The role of nicotinic acetylcholine receptors in lymphocyte development

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

The role of nicotinic acetylcholine receptors in lymphocyte development

Marina Skok a,*, Regis Grailhe b,1, Fabien Agenes c, Jean-Pierre Changeux b

a Department of Molecular Immunology, Palladin Institute of Biochemistry, 01601 Kiev, Ukraineb Receptors and Cognition Unit, Institut Pasteur, 75424 Paris, France

c Joseph Fourier University, 38054 Grenoble, France

Received 26 May 2005; accepted 21 September 2005

Abstract

The sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type)

mice were studied by flow cytometry. The absence of h2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the

bone marrow. In chimera mice, either h2 or a7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen,

only a7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We

conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Nicotinic acetylcholine receptor; Lymphocytes; Development; Knockout mice; Chimera mice

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) belong to the

superfamily of oligomeric proteins that transduce electric

signals across the cell membrane upon binding of neuro-

transmitters. These receptors may have different kinetic and

pharmacological properties according to their structural

differences (Lindstrom et al., 1998). nAChRs are found in

two primary categories, homopentameric ((a7)5, (a8)5,

(a9)5) or heteropentameric ((a1)2h1gy, (a3)2(h4)3, (a3)2a5(h4)2, (a4)2(h2)3, etc.) forms.

Neuronal nAChRs are expressed in the brain (Changeux

et al., 1998; Paterson and Nordberg, 2000), the autonomic

ganglia (Skok, 2002) and in other tissues, such as skin

(Grando, 1997), respiratory epithelium (Maus et al., 1998),

vascular endothelium (Macklin et al., 1998) and immune

organs (Toyabe et al., 1997; Kuo et al., 2002). The presence

of nAChRs in immune cells is of interest for many reasons.

0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2005.09.011

* Corresponding author. Tel.: +38 44 234 33 54; fax: +38 44 279 63 65.

E-mail address: [email protected] (M. Skok).1 Present address: Institut Pasteur Korea, 39-1 Hawolgok-dong, Sunguk-

gu, 136-79, Seoul, Korea.

First, many lymphoid organs are innervated with cholinergic

fibers and nAChR may mediate neuro-immune interactions

(Bellinger et al., 1993; Singh and Fatani, 1988; Artico et al.,

2002). Second, lymphocytes produce endogenous acetyl-

choline, which may be an autocrine/paracrine functional

regulator (Kawashima and Fujii, 2003). Finally, nAChRs

mediate the pathogenic effects of nicotine inhaled upon

smoking (Conti-Fine et al., 2000). The role of nAChRs was

demonstrated mainly for T lymphocytes where nicotine

affected both cell maturation (Middlebrook et al., 2002) and

activation (Kalra et al., 2000). Recently we reported the

presence of a4h2 and a7 nAChRs in B lymphocyte-derived

cell lines and showed that activation of a7 nAChR

stimulated cell proliferation (Skok et al., 2003). Subse-

quently, we used nAChR-knockout mice to determine that

a4h2 nAChRs expressed in normal B lymphocytes are

involved in regulating their activation and antibody immune

responses (Skok et al., 2005). These results suggested that

nAChR signaling is also involved in forming the pre-

immune repertoire of B cells. In this study, we investigated

the role of nAChRs in B lymphocyte development using

both nAChR knockout and chimera (knockout/wild-type)

mice. We show that a4h2 and a7 nAChRs are involved in

the development of both T and B lymphocytes. In particular,

gy 171 (2006) 86 – 98

M. Skok et al. / Journal of Neuroimmunology 171 (2006) 86–98 87

they affect B lymphocyte survival, possibly, by influencing

IgM and CD40 expression.

2. Materials and methods

2.1. Mice

The generation of both a7- and h2-knockout (� /� or KO)

mice has been previously described (Piccioto et al., 1995;

Orr-Urtreger et al., 1997). Iffa-Credo (France) supplied male

C57BL/6J wild-type control mice and h2� /� and a7� /�

mice from parents, respectively, backcrossed 17 times on a

C57BL/6J background. Two groups of wild-type and h2� /�

mice obtained free-base nicotine in drinking water (200 Ag/ml) during 10 months. All knockout and chimera mice were

kept in the animal facility of Pasteur Institute, Paris. Some

wild-type mice were analysed in the Institute for Genetics,

University of Cologne, Germany.

2.2. Generation and treatment of chimera mice

Thirty-seven-week-old male C57BL/6J mice (genotype

Ly 5.2) were irradiated by a Cesium source irradiator (Cis

Bio International, France) at 800 Rads for 2 min. Bone

marrow cells were harvested from 4 groups of mice, 4 or 5

mice per group: wild-type C57BL/6J Ly 5.1-positive mice

(hereafter WT 5.1), wild-type C57BL/6J Ly 5.2-positive

mice (WT 5.2), a7 knockout Ly 5.2-positive mice (a7� /�

5.2) and h2 knockout Ly 5.2-positive mice (h2� /� 5.2). The

harvested bone marrow cells were injected intraorbitally into

irradiated hosts. Each irradiated mouse received 6�106 cells

in 200 Al of medium. The cells of Ly 5.2 and Ly 5.1

phenotypes were mixed for injection in proportion 20 :80

determined by flow cytometry using Ly 5.1- and Ly 5.2-

specific antibodies (see below). Each cell combination: (WT

Mixtures of bonemarrow cells

from donor miceIrradiated hosts

WT.Ly5.1β2−/−.Ly5.2

WT.Ly5.1WT.Ly5.2

B6.Ly5.2

B6.Ly5.2

WT.Ly5.1α7−/−.Ly5.2

B6.Ly5.2

Fig. 1. The schematic diagram of chime

5.1+WT 5.2); (WT 5.1+h2� /� 5.2); (WT 5.1+a7� /� 5.2),

was injected into ten mice resulting in three groups of

chimeras.

Nicotine treatment began 1 week after bone marrow cell

injections. Each group of chimeras was divided into two

subgroups of five animals. One subgroup was given pure

drinking water, while the matched subgroup was given

water with gradually increasing concentrations of nicotine:

100 Ag/ml for 10 days, 200 Ag/ml for 1 week, 300 Ag/ml

for 1 week and 500 Ag/ml for 1 month. Then the mice

were killed by cervical dislocation and their bone marrow,

spleen and thymus cells were analysed by flow cytometry.

