Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

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The International Journal of Biochemistry & Cell Biology 40 (2008) 980–990

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow Lyudmyla M. Koval a , Alla S. Zverkova b , Regis Grailhe c,1 , Yuriy N. Utkin d , Victor I. Tsetlin d , Sergiy V. Komisarenko a , Maryna V. Skok a,∗ a

Palladin Institute of Biochemistry, 9 Leontovicha Str., 10601 Kyiv, Ukraine Institute of Hematology and Transfusiology, 12 Maxima Berlinskogo Str., 04060 Kyiv, Ukraine c Pasteur Institute, 25-28 Rue Docteur Roux, 75024 Paris, France Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 Miklukho-Maklaya Str., 117997 Moscow, Russia b

d

Received 10 September 2007; received in revised form 31 October 2007; accepted 2 November 2007 Available online 17 November 2007

Abstract Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing ␣4(␤2) and ␣7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125 I]-␣-bungarotoxin or [3 H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluoresceinlabeled ␣-cobratoxin. Both TER119+ (erythroid) and CD16+ CD43med (myeloid) progenitor cells bound more ␣4-specific antibodies than their mature forms, while the binding of ␣-cobratoxin and ␣7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of ␣7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of ␤2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through ␣7- and ␣4␤2-containing nicotinic receptors, which play different roles in the course of the cell maturation. © 2007 Elsevier Ltd. All rights reserved. Keywords: Nicotinic acetylcholine receptor; Hematopoiesis; Bone marrow; Knockout mice

1. Introduction ∗

Corresponding author at: Palladin Institute of Biochemistry, 9 Leontovicha Str., 01601 Kyiv, Ukraine. Tel.: +380 44 234 33 54; fax: +380 44 279 63 65. E-mail addresses: [email protected] (L.M. Koval), [email protected] (R. Grailhe), [email protected] (V.I. Tsetlin), [email protected] (S.V. Komisarenko), [email protected] (M.V. Skok). 1 Present address: Institut Pasteur Korea, 39-1 Hawolgok-dong, Sunguk-gu, 136-79 Seoul, Republic of Korea. 1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.11.006

The processes of hematopoiesis in the bone marrow are regulated by a variety of factors including those produced by the stromal elements, the developing hematopoietic cells themselves, and by exogenous substances, such as hormones and neurotransmitters, like acetylcholine. In particular, cutting nerves which enter the hip bones influenced erythropoiesis, while injecting nicotine inside the bone induced changes in

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the processes controlled by the autonomic nervous system (Chernigovskiy, Shehter, & Yaroshevskiy, 1967). These data demonstrated the role of the bone marrow innervation in hematopoiesis and the presence of nicotinic acetylcholine receptors within the bone marrow. This view was further supported by the recent studies of nicotine-stimulated changes in hematopoiesis (Khaldoyanidi et al., 2001; Serobyan, Orlovskaya, Kozlov, & Khaldoyanidi, 2005). Nicotinic acetylcholine receptors (nicotinic receptors) are ligand-gated ion channels mediating synaptic transmission in nerve and muscle cells. They are composed of several types of alpha and beta subunits forming either homomeric or heteromeric functionally distinct receptor subtypes (Paterson & Nordberg, 2000). Nicotinic receptors are also present in many non-excitable cells, such as skin keratinocytes (Arrendolo et al., 2003), respiratory tract epithelial cells, vascular endothelium (Conti-Fine, Navaneetham, Lei, & Maus, 2000) and most of the blood cells: leukocytes (Cormier et al., 2004), lymphocytes (Kawashima & Fujii, 2003), macrophages (Wang et al., 2003) and erythrocytes (Bennekou, 1993), where their functions are quite different from those in muscles or neurons. Previously we found that mouse lymphocytes express two nicotinic receptor subtypes containing ␣4␤2 and ␣7 subunits, respectively (Skok et al., 2003). These receptors regulate both mature B lymphocyte activation and their development in the bone marrow (Skok, Grailhe, & Changeux, 2005; Skok, Grailhe, Agenes, & Changeux, 2006; Skok, Grailhe, Agenes, & Changeux, 2007). The aim of the present study was to determine whether and how these two nicotinic receptor subtypes affect generation and maturation of myeloid and erythroid cells. 2. Materials and methods

