴱ, p ⬍ 0.01 vs PBS).
present in the sera of animals 11 days after immunization and
initiation of control or nicotine exposure. IgG2b production was
halved in nicotine-treated mice, whereas levels of IgG1 and IgA
were significantly increased ⬎3-fold and ⬎25%, respectively
(Fig. 6). The decrease in IgG2b is consistent with the decrease
in IFN-␥ with nicotine exposure and a blunted Th1 cell
response.
FIGURE 7. Nicotine alters lymphocyte subpopulations in the CNS during EAE. Mice were immunized to induce acute EAE and treated with PBS
(control; solid bars) or with nicotine (cross-hatched bars) at a dose of ⬃13
mg/kg daily for a total of 7 days. Mice were sacrificed on day 11 after
immunization, and mononuclear cells were isolated from the CNS as described in Materials and Methods. The dot plots generated after gating on
mononuclear cells (by forward vs side scatter) are shown for T and B cells.
A, Representative dot plots are shown for CD3⫺CD19⫹ cells (upper) and
CD4⫹CD8⫹ cells (lower). B, Absolute numbers, calculated as described in
Materials and Methods, appear for CD3⫹, CD3⫺, CD19⫹, CD4⫹, and
CD8⫹ cells.
Nicotine exposure alters lymphocyte subpopulations in the CNS
during EAE
As described earlier, histopathological studies revealed that there
are less inflammatory infiltration in CNS tissues from nicotinetreated animals than in spinal cord tissues from PBS-treated
control animals. To assess the impact of nicotine exposure on the
CNS immune cell profile, animals at day 11 after EAE induction
and initiation of control or nicotine treatment were cleared of
blood cells in the CNS and used to isolate CNS mononuclear cells
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with ⬃34 ⫾ 12 ng/ml in controls; p ⬍ 0.05 at day 26) (Table I). The
reduction in secreted IFN-␥ by cells isolated from nicotine-exposed
animals was most marked in CD8⫹ compared with CD4⫹ cells (Table II). There was no significant effect of nicotine exposure in IL-17
secretion (Table ⌱ and Table ⌱⌱); secreted IL-2 was reduced (Table I).
However, secreted IL-10 or TGF-␤1 levels were increased from cells
derived from nicotine-treated animals (Table I and Table II). Production of IL-4 was undetectable. The results imply nicotine treatment
induced a shift from Th1 toward Th2 or Th3 responses.
The Journal of Immunology
Discussion
Expression of nicotinic acetylcholine receptors on nonneuronal
cells such as APCs and other immune system cells underscores the
idea that nAChRs have functions well beyond mediation of nicotinic cholinergic neurotransmission. Although considerable evidence indicates that nicotine exerts anti-inflammatory and immune
modulatory effects in vivo (2), its specific roles in modulating CNS
inflammation and autoimmune responses are not well defined. Using the EAE model, we have now demonstrated that nicotine exposure can dramatically delay disease onset, attenuate disease severity, and facilitate recovery. This occurs whether nicotine
treatment begins before, at the time of, or after immunization with
myelin Ags to induce EAE. Moreover, nicotine exposure also suppresses disease development on adoptive transfer of autoimmune T
cells. At the histopathological level, nicotine exposure curtails the
infiltration of inflammatory cells into the CNS and the destruction
of myelin and axons. These results thus reveal new aspects of
nicotine’s functions as a potent immunomodulator, suggest novel
roles of nAChRs in those processes, and highlight the importance
of understanding interactions between nervous and immune systems, especially in the context of development and treatment of
CNS inflammatory disorders.
The brain and spinal cord are considered immunologically privileged organs in that, under normal physiological circumstances,
entry of lymphocytes and inflammatory mediators into the CNS is
blocked by the blood/brain barrier (27). Nevertheless, the initiation
of EAE, and perhaps MS, requires activation of T cells in peripheral lymphoid organs and homing of myelin-reactive T cells as
well as APCs to the CNS. Aided by local APCs, such as microglia
and astrocytes, infiltrating T cells undergo reactivation and, in concert with other cellular and soluble components of the immune
system, orchestrate the induction of CNS pathology (16, 17).