The methodologic diagram is schematically presented in

Fig. 1.

2.3. Antibodies

The following monoclonal antibodies purchased from

PharMingen, BD Biosciences, were used for FACS analy-

sis: PE-Texas red- or Alexa fluor-conjugated rat anti-mouse

CD45R/B220 (clone RA3-6B2); allophycocyanin (APC)- or

fluorescein-isothiocyanate (FITC)-conjugated rat anti-

mouse IgM (clone II/41); FITC-conjugated rat anti-mouse

CD21/CD35 (clone 7G6); R-phycoerythrin (R-PE)-conju-

gated rat anti-mouse CD23 (clone B3B4); R-PE-conjugated

rat anti-mouse CD43 (clone S7); FITC-conjugated rat anti-

mouse CD45RB (clone 16A); APC-C7-conjugated rat

anti-mouse CD11b (clone M1/70); R-PE-conjugated rat

anti-mouse CD44 (clone IM7); APC-C7-conjugated rat anti-

mouse CD4 (clone GK1.5); APC-conjugated rat anti-mouse

CD8a (clone 53-6.7); APC-conjugated rat anti-mouse

CD25(clone PC61); PE-conjugated rat anti-mouse CD5;

FITC-conjugated rat anti-mouse CD62L (clone MEL-14);

PerCP-CY5.5-conjugated mouse anti-mouse CD45.2 (clone

104); R-PE-conjugated mouse anti-mouse CD45.1 (clone

A20); Biotin-conjugated rat anti-mouse CD40 (clone

Chimeras

FA

CS

an

alys

is (

BM

/SP

L)

Nicotinetreatment

-

+

-

+

-

+

ra mice generation and analysis.

M. Skok et al. / Journal of Neuroimmunology 171 (2006) 86–9888

HM40-3) and Streptavidin conjugated to either R-PE or

CyChrome. For apoptosis studies we used Annexin V-

Biotin (Sigma) and Streptavidin-PE. Rabbit affinity puri-

fied polyclonal antibodies against a4 and a7 nAChR

subunits were obtained and biotinylated as previously

described (Skok et al., 1999).

2.4. Study of B lymphocyte populations in b2� /� mice

The bone marrow cells were separated by perfusion

from two femurs, while spleen cell suspension was

prepared by squeezing the spleen through a nylon mesh.

The number of viable cells was calculated with hemocy-

tometer using Trypan blue exclusion. The cells (0.5–

1�106 per sample) were resuspended in PBS containing

5% fetal bovine serum and 10 mM sodium azide and

stained with a cocktail of anti-B220, anti-IgM and anti-

CD43 antibodies (bone marrow) or anti-B220 and anti-

IgM antibodies (spleen) and analysed using FACS Calibur

flow cytometer and Cell Quest software (Beckton Dick-

inson). Before analysing, 1 Ag/ml of propidium iodide (PI)

was added. The results were expressed as absolute

numbers of cells within various lymphocyte populations

calculated as a percentage of total viable cells counted

with hemocytometer.

2.5. Study of lymphocyte populations in chimera mice

The bone marrow and spleen cells of chimera mice

were prepared as described above, the thymus cell

suspension was prepared by a similar method as the

splenocytes. The cells were stained with the following

antibody cocktails.

Bone marrow: anti-B220-Alexa fluor; anti-IgM-APC;

anti-CD43-PE; anti-CD11b-APC-C7; anti-CD45.2-

PerCP-CY5.5;

Thymus: anti-CD4-APC-C7; anti-CD8-APC; anti-CD44-

PE; anti-CD25-FITC; anti-CD45.2-PerCP-CY5.5;

Spleen I: anti-B220-TexasRed; anti-CD23-PE; anti-

CD21/35-FITC; anti-IgM-APC; anti-CD45.2-PerCP-

CY5.5;

Spleen II: anti-CD4-APC-C7; anti-CD25-APC; anti-

CD45Rb-FITC; anti-CD44-PE; anti-CD45.2-PerCP-

CY5.5;

Spleen III: anti-CD8-APC; anti-CD44-PE; anti-CD62L-

FITC; anti-CD4-APC-C7; anti-CD45.2-PerCP-CY5.5;

Spleen IV: anti-B220-Alexa fluor; anti-CD4-APC-C7;

anti-CD8-APC; anti-CD45.2-PerCP-CY5.5; anti-

CD45.1-PE.

The stained cells were analysed with BD LSR flow

cytometer and Cell Quest software. The results were

expressed as the ratio of CD45.2 (Ly 5.2)-positive to

CD45.2-negative (CD45.1 (Ly 5.1)-positive) cells within

separate cell populations.

2.6. Study of nAChR subunits expression in the course of B

lymphocyte development

The bone marrow and spleen cells of the wild-type mice

were stained with the following antibody cocktails.

Bone marrow: anti-B220-FITC; anti-IgM-PE; anti-a4/a7

nAChR-Biotin followed by Streptavidin-Cychrome.

Spleen: anti-IgM-PE; anti-CD23-FITC; anti-a4/a7

nAChR-Biotin followed by Streptavidin-Cychrome.

The peritoneal cells were obtained by perfusing the

mouse peritoneal cavity with PBS containing 5% fetal

bovine serum and 10 mM sodium azide. They were stained

with anti-IgM-FITC; anti-CD5-PE and anti-a4/a7 nAChR-

Biotin followed by Streptavidin-Cychrome.

The stained cells were analysed with FACScan flow cyto-

meter and Cell Quest software. The results were expressed as

a mean of fluorescence intensity (nAChR-specific antibody

binding) within separate PI-negative cell populations.

2.7. Study of apoptosis and IgM/CD40 expression

The bone marrow and spleen cells of the wild-type, h2� /�

and a7� /� mice were resuspended in Annexin V binding

buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES, pH

7.4) and treated with Annexin V-Biotin (1 :10) for 15 min at 4

-C. Then the cells were washed once with the binding buffer,briefly centrifuged and further stained with anti-B220-Alexa

fluor, anti-IgM-PE and Streptavidin-APC as described above.