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Palladin Institute Animal Care and Treatment Committee. 2.2. Antibodies Rabbit affinity purified antibodies against nicotinic receptor subunits were obtained, biotinylated and characterized by us previously (Skok et al., 1999). The following antibodies against mouse cell-specific markers were used in flow cytometry: Fluorescein-labeled antiCD19, anti-CD11b, and anti-Ly 6C-6G, phycoerythrinelabeled anti-CD43 (all from PharMingen, BD) and biotinylated anti-TER 119 (a kind gift of Dr. A. Cumano, Pasteur Institute, Paris). Biotinylated antibodies were revealed with phycoerythrine-conjugated Streptavidin (PharMingen, BD). 2.3. Fluorescein-labeled α-cobratoxin ␣-Cobratoxin was purified from Naja siamensis venom as described previously (Kukhtina et al., 2000). To obtain a fluorescent derivative, a solution of fluorescein isothiocyanate (0.4 mg in 1 ml of dimethyl sulfoxide) was added slowly to 1 ml of ␣-cobratoxin solution (7 mg/ml) in 0.2 M sodium phosphate buffer (pH 10.5, 0.175 mM EDTA) with continuous stirring. The reaction mixture was incubated for 105 min at room temperature and then desalted by gel filtration on a Sepadex G-15 column (2.5 cm × 40 cm) equilibrated with 0.1 M acetic acid. The protein fraction was freeze–dried and separated by reverse-phase HPLC on a Vydac C18 column (10 mm × 250 mm) with a gradient of aqueous acetonitrile from 20 to 30% (v/v) in 0.1% (v/v) trifluoroacetic acid over 20 min at a flow rate of 2 ml/min. The fraction containing singly labeled ␣-cobratoxin (as determined by matrix-assisted laser-desorption ionization massspectrometry) was freeze–dried and used for further studies.

2.1. Animals 2.4. Radioligand binding Experiments were performed in age-matched male wild-type and mutant mice with common C57BL/6J background. Two different knock-out mice were used, lacking either ␤2 (Piccioto et al., 1995) or ␣7 (OrrUrtreger et al., 1997) nicotinic receptor subunits. The mice were kept in the animal facility of Pasteur Institute, Paris. Two groups of wild-type mice: control ones and those receiving nicotine with the drinking water (200 ␮g/ml during 10 months) were kept and analyzed in Palladin Institute of Biochemistry, Kyiv. All procedures conformed to the guidelines of the Centre National de la Recherche Scientifique and of

Bone marrow cells were washed out from the hip bones of mice. The cells (3 × 105 per sample) were incubated with 20 nM either [125 I]-␣-bungarotoxin or [3 H]-Epibatidine (Amersham Bioscience) for 1 h at 4 ◦ C. To evaluate the level of non-specific binding, 2 mM nicotine was added to a half of the samples. After incubation, the cells were harvested onto glass-fiber filters (GF-C, Whatman) pre-saturated with 1% dry milk, washed with cold PBS and counted in either beta- (for epibatidine) or gamma- (for ␣bungarotoxin) counter. The number of binding sites per