Mechanisms underlying the dramatic effects of nicotine in our
model can be multifactorial. First, we demonstrated that the expansion of MOG-reactive T cells from the spleen in nicotinetreated mice was significantly dampened. In these animals, MOGreactive Th cells produced less IFN-␥ and IL-2 than cells from
PBS-treated controls, whereas production of IL-10, and particularly TGF-␤, was augmented. A marginal, but not significant reduction of IL-17 was observed in mice that received nicotine. Our
observation is somewhat surprising given the augmentation of
TGF-␤ in nicotine-exposed mice. We reasoned that the timing of
TGF-␤ production might contribute to lack of effect of Th-17 development. Nicotine also did not appear to induce apoptosis in
autoreactive T cells. This outcome invites the prediction that the
immunological effects induced by nicotine may have contributed
to the decreased T cell proliferation and altered cytokine profile.
Increased production of TGF-␤, by itself, can suppress MOG-reactive T cells. In contrast, TGF-␤ may promote the generation of
CD4⫹CD25⫹ regulatory T cells (28, 29). Indeed, we observed
that, although the absolute numbers of CD4⫹CD25⫹ regulatory T
cells were not dramatically altered by nicotine exposure, expression of FoxP3 was significantly up-regulated. These regulatory T
cells with enhanced FoxP3 expression may contribute to the suppression of T effector/autoreative cells.
The other factor that contributes to the decreased expansion and
a shift in cytokine profile of MOG-reactive T cells is an altered
APC phenotype in nicotine-treated animals. Consistent with previous studies (30), we found that nicotine significantly reduced
levels of MHC class II, CD80, and CD86 expression on peripheral
CD11c⫹ and CD11b⫹ cells. Notably, these changes were more
dramatic for CD11b⫹ cells. In an experimental sepsis model,
Wang and colleagues reported that nicotine prevented activation of
the NF-␬B pathway and inhibited HMGB1 secretion from macrophages, which appear to be responsible for improved survival of
the animals (8). It has been well established that a specific type of
APC can direct differentiation of Th cells to produce regulatory
cytokines and moderate immune responses (31–34). It is highly
likely that the altered APC phenotype in nicotine-treated animals
may, at least in part, reduce the encephalitogenic capacity of
MOG-reactive T cells in the EAE model.
In sharp contrast with control EAE mice, nicotine-treated animals had relatively few cellular infiltrates in CNS. Further, flow
cytometry analysis of the cellular infiltrates showed a significant
reduction of CD4⫹, CD8⫹, CD19⫹, CD11c⫹, CD11b⫹, and
CD11b⫹CD45⫹cell populations, and the reductions in CD19⫹ B
cells and CD11c⫹ dendritic cells seem to reflect diminished migration into the CNS from the periphery. It is presently unclear
whether the reduction of CD4⫹ and CD8⫹ cells in the CNS stems
from reduced influx from the periphery, impaired expansion in the
CNS after migration, or both. Whatever the mechanism, it is clear
that there is significantly reduced expression of Ag presentation
machinery by resident or infiltrating CD11c⫹ and CD11b⫹ cells.
There are several possible ways for nicotine exposure to affect
disease symptoms, even when applied after EAE has been initiated
and initial presentation of CNS symptoms recover after the peak
stage. First, nicotine may inhibit myelin-reactive T cell determinant spreading when T cells migrating from the periphery encounter CNS Ags. Second, a large percentage of C57BL/6 mice will
spontaneously recover after peak stage of neurological deficit via
unknown mechanisms that may be particularly sensitive to nicotinic action. Third, nicotine may directly or indirectly facilitate
function of oligodendrocytes, myelin-forming cells, to stimulate
regeneration after inflammatory insults. These possibilities are currently under investigation in our laboratory.
In agreement with previous studies that nicotine could suppress
the migration of leukocytes to the inflammation/infection site (35),
we also demonstrated that there was a dramatic reduction in overall inflammatory cell numbers, including monocytes that migrated
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(i.e., resident microglial cells and monocytes that had migrated
into the CNS from the blood). Cells were then stained for surface
markers to define absolute numbers of and percentages of specific,
CNS mononuclear cell subsets using flow cytometry.
CD3⫹, CD4⫹, and CD8⫹ T cells and CD3⫺CD19⫹ B cells were
abundant in the CNS from PBS-treated EAE mice, but numbers of
all of these cells were drastically (ⱖ4-fold) reduced in central tissues from nicotine-treated animals (Fig. 7).