Before analysing, PI was added to each sample. The stained

cells were analysed with BD LSR flow cytometer and Cell

Quest software. The results were expressed as the part (%) of

Annexin V+PI� cells within separate cell populations.

The bone marrow and spleen cells of the wild-type, h2� /�

and a7� /� mice were stained with anti-B220-Alexa fluor,

anti-IgM-PE and anti-CD40-Biotin followed by Streptavidin-

PE. The results were expressed as a mean of CD40/IgM-

specific antibody binding within separate cell populations of

PI-negative cells.

2.8. Statistical analysis

All experiments have been performed at least in

triplicates (unless indicated otherwise) using Student’s

criterion. The differences P <0.05 (*), P <0.005 (**) and

P <0.0005 (***) were considered statistically significant.

3. Results

3.1. Study of B cell populations in nAChR-deficient and

wild-type mice chronically treated with nicotine

The first set of experiments was performed in wild type

and h2-knockout mice as well as in chronically nicotine-


treated mice. Using fluorescent flow cytometry and cell

counting, we measured the absolute numbers of B lympho-

cytes in their bone marrow and spleen. As shown in Fig. 2A,

the number of B lymphocytes in the bone marrow, but not in

the spleen, of h2� /� mice was lower, while higher in

nicotine-treated mice when compared to control animals.

The effect of knockout was observed starting from the early

stage of B cell development, for the B220+CD43+IgM�

(pro/preB) cells, and was most obvious at the stage of

immature newly generated B220+IgM+B cells (Fig. 2B).

Nicotine favored generation of B220+IgM�CD43� (preB)

cells. These data indicate that h2-containing nAChRs, as

well as nicotine, can regulate B lymphocyte development

from the very early precursors.

3.2. Study of B and T cell populations in chimera mice

The h2-knockout mice lacked corresponding nAChRs

not only in lymphocytes, but in many other tissues,

including the brain. Previously it was found that aged

h2� /� mice had increased corticosteroid levels in the blood

compared to the wild-type (Zoli et al., 1999). Therefore, we

could not exclude the putative role of nAChR on B

lymphocyte development mediated through, for example,

NG+Rc

+Nic

Pre

+Nic -Nic

* * *

40

30

20

10

0

X 104

A

B

WT+Nic WT W0

10

20

30

40

50

60

β2KO

*

*Bone Marrow, x104

Ab

solu

te B

cel

l nu

mb

ers

Fig. 2. Absolute numbers of total B220+ cells in the bone marrow (104) and splee

C57BL/6J (WT) and h2KO mice, either treated (+Nic) or non-treated (�Nic) w

NG+Rc—immature newly generated and re-circulating B cells.

neuro-endocrine pathways. To address this question, chi-

mera mice were generated in which mixtures of B-

lymphocyte progenitor cells lacking h2- or a7-nAChRs

and the wild-type ones developed in irradiated wild-type

hosts. The cells could be distinguished by the genetic

variant (allotype) of the leukocyte-specific marker Ly 5

(CD45): Ly 5.1 or Ly 5.2.

The host mice were injected with the mixture of bone

marrow cells from Ly 5.1-positive wild-type (WT 5.1) mice

and Ly 5.2-positive control C57Bl/6J mice (WT 5.2), h2-knockout mice (h2� /� 5.2) or a7-knockout mice (a7� /�

5.2); half of each group was then chronically treated with

nicotine. The scope of the study was to determine whether

the nAChR-deficient Ly 5.2 lymphocytes have an advantage

or disadvantage when competing with the cells of Ly 5.1

wild-type genotype and whether nicotine affects this com-

petition. The results were evaluated as a ratio of Ly 5.2 to Ly

5.1 cells in different lymphocyte populations analysed using

marker-specific antibodies by FACS analysis.

We used the 20:80 ratio of Ly 5.2 to Ly 5.1 cells in the

injected mixture (20.4T0.67 to 79.6T0.67, as calculated by

flow cytometry). Thus, their expected ratio in WT 5.2/WT

5.1 chimeras was about 0.25. Instead, we found much

higher ratios varying considerably among different cell

Pro/pre

-Nic +Nic -Nic

*

*

T+Nic WT

WT

β2KO

β2KO

Spleen, x106

n (106) (A) and of B lymphocyte subpopulations in the bone marrow (B) of

ith nicotine. FACS analysis performed as shown in Fig. 3A–B and 4A.


populations. In fact, the expected values were found only for

thymic CD8+ T cells; in general, the ratios for T cells were

much lower than for B cells. These data indicated the

incomplete depletion of host Ly 5.2 lymphocytes upon

irradiation and therefore, the admixture of host cells in the

resultant Ly 5.2 to Ly 5.1 ratio. However, having a control

of WT 5.2/WT 5.1 chimera, we could compare it with the

h2� /� 5.2/WT 5.1 and a7� /� 5.2/WT 5.1 groups to

evaluate the advantage or disadvantage of nAChR-deficient

cells in the development.

Nicotine affected similarly the cells of Ly 5.1 and of Ly 5.2

genotypes, since the ratios found for each cell population in

B22

0

IgM

IgM

Ly 5.2

50.2%

Pre/pro NG+Rc

A

C

0

1

2

3

4

5

6PreBPro/PreB

**

*

Ly

5.2

/ Ly

5.1

WT

β2KOα7K

O WT

β2KOα7K

O

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

100 101 102 103 104

Fig. 3. FACS analysis of bone marrow cells (A–D) and the ratios of Ly 5.2 to Ly 5.

(WT), h2� /� 5.2/WT 5.1 (h2KO) and a7� /� 5.2/ WT 5.1 (a7KO) chimera nicot

cells; Mac—macrophages. (C) An example of Ly 5.2+ and Ly 5.2� (Ly 5.1+) cell se

(E) represents a meanTSE for five mice studied in three individual experiments.

nicotine-treated and non-treated WT 5.2/WT 5.1 chimeras

were almost the same (data not shown). In contrast,

significant difference in Ly 5.2 to Ly 5.1 ratios for WT 5.2/

WT 5.1 and KO 5.2/WT 5.1 chimeras was found (Figs. 3–6).