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cell was calculated as described previously (Skok et al., 2003). 2.5. Flow cytometry The bone marrow cell suspensions were stained with either single cell marker-specific antibody or a combination of nicotinic receptor subunit-specific antibodies or fluorescein-labeled ␣-cobratoxin with cell marker-specific antibodies. For double staining, cell suspensions were treated with either biotinylated TER 119-specific antibody and ␣4-specific rabbit antibody followed by Streptavidin–phycoerythrine conjugate and fluorescein-labeled anti-rabbit antibody or with biotinylated TER 119-specific antibody followed by Streptavidin–phycoerythrine conjugate and fluorescein-labeled ␣-cobratoxin. Alternatively, they were stained with either ␣4-specific rabbit antibody followed by phycoerythrine-labeled CD43-specifc antibody and fluorescein-labeled anti-rabbit antibody or phycoerythrine-labeled CD43-specific antibody and fluorescein-labeled ␣-cobratoxin. The treated cells were analyzed in EPICS-XL fluorescent flow cytometer (Coulter-Beckman) with appropriate software. 2.6. Morphological analysis For morphological analysis, the bone marrow cells were printed onto the slides directly from the broken bone. Spleens were cut crosswise and also printed out onto the slide. The blood smears were prepared by conventional procedure. Slides were dried at room temperature, stained according to Pappenheim and analyzed by light microscopy. The results were statistically worked out according to Student’s test and were expressed as a part (%) of morphologically distinct cell type within the whole mixture. 3. Results The presence of nicotinic receptors in the bone marrow of mice was at first demonstrated by the binding of radioactive nicotinic receptor ligands: [3 H]Epibatidine (specific agonist of heteromeric nicotinic receptors) and [125 I]-␣-Bungarotoxin (specific antagonist of ␣7-containing nicotinic receptors). As shown in Fig. 1, total bone marrow cells bound both epibatidine and ␣-bungarotoxin. The cells of ␣7−/− mice lost ␣-bungarotoxin binding completely, while that of epibatidine remained unchanged. According to calculations, total bone marrow cells of the wild-type C57Bl/6J mice contained about 4000 of epibatidine binding sites and

Fig. 1. Specific binding of [125 I]-␣-Bungarotoxin (A) and [3 H]Epibatidine (B) to the total bone marrow cells of the wild-type C57Bl/6J (WT) or ␣7-knockout (␣7−/− ) mice; n = 3. The non-specific binding observed in the presence of 2 mM nicotine has been subtracted. Mean values ± S.E.M. are represented.

about 7000 of ␣-bungarotoxin binding sites per cell. The number of epibatidine sites was much less, while that of ␣-bungarotoxin sites was significantly more in the bone marrow compared to previously studied spleen B lymphocytes (12 000 and 4000, respectively, Skok et al., 2005). Since lymphocytes comprise only a part of total bone marrow cells (up to 8%, according to morphological analysis), we concluded that other cell types present in the bone marrow also expressed nicotinic receptors. Their subunit composition was further studied by flow cytometry using subunit-specific antibodies and fluorescein-labeled ␣-cobratoxin (specific ligand for ␣7-containing nicotinic receptors). As shown in Fig. 2, either ␣-cobratoxin or the antibody against ␣7 subunit stained the bone marrow cells as a single peak, while ␣4-specific antibody bound a separate cell population. This finding suggested that ␣4-containing nicotinic receptors were expressed in limited number of cells, while ␣7-containing receptors were present, in more or less extent, in all cells studied. Then, total bone marrow

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Fig. 2. Binding of nicotinic receptor subunit-specific antibodies (␣4 Ab and ␣7 Ab), non-specific rabbit immunoglobulins (IgG) or ␣-cobratoxin (␣-CTX) to the total live bone marrow cells in flow cytometry. The numbers correspond to the part (%) of total cells found within the gate indicated. The gate was established to exclude the non-stained cells (for ␣-CTX) or cells treated with anti-rabbit IgG alone (for the antibodies). A typical result of three independent experiments is represented.

cells were fractionated according to their size and granularity. As shown in Fig. 3, four different gates have been established. Gate A contained up to 90% of TER 119+ cells and about 10% of CD19+ /B220+ CD16+ cells. The neighboring gate B contained less granular TER 119+ cells (about 60%) and CD19+ /B220+ CD16+ cells (about 30%). Gate C contained CD43med CD16+ B220− /CD19− Ly6C− CD11b− cells (80–90%) and up to 20% of the large TER119+ cells. Finally, Gate D included CD11b+ Ly6C+ CD16+ CD43hi B220− TER119− cells. Since the cells within the gates established were not homogeneous, double staining for nicotinic receptor subunits and specific cellular markers was further performed. TER 119 is a specific marker of erythroid cells (Asari et al., 2005). Fig. 4 demonstrates that TER 119+ cells from gate C were almost totally ␣4-positive, TER 119+ cells from gate B contained two distinct subpopulations: ␣4-positive and negative, while those from gate A were mainly negative. In contrast, ␣-cobratoxin binding was not so different: the major part of TER 119+ cells in