We also examined APCs in the CNS during EAE, including
CD11c⫹ dendritic cell and CD11b⫹ macrophage/microglial cell
(CD11b⫹CD45⫹) populations. CD45 was initially used to distinguish microglia from infiltrating macrophages. However, upon activation, expression of CD45 no long clearly distinguishes the two
cell types (26). CD11b⫹CD45⫹ and CD11c⫹ cells were dramatically (⬃10-fold) reduced in CNS tissues from nicotine-exposed
animals (Fig. 8, A and B). There also were reductions (⬃2– 4-fold
in proportions and ⱖ10-fold in absolute numbers) of MHC class
II⫹, CD80⫹, and CD86⫹ levels on CD11b⫹ cells and ⬎10-fold
reductions in levels of those markers on CD11c⫹ cells with nicotine treatment (Fig. 8, C–F). Importantly, the magnitude of reduction in nicotine-exposed animals of CD80 and CD86 levels on
CD11b⫹ or CD11c⫹ cells was much greater for CNS than for
peripheral monocytes (see Fig. 4). This suggests that nicotine exposure may have distinct immunomodulatory effects in different
anatomic compartments, perhaps reflecting different mechanisms
beyond different magnitudes of effect.
1737
1738
Acknowledgments
We thank X. Bai for histological analysis; J. Wu, A. Simard, and P. Whiteaker for fruitful discussions and advice; S. Miller, L. Lucero, and S. Rhodes
for technical assistances; and P. Minick for editing the manuscript.
Disclosures
The authors have no financial conflict of interest.
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from the periphery and CNS microglial cells. Considerable evidence suggests that microglia have multiple and sometimes contrasting functions in inflammatory and degenerative disorders of
the CNS (36 – 40); not only can microglia only serve as APCs, but
they also possess neurotoxic or neuroprotective activities (17, 41–
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Nicotine dosing in the current study was designed to mimic in
mouse plasma levels of nicotine achieved in the typical human
cigarette smoker, and the EAE model is a surrogate for at least
some forms of MS. More specifically, EAE most closely resembles
the human demyelinating disorder, acute disseminated encephalomyelitis, one of the subforms of MS. Further studies are required
to verify whether nicotine’s effects on EAE can be generalized to
other forms CNS disorders, including MS. The literature on relationships between tobacco product use and autoimmune MS is
mixed. Some epidemiological studies suggested that smoking was
associated with aggravation of MS symptoms (44 – 47), whereas
other studies documented no influence of smoking on disease progression and MS severity (48). It is only recently that a consensus
developed that the risk for developing Parkinson’s disease is about
one half in smokers of the risk in nonsmokers (19, 20), and perhaps
similar studies should be initiated with regard to risks for developing MS. It is important to note that nicotine exposure is not the
only factor influencing health in smokers; many other elements in
tobacco could alter nervous and immune system function (2, 49 –
51). For example, acrolein affects neutrophil function and decreases the resistance of the lungs to infections (52, 53). Hydroquinone has been shown to inhibit the activation and proliferation
of T cell (54, 55). Chronic exposure to benzo[a]pyrene induces
dose-related decreases in the mass and cellularity of lymphoid tissues, and maternal exposure to benzo[a]pyrene alters the development of T cells and immune responses in the offspring (55, 56).
Many compounds are associated with brain toxicity including vinyl chloride, arsenic, and hydrogen cyanide (57, 58). Also, the
timing of nicotine exposure or smoking behavior with regard to
disease stage has not been considered. However, it does make
sense that if pure nicotine acts as an immunosuppressive agent,
under the right circumstances, that activity could be leveraged.
Our study provides evidence that nicotine exposure can prevent
the loss of tolerance to myelin Ags. Nicotine may have these effects by acting on multiple steps in the autoimmune response. Expression of ␣7 nicotinic acetylcholine receptors by CNS microglia
and/or astrocytes invites the prediction that this receptor subtype
may mediate the effects of nicotine observed in the EAE model.
Further elucidation of roles played by ␣7 nicotinic receptors and
other potential entities involved in mediating beneficial effects of
nicotine on EAE promises to illuminate novel and potentially superior strategies for treatment of human neuroinflammatory and
neurodegenerative diseases.
NICOTINE IN CNS INFLAMMATION
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