On the other hand, for some cells, the results were

different in nicotine-treated compared to non-treated mice.

In particular, in the spleen the difference in Ly 5.2 to Ly 5.1

ratios of both B lymphocytes and CD4+ T lymphocytes was

found in nicotine-treated animals only (Fig. 4). The same

was true for preB cells in the bone marrow (that corresponds

to the data presented in Fig. 2B) and late double-negative

precursors in the thymus. These data indicate that nicotine

B22

0B

220

CD11b

E

CD43

PreII Pro/preI

Mac

B

D

MacNG/Rc B

*

*

WT

β2KOα7K

O WT

β2KOα7K

O

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

100 101 102 103 104

1 cells in various cell populations in the bone marrow (E) of WT 5.2/WT 5.1

ine-treated mice. NG+Rc—immature newly generated and re-circulating B

paration within NG+Rc B cells (chimera a7� /� 5.2/WT 5.1). Each column

B22

0

IgM

CD

21

CD23

C

Total B

Trans B

MZ B

Fol B

0

1

2

3

4

5

MZ BFol BTrans BTotal B

**

**

***

+Nic-Nic

Ly

5.2

/ Ly

5.1

A B

WT

α7KO

β2KO W

Tα7K

Oβ2

KO WT

α7KO

β2KO W

Tα7K

Oβ2

KO WT

α7KO

β2KO

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

100

101

102

103

104

Fig. 4. FACS analysis of spleen B lymphocytes (A–B) and the ratios of Ly 5.2 to Ly 5.1 cells in various B cell subpopulations in the spleen (C) of WT 5.2/WT

5.1 (WT), h2� /� 5.2/WT 5.1 (h2KO) and a7� /� 5.2/WT 5.1 (a7KO) chimera mice. All data, except especially indicated (Total B cells in the spleen), are from

nicotine-treated mice. Trans B—transitional B cells; Fol B—follicular B cells; MZ B—marginal zone B cells. Each column (C) represents a meanTSE for five

mice studied in three individual experiments.


indeed affected lymphocyte expansion/survival through h2-and a7-containing nAChRs. They demonstrate that signal-

ing through nAChR (and not only its expression) is

necessary to regulate lymphocyte expansion.

The results presented in Fig. 3 demonstrate that in the

bone marrow the expansion of B lymphocytes was affected

with both h2- and a7-containing nAChRs. The disadvantageof KO cells appeared starting from B220+IgM�CD43+ pro/

preB cells, became significant in B220+CD43�IgM� preB

cells and was also evident in immature newly generated and

re-circulating B220+CD43�IgM+ B cells. In contrast, the

development of bone-marrow macrophages (CD11b+) was

not affected by nAChR deficiency.

In the spleen, only a7� /� cells were under-represented,

mainly within the CD21+CD23+ follicular B cell and the

CD21+CD23� marginal zone B cell compartments (Fig. 4).

In contrast to the bone marrow, the effect of a7KO was

observed solely in the spleens of nicotine-treated mice. This

means that nicotine stimulated the expansion of B lympho-

cytes through a7-containing nAChRs, while the endogenous

nAChR agonist was absent in the spleen. The close effect of

nAChR-deficiency on follicular and marginal zone B cells

indicates that nAChR signaling is more important for early B

lymphocyte development than for their further functional

differentiation.

A very similar picture was found for T lymphocytes. As

shown in Fig. 5, both h2- and a7-containing nAChRs were

important for T cell development in the thymus. Their effect

was seen starting from the very early double-negative

precursors (DN1) and was mostly pronounced in late

double-negative precursors (DN4). In single-positive CD4+

and CD8+ thymic T cells the effect of h2 KO was still

observed, but only that of a7KO was statistically significant.

In the spleen (Fig. 6), only the effect of a7KO was found.

For both CD4+ and CD8+ T lymphocytes, a7 nAChR played

a role in the expansion of total and naı̈ve cells and less of

regulatory/effector ones. This means that, as with B

lymphocytes, a7 nAChRs were more important for develop-

ment of basic T lymphocyte subpopulations (CD4+ and

CD8+) than for their further differentiation.

According to the data described above, the common rule

for B and T lymphocytes is that both a4h2 and a7 nAChRs

CD

4

CD8

CD

25

CD44

CD

4

CD

8

Ly 5.2 Ly 5.2

E

73% 27% 88% 12%

A

C

B

D

DN1

DN2-3

DN4DN CD8

CD4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

CD8+CD4+DN4DN2-3DN1

**

**

***

*

*

Ly

5.2

/ Ly

5.1

WT

α7KO

β2KO W

Tα7K

Oβ2

KO WT

α7KO

β2KO W

Tα7K

Oβ2

KO WT

α7KO

β2KO

100 101 102 103 104100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

100 101 102 103 104100 101 102 103 104

Fig. 5. FACS analysis of thymocytes (A–D) and the ratios of Ly 5.2 to Ly 5.1 cells in various cell populations in the thymus (E) of WT 5.2/WT 5.1 (WT), h2� /�

5.2/WT 5.1 (h2KO) and a7� /� 5.2/WT 5.1 (a7KO) chimera nicotine-treated mice. DN—double-negative T lymphocyte precursors. (C)– (D) Examples of Ly

5.2+ and Ly 5.2� (Ly 5.1+) cell separation within CD4+ and CD8+ T cells in a7� /� 5.2/ WT 5.1 chimera mice. Each column (E) represents a meanTSE for five

mice studied in three individual experiments.


regulate their development in the primary lymphoid organs

(bone marrow and thymus), while in the periphery (spleen)

only a7 nAChRs continue to exert an effect.