gate C and about a half of them in gates A and B were positive. Binding of non-specific rabbit immunoglobulins was three to fivefold weaker than that of nicotinic receptor-specific antibodies (data not shown). According to morphological analysis, the development of erythroid cells is accompanied with the decrease in size and the increase in granularity. Therefore, we qualified TER 119+ cells in gate C as the earliest, while those in gate A as the most mature erythroid cells. Consequently, our data indicated that ␣4-containing nicotinic receptors were present in the early erythroid precursor cells and gradually disappeared along with their maturation, while ␣7-containing nicotinic receptors remained in more mature forms of these cells. When we tested TER 119+ cells in gate B with a wide panel of nicotinic receptor subunit-specific antibodies, it was found that antibodies against ␣4 and ␤2 subunits bound 19.9 ± 0.35% of cells, while antibodies against ␣7 and ␤4 subunits stained 37.1 ± 2.35% of cells. These data were in accord with our previous suggestion that in B lymphocytes ␣4 subunits were com-

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Fig. 3. Flow cytometry dot plot demonstrating the distribution of total bone marrow cells according to their size and granularity (forward and side scatters, respectively). Gate A: up to 90% of TER 119+ cells and about 10% of CD19+ /B220+ CD16+ CD43− Ly6C− CD11b− cells; gate B: about 60% of TER 119+ cells and about 30% of CD19+ /B220+ CD16+ Ly6C− CD11b− cells; gate C: CD43med CD16+ B220− /CD19− Ly6C− CD11b− cells (80–90%) and up to 20% of TER119+ cells; gate D: CD11b+ Ly6C+ CD16+ CD43hi B220− TER119− cells.

bined with ␤2 to form ␣4␤2 (or ␣4(␣5)␤2) receptors, while ␣7 subunit could be combined with ␤4 (and ␣5) to form heteromeric ␣7(␣5)␤4 receptors (Skok et al., 2007). The data on their differential expression in the course of erythroid cell development corresponded to our results obtained for T and B lymphocytes, where both expression and functional role of ␣4␤2 nicotinic receptors decreased along with the cell maturation, while that of ␣7-containing nicotinic receptors increased or remained unchanged (Skok et al., 2006). The cells within gate D were large and granular; they expressed the set of granulocyte–monocyte-specific markers (CD11b, CD16, CD43hi and Ly6C-6G) and were qualified as neutrophils. As shown in Fig. 5, these cells bound both ␣-cobratoxin and ␣4-specific antibody, therefore, they expressed ␣7-, as well as ␣4-containing nicotinic receptors. Interestingly, CD43med cells within gate C bound more ␣4-specific (and ␤2-specific) antibody than CD43hi cells from gate D, i.e. they expressed more of ␣4␤2 nicotinic receptors than neutrophils. The binding of ␣-cobratoxin was almost unchanged, while that of ␣7-specific antibody was even less in CD43med cells compared to CD43hi (data not shown). CD43med cells were much less granular and a bit smaller than neutrophils, but larger than lymphocytes. They did not express CD11b and Ly6C-6G and comprised up to 10% of the total cells. According to the literature data (De