3.3. Study of nAChR subunits expression in the course of B

lymphocyte development

We studied the expression of either a4 or a7 nAChR

subunits in different stages of B lymphocyte development

using subunit-specific antibodies and FACS analysis. As

shown in Fig. 7, a4-specific antibody bound starting from

B220+IgM� pro/pre B cells in the bone marrow, the highest

binding being found in the newly generated B220+IgM+

cells. It decreased upon B cell maturation in the spleen, being

the highest in IgM+CD23� (transitional type 1+ marginal

zone) B cells (Su et al., 2004). The level of a4-specific

antibody binding in re-circulating B cells in the bone marrow

was comparable to that of mature B cells in the spleen. In

contrast, the binding of a7-specific antibody increased upon

B lymphocyte maturation, the highest value being observed

C(CD4)

D(CD8)

CD

4

CD25

CD

8

CD44

A BNaïve Regulatory Naïve Effector

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 RegulatoryNaiveTotal

*

*

Ly

5.2

/ Ly

5.1

0.0

0.5

1.0

1.5

2.0

2.5EffectorNaiveTotal

*

Ly

5.2

/ Ly

5.1

WT

α7KO

β2KO W

Tα7K

Oβ2

KO WT

α7KO

β2KO

WT

α7KO

β2KOW

Tα7K

Oβ2

KOWT

α7KO

β2KO

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

100

101

102

103

104

Fig. 6. FACS analysis of T lymphocytes in the spleen (A–B) and the ratios of Ly 5.2 to Ly 5.1 cells in various cell populations (E) of WT 5.2/WT 5.1 (WT),

h2� /� 5.2/WT 5.1 (h2KO) and a7� /� 5.2/ WT 5.1 (a7KO) chimera nicotine-treated mice.


in the most mature CD23+IgMlow (follicular) B cells in the

spleen. These data correlated well with the role of h2- anda7-containing nAChRs in B lymphocyte development found

with the chimera mice (Figs. 3 and 4). They indicate that the

expression of two nAChR subtypes exhibits variations

corresponding with the B lymphocyte maturation: a4h2subtype has maximal expression (and significance for

development) in the bone marrow and early in the spleen,

while the expression of a7 subtype rises upon further

maturation. Consequently, this subtype determines B cell

expansion in the spleen. These data correlate with the

differential expression of nAChR subunits along with the T

lymphocyte maturation (Kuo et al., 2002). Taking into

account the heterogeneity of B lymphocyte lineage (Stall et

al., 1992), we also compared the nAChR expression in B1

and B2 cells of peritoneal cavity (where most of B1 cells are

found). Both B1a (IgM+CD5+) and B1b (IgMhighCD5�)

cells demonstrated much higher binding of a4-specific and

a7-specific Abs than B2 cells (IgMlowCD5�): 116.0 and

107.2 vs. 72.4 (for a4) and 304.9 and 160.3 vs. 64.2 (for a7)

Bone marrow Spleen

B22

0

IgM

IgM CD23

A

B

Pro/pre Rc

NG

MZ+T1

Fol

T2

Pro/p

re Pre NG

T1+M

Z T2 Fol

Rc

10

20

30

40

50

60

70

80

90Bone marrowSpleenBone marrow

Ab

bin

din

g, m

ean

Ab4 Ab7

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

100

101

102

103

104

Fig. 7. Expression of a4 and a7 nAChR subunits in the course of B lymphocyte development in the wild-type mouse. (A) FACS analysis of the bone marrow

and spleen B lymphocytes; (B) fluorescence intensity means of a4-specific (Ab4) and a7-specific (Ab7) antibody binding within identified cell subpopulations

(shown are the data of one representative experiment out of three). NG—immature newly generated; T1 and T2—transitional type 1 and 2; MZ—marginal

zone; Fol—follicular; Rc—re-circulating B cells.


units of mean fluorescence intensity, respectively. Possibly,

the high level of nAChR expression is connected with the

self-renewing capacity of B1 cells.

3.4. Study of lymphocyte survival

The fact that nicotine favored, while nAChR deficiency

disfavored the lymphocyte expansion in the course of

development could mean that signaling through nAChR

affects either their proliferation or survival. To check whether

nAChRs are involved in regulating the lymphocyte survival,

we compared the number of apoptotic B cells in the bone

marrow and spleen of the wild-type and nAChR-knockout

mice. As shown in Fig. 8, the part of annexin V+PI� cells

within the preB and immature B lymphocytes was signifi-

cantly higher in both h2KO and a7KO mice compared to the

WT. In contrast, splenic B lymphocytes of a7KO mice did

not contain more annexin V+PI� cells than those ofWTmice.

Therefore, the decrease in B lymphocyte numbers in the

immature B cells of knockout mice was due to their worse

survival in the course of development. In the spleen, the

decrease in B cell expansion did not depend on their survival

any more, but was due to either decreased migration from the

bone marrow or weaker proliferation within the spleen.

4. Discussion

The pro-proliferative role of nicotine was established in

several types of cells (Quik et al., 1994, Heeschen et al.,

2001). In particular, nAChRs expressed in the lung

epithelium were found connected with the development of

small cell lung carcinoma in smokers (Codignola et al.,

1994; Sciamanna et al., 1997). We reported that nicotine

AnnexinV

B22

0

A

B

WT

6.75% 17.3%

0

2

4

6

8

10

12

14

16

18

Total B, SplNG+Rc B, BMPro/Pre B, BM

***

***

**

An

nex

in V

+ P

I- cel

ls, %

WT

WTα7K

O WTα7K

O WTα7K

Oβ2

KO WTβ2

KO

β2KO

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

100

101

102

103

104

Fig. 8. FACS analysis of the newly generated and re-circulating (NG+Rc) bone marrow B lymphocytes of the wild-type (WT) and h2KO mice (A) and the part

of early apoptotic cells within various B lymphocyte subpopulations in the bone marrow (BM) and spleen (Spl) of the WT, h2KO and a7KO mice. PI—

propidium iodide. Each column represents a meanTSE for 3 to 8 mice.


favored proliferation of B-lymphocyte-derived hybridoma

(Skok et al., 2003). In the bone marrow, B lymphocyte

B220+IgM� precursor cells proliferate, while the immature

newly generated cells do not divide but are subjected to

selection process (Rolink et al., 2001). Positive selection

preserves cells expressing correctly assembled B cell

receptor (BCR), while negative selection eliminates poten-

tially autoreactive B cells (Melchers et al., 2000). Our data

demonstrate that nAChR deficiency affected B lymphocyte

survival in the bone marrow. Moreover, the effect of nAChR

deficiency on both T and B lymphocyte development was

most pronounced at the stages when the premature antigen-

specific receptors (preTCR and preBCR) start to be

expressed (Germain, 2002; Melchers et al., 2000.). The

connection between TCR and nAChR signaling has been

clearly demonstrated by others (Geng et al., 1995; Kalra et

al., 2002), while we found some connection of a4h2

nAChR with CD40 in mature B cells (Skok et al., 2005).

Both BCR and CD40 signaling are critical for the survival

of developing B lymphocytes. We observed the small but

obvious increase (10–12%) in the IgM- and CD40-specific

antibody binding in the newly generated B lymphocytes of

nAChR-deficient compared to the wild-type mice. The

small difference (5–8%) was still observed in spleen B cells

of both h2KO and a7KO (data not shown). These results

are in accord with the more intensive proliferation of B cells

from h2-deficient mice in response to anti-CD40 antibody,

compared to the wild-type mice (Skok et al., 2005). They

indicate that nAChR signaling may affect the expression

and/or signaling through BCR/CD40 in both premature and

mature B cells. The stronger signaling, as well as the

increased avidity of binding to self antigen-expressing cells

due to higher BCR numbers, could account for increased

negative selection of nAChR-deficient compared to the


wild-type cells. This is in agreement with the increased

numbers of apoptotic cells found in the bone marrow of

nAChR-deficient mice (Fig. 8). Previously we found the

smaller serum IgG levels and IgG-secreting cell numbers in

the spleens of h2-knockout mice and suggested that these

mice had smaller pre-immune B cell repertoires than the

wild-type mice (Skok et al., 2005). This suggestion is also in

accord with the idea of stronger negative selection of

nAChR-deficient B cells in the course of their development.

The presence of nAChRs in both thymus and mature

peripheral T lymphocytes was clearly demonstrated (Wak-

kach et al., 1996;Mihovilovic et al., 1997). It was also shown

that cholinergic signals regulate thymic differentiation and

selection (Rinner et al., 1999). At first, this effect was

considered due to nAChRs present in thymic epithelial cells,

but then nAChRs expressed in developing thymocytes were

shown to be involved (Middlebrook et al., 2002). These data

are consistent with our results (Fig. 5). Our data demonstrate

for the first time that both a4h2 and a7 nAChRs are

involved in B lymphocyte development in the bone marrow.

They affect the survival/expansion of developing B cells. As

in the thymus, the effect is observed when premature forms

of antigen-specific receptors begin to be expressed. In the

spleen, the signal through a7 nAChR favors both B and T

cell expansion. In this case, the processes of cell division

and not survival seem to be involved. Cell proliferation, as

well as T cell maturation in the thymus are mediated, in

particular, through MAPK pathway (Germain, 2002), which

was also shown critical for nAChR signaling in PC-12 cells

(Tang et al., 1998). These data suggest that nAChR signa-

ling intersects the antigen-specific receptor signaling path-

way at the level of MAP kinase. This suggestion is

supported by the fact that nAChR deficiency resulted in

significantly more CD4+ than CD8+ thymic T cell depletion

(Fig. 5), as observed when MAPK1 is blocked (Germain,

2002). Our results also indicate that, in general, a4h2 and

a7 nAChRs function in a similar way. However, the absence

of a7 nAChR subtype affected T and B lymphocyte

development more than h2-deficiency (Figs. 3–6); in fact,

h2-containing nAChRs played a role mainly in the very

early stages of maturation. This may be due to different ion

selectivity of the two nAChR subtypes, in particular, the

higher permeability of a7 nAChRs for Ca ions (Lindstrom

et al., 1998), which are critical for many intracellular signa-

ling events.

The data on the role of nAChRs in the course of

lymphocyte development and expansion raises a question on

the source of acetylcholine in the lymphoid organs.

Cholinergic innervation of the bone marrow and thymus,

but not the spleen, has been demonstrated (Bellinger et al.,

1993; Singh and Fatani, 1988; Artico et al., 2002). More-

over, it was shown that both cutting and stimulating the

cholinergic nerves affected the hemopoiesis in the bone

marrow and thymus (Chernigovskiy et al., 1967; Singh and

Fatani, 1988). Therefore, nAChRs expressed in the lym-

phocytes and their precursors may mediate the neural

regulation of lymphopoietic processes. In neurons, the level

of nAChR expression strongly depends on synaptic con-

tacts: for example, axotomy of superior cervical ganglion

neurons resulted in significant decrease in both a7- and h4-containing nAChR numbers, the number of a7 nAChRs

being restored quickly (Zhou et al., 2001). It seems likely

that the decrease in a4h2 nAChR expression found in the

lymphocytes that leave the bone marrow and thymus is due

to cessation of nerve signals. In this case, it is reasonable to

suggest that these are a4h2nAChRs, which mediate nerve

regulation of lymphocyte development.

In addition, endogenous acetylcholine production was

found both in the thymus (Rinner et al., 1999) and in the

blood, creating a constant concentration in the blood plasma

(about 140 pg/ml; Kawashima et al., 1982). Both T and B

lymphocytes were shown to produce acetylcholine when

activated (Rinner et al., 1998). At present, the non-neuronal

cholinergic system in lymphocytes is considered important in

regulating many immune functions (Fujii, 2004). Therefore,

acetylcholine produced by either lymphoid cells themselves

or by surrounding stromal elements might be also regarded as

an autocrine/paracrine regulator of lymphocyte development.

Our finding that, in contrast to the thymus and bone marrow,

only a7-containing nAChRs were involved in regulating

lymphocyte propagation in the spleen makes it possible to

suggest that this nAChR subtype mediates the effect of

endogenous acetylcholine. In neurons, a7-containing

nAChRs are often found extrasynaptically and are thought

to respond to acetylcholine leaking from the synapse

(Lindstrom, 1996). In addition, we can not exclude the effect

of dietary choline, since a7-containing nAChRs were shown

to be activated by this agonist (Alkondon et al., 1997).