Bruyn, Delforge, & Bron, 2003; Elghetany, Ge, Patel, Martinez, & Uhrova, 2004; Sykes, Scheele, Pasillas, & Kamps, 2003; Szilvassy & Cory, 1993) and those of morphological analysis (Fig. 6), such characteristics fit to myeloid progenitors: myeloblasts and promyelocytes. Then it appears that ␣4-containing nicotinic receptors are inherent to early myeloid cells and are down-regulated upon their maturation, similarly to the nicotinic receptors of lymphocytes (Skok et al., 2006) and erythroid cells (Fig. 3). To test the role of two nicotinic receptor subtypes in the generation and maturation of hematopoietic cells we performed morphological analysis of the bone marrow cells of wild-type C57Bl/6J mice compared to ␤2−/− , ␣7−/− ones or wild-type mice chronically treated with nicotine. As shown in Fig. 6A, the bone marrow cells of ␤2−/− mice contained more early and junior myeloid cells (myeloblasts, promyelocytes, myelocytes and metamyelocytes), but less relatively mature erythroid cells (normoblasts) than cells of the wild-type mice. The parts of mature (band and segmental) neutrophils, lymphocytes and erythroid progenitors (erythroblasts and pronormocytes) were similar. These data indicated that the absence of ␤2-containing nicotinic receptors favored generation of myeloid cell precursors. Since the level of erythroid cell precursors was not affected, while the part of normoblasts decreased, we suggested that ␤2-containing nicotinic receptors regulated further normoblast maturation (loss of nucleus and transformation into reticulocytes, which leave the bone marrow for the blood circulation). The bone marrow cells of ␣7−/− mice contained significantly less myeloblasts, metamyelocytes, band neutrophils, erythroblasts and pro-normoblasts than the wild-type cells (Fig. 6B). The part of normoblasts and mature neutrophils was not changed, while the part of lymphocytes increased significantly. The bone marrow cells of mice chronically treated with nicotine contained significantly less myeloblasts, promyelocytes and erythroblasts, but more segmental neutrophils than the cells of untreated mice (Fig. 6C). The parts of other cell types remained unchanged. So, the effect of chronic nicotine resembled that of ␣7 knockout and also favored mature neutrophils accumulation. In contrast to the bone marrow, no significant changes were found in the cell content of either spleens or blood of mice examined (Tables 1 and 2). This means that ␣7and ␤2-containing nicotinic receptors are important for the processes of blood cells generation and maturation, but not for their migration to and accumulation in the periphery.

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Fig. 4. Binding of ␣4-specific antibody (␣4 Ab) or ␣-cobratoxin (␣-CTX) to mouse erythroid cells (TER 119+ ) within gates A, B and C shown in Fig. 3. The numbers correspond to the part (%) of TER 119+ cells stained with either ␣4 Ab or ␣-CTX. The gates were established to limit the negative (non-stained) cells within the left upper quadrant. A typical result of three independent experiments is represented. Table 1 Relative amounts (%) of morphologically different cell types in the spleens of ␤2−/− , ␣7−/− or nicotine-treated (Nic) mice compared to their wild-type (WT) or non-treated (NT) counterpartsa Cell type

WT

Lymphoblasts Prolymphocytes Lymphocytes Neutrophils (band) Neutrophils (segmental) Erythrocytes

1.67 13.3 55.3 1.67 5.0 20.3

␤2−/− ± ± ± ± ± ±

0.3 0.9 1.3 0.3 1.0 1.8

1.33 14.7 55.0 2.3 3.3 20.7

± ± ± ± ± ±

␣7−/−

WT 0.3 2.4 2.1 0.9 0.7 3.5

1.5 13.5 41.8 3.0 3.25 35.2

± ± ± ± ± ±

0.6 2.1 5.4 0.6 0.6 5.8

1.0 10.5 50.3 1.7 3.0 33.0

± ± ± ± ± ±

NT 0.6 0.5 7.9 0.3 0.6 3.0

0.88 12.2 64.5 3.5 4.6 9.8

Nic ± ± ± ± ± ±

0.1 4.6 2.7 1.8 0.9 3.0

1.25 17.8 63.2 2.0 2.5 12.5

± ± ± ± ± ±

0.2 2.8 3.9 0.6 0.6 3.7

Separate age-matched control groups were used for each experiment; mean values ± S.E.M. are represented; n = 4 for each group. No statistically significant differences between corresponding groups were observed. a

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Fig. 5. Binding of ␣4-specific antibody or ␣-cobratoxin to CD43+ bone marrow cells within gates C and D shown in Fig. 3. The numbers correspond to the part (%) of CD43+ cells stained with either ␣4 Ab or ␣-CTX. The gates were established to limit the negative (non-stained) cells within the left upper quadrant. A typical result of three independent experiments is represented. Table 2 Relative amounts (%) of morphologically different cell types in the blood of ␤2−/− (n = 8), ␣7−/− (n = 8) or nicotine-treated mice (Nic, n = 4) compared to their wild-type (WT) or non-treated (NT) counterpartsa Cell type