Altogether, the data presented here demonstrate the

importance of cholinergic signaling for lymphocyte devel-

opment and suggest the different roles of h2- and a7-

containing nAChR subtypes. In addition, they suggest that

nicotine inhaled upon smoking can affect not only

lymphocyte activation (Geng et al., 1995; Kalra et al.,

2000), but also lymphopoiesis.

Acknowledgements

We are grateful to Prof. A. Tarakhovsky and to Dr. A.

Cumano for the help in performing FACS analysis and for

fruitful discussions and to Dr. B. Molles for editing the

manuscript. The work was supported with Alexander von

Humboldt fellowship, two EMBO short-term fellowships

and French–Ukrainian program of collaboration ‘‘Dnipro’’.

References

Alkondon, M., Pereira, E.F.R., Cortes, W.S., Maelicke, A., Albuquerque,

E.X., 1997. Choline is a selective agonist of a7 nicotinic acetylcholine

receptors in the rat brain neurons. Eur. J. Neurosci. 9, 2734–2742.


Artico, M., Bosco, S., Cavallotti, C., Agostinelli, E., Giuliani-Piccari, G.,

Sciorio, S., Cocco, L., Vitale, M., 2002. Noradrenergic and cholinergic

innervation of the bone marrow. Int. J. Mol. Med. 10, 77–80.

Bellinger, D.L., Lorton, D., Hamill, R.W., Felten, S.Y., Felten, D.L., 1993.

Acetylcholinesterase staining and choline acetyltransferase activity in

the young adult rat spleen: lack of evidence for cholinergic innervation.

Brain Behav. Immun. 7, 191–204.

Changeux, J.-P., Bertrand, D., Corringer, P.-J., Dehaene, S., Edelstein, S.,

Lena, C., Le Novere, N., Marubio, L., Picciotto, M., Zoli, M., 1998.

Brain nicotinic receptors: structure and regulation, role in learning and

reinforcement. Brain Res. Rev. 26, 198–216.

Chernigovskiy, V.N., Shehter, S.Y., Yaroshevskiy, A.Y., 1967. Regulation

of Erythropoiesis. Nauka Publishing Corporation, Leningrad. (in

Russian).

Codignola, A., Tarroni, P., Cattaneo, M.G., Vicentini, L.M., Clementi, F.,

Sher, E., 1994. Serotonin release and cell proliferation are under the

control of a-bungarotoxin-sensitive nicotinic receptors in small-cell

lung carcinoma cell lines. FEBS Lett. 342, 286–290.

Conti-Fine, B.M., Navaneetham, D., Lei, S., Maus, A.D.J., 2000. Neuronal

nicotinic receptors in non-neuronal cells: new mediators of tobacco

toxicity? Eur. J. Pharmacol. 393, 279–294.

Fujii, T., 2004. An independent, non-neuronal cholinergic system in

lymphocytes and its roles in regulation of immune function. Nippon

Yakurrigaku Zasshi 123, 179–188.

Geng, Y., Savage, S.M., Johnson, L.J., Seagrave, J.-C., Sopori, M.L., 1995.

Effects of nicotine on the immune response: I. Chronic exposure to

nicotine impairs antigen receptor-mediated signal transduction in

lymphocytes. Toxicol. Appl. Pharmacol. 135, 268–278.

Germain, R., 2002. T cell development and the CD4–CD8 lineage

decision. Nat. Rev. Immunol. 2, 309–322.

Grando, S.A., 1997. Biological functions of keratinocyte cholinergic

receptors. J. Inv. Dermatol. 2, 41–48.

Heeschen, C., Jang, J.J., Weis, M., Pathak, A., Kaji, S., Hu, R.S., Tsao,

P.S., Johnson, F.L., Cooke, J.P., 2001. Nicotine stimulates angio-

genesis and promotes tumor growth and atherosclerosis. Nat. Med. 7,

775–777.

Kalra, R., Singh, S.P., Savage, S.M., Finch, G.L., Sopori, M.L., 2000.

Effects of cigarette smoke on immune response: chronic exposure

to cigarette smoke impairs antigen-mediated signaling in T cells

and depletes IP3-sensitive Ca2+ stores. J. Pharmacol. Exp. Ther. 293,

166–171.

Kalra, R., Singh, S.P., Kracko, D., Matta, S.G., Sharp, B.M., Sopori, M.L.,

2002. Chronic self-administration of nicotine in rats impairs T cell

responsiveness. J. Pharmacol. Exp. Ther. 302 (3), 935–939.

Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its

biological function. Life Sci. 72, 2101–2109.

Kawashima, K., Oohata, H., Fujimoto, K., Suzuki, T., 1982. Extraneuronal

localization of acetylcholine and its release upon nicotinic stimulation in

rabbits. Neurosci. Lett. 104, 336–339.

Kuo, Y., Lucero, L., Michaels, J., DeLuca, D., Lukas, R.J., 2002.

Differential expression of nicotinic acetylcholine receptor subunits in

fetal and neonatal mouse thymus. J. Neuroimmunol. 130, 140–154.

Lindstrom, J., 1996. Neuronal nicotinic acetylcholine receptors. In:

Narahashi, T.Ion Channels vol. 4. Plenum, New York, pp. 377–450.

Lindstrom, J., Peng, X., Kuryatov, A., Lee, E., Anand, R., Gerzanich, V.,

Wang, F., Wells, G., Nelson, M., 1998. Molecular and antigenic

structure of nicotinic acetylcholine receptors. Ann. N.Y. Acad. Sci.

841, 71–86.

Macklin, K.D., Maus, A.D.J., Pereira, E.F.R., Albuquerque, E.X., Conti-

Fine, B.M., 1998. Human vascular endothelial cells express func-

tional nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 287,

435–439.

Maus, A.D.J., Pereira, E.F.R., Karachunski, P.I., Horton, R.M., Navanee-

tham, D., Macklin, K., Cortes, W.S., Albuquerque, E.X., Conti-Fine,

B.M., 1998. Human and rodent bronchial epithelial cells express

functional nicotinic acetylcholine receptors. Mol. Pharmacol. 54,

779–788.

Melchers, F., ten Boekel, E., Seidl, T., Kong, X.C., Yamagami, T., Onishi,

K., Shimizu, T., Rolink, A.G., Andersson, J., 2000. Repertoire selection

by pre-B-cell receptors and B-cell receptors, and genetic control of B-

cell development from immature to mature B cells. Immunol. Rev. 175,

33–46.

Middlebrook, A.J., Martina, C., Chang, Y., Lukas, R.J., DeLuca, D.,

2002. Effects of nicotine exposure on T cell development in fetal

thymus organ culture: arrest of T cell maturation. J. Immunol. 169,

2915–2924.

Mihovilovic, M., Denning, S., Mai, Y., Whichard, L.P., Patel, D.D., Roses,

A.D., 1997. Thymocytes and cultured thymic epithelial cells express

transcripts encoding alpha-3, alpha-5 and beta-4 subunits of neuronal

nicotinic acetylcholine receptors:preferential transcription of the alpha-3

and beta-4 genes by immature CD4+ 8+ thymocytes. J. Neuroimmunol.

79, 176–184.

Orr-Urtreger, A., Goldner, F.M., Saeki, M., Lorenzo, I., Goldberg, L., de

Biasi, M., Dani, J.A., Patrick, J.W., Beaudet, A.L., 1997. Mice deficient

in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-

bungarotoxin binding sites and hippocampal fast nicotinic currents. J.

Neurosci. 17, 9165–9171.

Paterson, D., Nordberg, A., 2000. Neuronal nicotinic receptors in the

human brain. Progr. Neurobiol. 61, 75–111.

Piccioto, M.R., Zoli, M., Lena, C., Bessis, A., Lallemand, Y., LeNovere, N.,

Vincent, P., Pich, E.M., Brulet, P., Changeux, J.P., 1995. Abnormal

avoidance learning in mice lacking functional high-affinity nicotine

receptor in the brain. Nature 374, 65–67.

Quik, M., Chan, J., Patrick, J., 1994. Alpha-bungarotoxin blocks the

nicotinic receptor mediated increase in cell number in a neuroendocrine

cell line. Brain Res. 655, 161–167.

Rinner, I., Kawashima, K., Schauenstein, K., 1998. Rat lymphocytes

produce and secrete acetylcholine in dependence of differentiation and

activation. J. Neuroimmunol. 81, 31–37.

Rinner, I., Globerson, A., Kawashima, K., Korsatko, W., Schauenstein,

K., 1999. A possible role for acetylcholine in the dialogue

between thymocytes and thymic stroma. Neuroimmunomodulation

6, 51–55.

Rolink, A.G., Schaniel, C., Andersson, J., Melchers, F., 2001. Selection

events operating at various stages in B cell development. Curr. Opin.

Immunol. 13, 202–207.

Sciamanna, M.A., Griesmann, G.E., Williams, C.L., Lennon, V., 1997.

Nicotinic acetylcholine receptors of muscle and neuronal (a7) types

coexpressed in a small cell lung carcinoma. J. Neurochem. 69,

2302–2311.

Singh, U., Fatani, J., 1988. Thymic lymphopoiesis and cholinergic

innervation. Thymus 11, 3–13.

Skok, V.I., 2002. Nicotinic acetylcholine receptors in autonomic ganglia.

Auton Neurosci. Basic Clin. 97, 1–11.

Skok, M.V., Voitenko, L.P., Voitenko, S.V., Lykhmus, E.Y., Kalashnik,

E.N., Litvin, T.I., Tzartos, S.J., Skok, V.I., 1999. Alpha subunit

composition of nicotinic acetylcholine receptors in the rat autonomic

ganglia neurons as determined with subunit-specific anti-a(181–192)

peptide antibodies. Neuroscience 93, 1427–1436.

Skok, M.V., Kalashnik, E.N., Koval, L.N., Tsetlin, V.I., Utkin, Y.N.,

Changeux, J.-P., Grailhe, R., 2003. Functional nicotinic acetylcholine

receptors are expressed in B lymphocyte-derived cell lines. Mol.

Pharmacol. 64, 885–889.

Skok, M.V., Grailhe, R., Changeux, J.-P., 2005. Nicotinic receptors regulate

B lymphocyte activation and immune response. Eur. J. Pharmacol. 517,

246–251.

Stall, A.M., Adams, S., Herzenberg, L.A., Kantor, A.B., 1992. Character-

istics and development of the murine B1b (Ly-1 B sister) cell

population. In: Herzenberg, L.A., Haughton, G., Rajewsky, K., editors.

CD5 B Cells in Development and Disease, Ann. New York Acad. Sci.

vol. 651. The New York Academy of Sciences, pp. 33–43.

Su, T.T., Guo, B., Wei, B., Braun, J., Rawlings, D.J., 2004. Signaling in

transitional type 2 B cells is critical for peripheral B cell development.

Immunol. Rev. 197, 161–178.


Tang, K., Wu, H., Mahata, S.K., O’Connor, D.T., 1998. A crucial role for

the mitogen-activated protein kinase pathway in nicotinic cholinergic

signaling to secretory protein transcription in pheochromocytoma cells.

Mol. Pharmacol. 54, 59–69.

Toyabe, S., Iiai, T., Fukuda, M., Kawamura, T., Suzuki, S., Uchiyama, M.,

Abo, T., 1997. Identification of nicotinic acetylcholine receptors on

lymphocytes in the periphery as well as thymus in mice. Immunology

92, 201–205.

Wakkach, A., Guyon, T., Bruand, C., Tzartos, S., Cohen-Kaminsky, S.,

Berrih-Aknin, S., 1996. Expression of acetylcholine receptor genes in

human thymic epithelial cells—implications for myasthenia gravis. J.

Immunol. 157, 3752–3760.

Zhou, Y., Deneris, E., Zigmond, R.E., 2001. Nicotinic acetylcholine

receptor subunit proteins alpha7 and beta4 decrease in the superior

cervical ganglion after axotomy. J. Neurobiol. 46 (3), 178–192.

Zoli, M., Picciotto, M.R., Ferrari, R., Cocchi, D., Changeux, J.-P., 1999.

Increased neurodegeneration during ageing in mice lacking high-

affinity nicotine receptors. EMBO J. 18, 1235–1244.

The role of nicotinic acetylcholine receptors in lymphocyte development

Documents