WT

␤2−/−

WT

␣7−/−

NT

Nic

Lymphocytes Neutrophils Monocytes

85.4 ± 1.25 10.1 ± 0.9 5.2 ± 0.3

80.5 ± 3.2 9.8 ± 2.4 6.5 ± 1.0

75.2 ± 2.4 20.7 ± 1.5 4.2 ± 1.3

70.1 ± 4.2 21.5 ± 3.4 7.2 ± 1.4

63.5 ± 5.7 29.5 ± 3.6 9.0 ± 1.7

64.9 ± 6.3 29.2 ± 3.9 5.2 ± 2.2

a Separate age-matched control groups were used for each experiment; mean values ± S.E.M. are represented. No statistically significant differences between corresponding groups were observed.

4. Discussion The data presented suggest an important role of nicotinic acetylcholine receptors for generation and maturation of myeloid and erythroid cells. They clearly indicate that the two nicotinic receptor subtypes influence hematopoietic processes in different ways. The absence of ␤2-containing nicotinic receptors results in so called “left shift” favoring early precursor cell generation, while ␣7-containing nicotinic receptors deficit results in the “right shift” to mature cell forms. So, in physiological conditions, ␤2-containing nAChRs limit the early progenitor generation, while ␣7 ones favor the expansion of immature cell forms. This finding is in accord with our data that ␣4␤2 nicotinic receptors are

inherent to progenitor cells, while ␣7-containing ones are expressed in mature cells as well. The apparent discrepancy between the increased or decreased numbers of progenitor cells in the bone marrow and their equal numbers found in the spleen and blood of knockout mice can be explained by the different life spans of immature vs. mature cell forms. This is clearly seen within the bone marrow, where the parts of immature forms are dozens of times less than those of mature cells and increased generation of myeloblasts and promyelocytes did not result in increased mature neutrophils numbers (Fig. 6A). This means that cholinergic regulation plays a role for the early stages of the blood cell development, probably, those requiring cell contacts with the bone marrow stroma. Later these changes are

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Fig. 6. Relative numbers of morphologically distinct cells in the bone marrows of ␤2 knockout (␤2−/− , A, n = 6), ␣7 knockout (␣7−/− , B, n = 4) or nicotine-treated C57Bl/6J mice (Nic, C, n = 4) compared to their wild-type/untreated counterparts. (1) myeloblasts; (2) promyelocytes; (3) myelocytes; (4) metamyelocytes; (5) band neutrophils; (6) segmental neutrophils; (7) lymphocytes; (8) erythroblasts; (9) pronormocytes; (10) normoblasts. Separate age-matched control groups were used for each experiment resulting in different control values in A, B and C graphs. Mean values ± S.E.M. are represented. A significant (p < 0.05) difference between the values of the control and knockout/nicotine-treated groups is shown by “*”.

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leveled by accumulation of the long-lived mature cells. Previously we found similar phenomenon for the lymphocytes. The nAChR presence played a role for the B lymphocyte expansion within the bone marrow, but did not affect the mature B lymphocyte numbers in the spleen (Skok et al., 2006). The decrease in early hematopoietic progenitor cells found upon chronic nicotine treatment is in accord with the published data (Serobyan et al., 2005). It also complements the data of Soreq et al. (1994) where inhibition of acetylcholine esterase (and correspondingly stronger nicotinic receptor activation) resulted in progenitor cell expansion and suppression of hematopoietic apoptosis. The latter finding corresponds to our data on mature neutrophils accumulation in the bone marrows of nicotine-treated mice. It was also reported that nicotine favored cultured neutrophils survival by decreasing the rate of their apoptosis (Aoshiba, Nagai, & Yasui Konno, 1996). ␣7-Containing receptors were shown to support the cell survival (Gimonet et al., 2003) that is also in accord with our data. Our studies demonstrated that the absence of ␣7 nicotinic receptors and chronic nicotine treatment resulted in similar changes in the bone marrow cell content (Fig. 6). Such similarity could not be explained by the compensatory increase of heteromeric ␣4␤2 receptors, which are the main target of nicotine in the brain (Marubio & Changeux, 2000), because no increase of epibatidine binding was found in ␣7 knockout mice (Fig. 1B). The ␣7 knockout-like effect observed in nicotine-treated mice could be due to receptor desensitization usually observed upon chronic nicotine treatment (Quitadamo, Fabbretti, Lamanauskas, & Nistri, 2005). Such explanation also indirectly indicates that there are ␣7-containing nicotinic receptors which play the major role in regulating hematopoiesis. The role of ␣4␤2 receptors seems to be limited to the control of early myeloid precursor cell generation and/or normoblast maturation (because these processes are antagonistic, Koh et al., 2005). The bone marrow cells of ␣7 knockout mice contained a significantly increased part of lymphocytes as compared to the wild-type cells (Fig. 6B). Our previous experiments showed the negative effect of ␣7 knockout on B lymphocyte expansion in the bone marrow, as well as T lymphocytes in the thymus (Skok et al., 2006). Therefore, we consider an increase of lymphocyte part as a compensatory reaction for the slowed generation of both myeloid and erythroid cells found in ␣7 knockouts, as it is described for the cases of aplastic anemia or myelodepression (Degtyareva, 1974). The found effect of either nicotinic receptor absence or desensitization on hematopoietic cell maturation indi-

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cates that in physiological conditions nicotinic receptors expressed in these cells are activated with endogenous ligand(s). The bone marrow is innervated with sympathetic fibers (Mignini, Strecioni, & Amenta, 2003) and choline acetyltransferase-immunoreactive nerve fibre-like structures were found around haematopoietic islets (Artico et al., 2002); therefore, neuronal acetylcholine can mediate the autonomic nervous system control of hematopoiesis. In addition, increasing evidence suggests an important role of extraneuronal cholinergic system (Fujii & Kawashima, 2001). Acetylcholine was shown to be synthesized in many tissues, such as skin (Grando, Kist, Qi, & Dahl, 1993), airway epithelium (Lips et al., 2005), bladder urothelium (Yoshida et al., 2006) and peripheral blood leukocytes including activated T lymphocytes (Rinner, Kawashima, & Schauenstein, 1998). Moreover, the components of acetylcholine-synthesizing and degrading systems (choline-acetyltransferase and acetylcholinesterase) were found in mesenchymal stem cells (Hoogduijn, Rakonczay, & Genever, 2006). This means that within the bone marrow acetylcholine can be released by the stromal elements (e.g. osteoclasts) affecting generation and differentiation of blood cells upon cell-stroma contacts. Additional information is provided by the data on nAChR expression in CD43med vs. CD43hi cells (Fig. 5). CD43 is a mucin-like molecule involved in cell-to-cell interactions (Rupniewska, Rolinski, & Bojarska-Junak, 2000). It is down-regulated on granulocytes upon inflammation (Remold-O’Donnell & Parent, 1994), which shifts these cells to less mature phenotype (Sunderkotter et al., 2004). This finding supports our suggestion for the less mature state of CD43med cells in gate C compared to gate D (Fig. 5). The observed connection between CD43 and ␣4 nicotinic receptor expression suggests the role of this nicotinic receptor subtype in regulating cell adhesion that was clearly shown for skin keratinocytes and ␣3␤2 nicotinic receptors (Grando, 2006). When produced by activated T lymphocytes recirculating in the bone marrow, acetylcholine may provide a feedback signal stimulating hematopoiesis upon the immune response. In addition, recent evidence suggests that nicotinic receptors can be activated with peptide ligands SLURP-1 and SLURP-2 produced by leukocytes (Arredondo, Chernyavsky, Webber, & Grando, 2005; Arredondo, Chernyavsky, Jolkovsky, Webber, & Grando, 2006). Such a finding can provide a completely new explanation for extraneuronal cholinergic regulation. Finally, the established role of nicotinic receptors helps to understand the influence of smoking on

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