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Nicotine • Bone marrow • Spleen • Hematopoiesis stem progenitor cells • Extramedullary ... The effect of sustained exposure to nicotine, a major constitu-.

TISSUE-SPECIFIC STEM CELLS Sustained Exposure to Nicotine Leads to Extramedullary Hematopoiesis in the Spleen TERLIKA S. PANDIT, LYUDMILA SIKORA, GIRIJA MURALIDHAR, SAVITA P. RAO, P. SRIRAMARAO Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California, USA Key Words. Nicotine • Bone marrow • Spleen • Hematopoiesis stem progenitor cells • Extramedullary hematopoiesis

ABSTRACT The effect of sustained exposure to nicotine, a major constituent of cigarette smoke, on hematopoiesis in the bone marrow (BM) and spleen was evaluated in a murine model. BALB/c mice were exposed to nicotine subcutaneously using 21-day slow-release pellets. Exposure to nicotine had no effect on the proliferation of long-term BM cultures or on their ability to form colonies. However, there was a significant decrease in the generation of lineage-specific progenitor cells, specifically eosinophil (colony-forming unit [CFU]-Eos) progenitors, in the BM of nicotine-exposed mice compared with control mice. Surprisingly, sustained exposure of mice to nicotine was found to induce significant hematopoiesis in the spleen. There was a significant increase in total colony formation as well as eosinophil-, granulocyte-macrophage-, and B-lymphocyte-specific

progenitors (CFU-Eos, CFU-GM, and CFU-B, respectively) in nicotine-exposed mice but not in control mice. Sustained exposure to nicotine was associated with significant inhibition of rolling and migration of enriched hematopoietic stem/progenitor cells (HSPCs) across BM endothelial cells (BMECs) in vitro as well as decreased expression of ␤2 integrin on the surface of these cells. Although sustained exposure to nicotine has only a modest effect on BM hematopoiesis, our studies indicate that it significantly induces extramedullary hematopoiesis in the spleen. Decreased interaction of nicotine-exposed HSPCs with BMECs (i.e., rolling and migration) may result in altered BM homing of these cells, leading to their seeding and proliferation at extramedullary sites such as the spleen. STEM CELLS 2006;24:2373–2381

INTRODUCTION

intrauterine exposure to nicotine results in inferior colonization of fetal BM by hematopoietic stem/progenitor cells (HSPCs), which may subsequently result in an imbalance of mature blood and immune cell production after birth [9]. In healthy adults, mature blood cells have a limited life span and are continuously replaced by the proliferation and differentiation of a very small population of pluripotent HSPCs found primarily in the BM. HSPCs have the ability to replenish themselves (i.e., demonstrate self-renewal) and to differentiate into mature blood cells of all lineages in the adult organism [10]. During periods of hematopoietic stress and certain pathological conditions as well as during fetal development, however, extramedullary organs such as the liver, spleen, and even the heart have been found to demonstrate renewed capability of producing progenitor cells of mature blood lineages [11–13]. Here, we have extended our previous in vitro studies [8] by evaluating the effect of sustained exposure to nicotine on hematopoiesis in a murine model with subcutaneously (s.c.) embedded nicotine pellets. Our studies demonstrate that nicotine exposure induces extramedullary hematopoiesis in the spleen while inhibiting the generation of lineage-specific progenitors, specifically eosinophil (colony-forming unit [CFU]-Eos) pro-

Cigarette smoking is a major risk factor for several diseases, including cancer and respiratory, cardiovascular, cerebral, and peripheral vascular diseases. The deleterious effects of cigarette smoke on the cellular components of peripheral blood were identified as early as three decades ago [1]. Exposure to nicotine, a major constituent of cigarette smoke, impairs immune and inflammatory responses [2, 3], alters leukocyte trafficking [4], and causes a reduction in the number of functionally active immunocompetent cells [5]. Chronic nicotine self-administration suppresses T-cell responsiveness [6] and is an important immunomodulator at the level of immune cell apoptosis, a process thought to contribute to autoimmunity, cardiovascular disease, and carcinogenesis [7]. In addition to these effects, previous studies from our laboratory have demonstrated that exposure of murine long-term bone marrow cultures (LTBMCs) to nicotine in vitro inhibited the formation of loci of active hematopoiesis (“cobblestone areas”) in a dose-dependent manner. Furthermore, in vitro exposure to nicotine decreased CD44 expression on stromal as well as lung- and bone marrow (BM)derived endothelial cell lines, and this decrease may interfere with stem cell homing [8]. Recent studies have shown that

Correspondence: P. Sriramarao, Ph.D., Division of Vascular Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Drive, San Diego, California 92121, USA. Telephone: 858-587-8788, ext. 101; Fax: 858-587-6742; e-mail: [email protected] Received September 13, 2005; accepted for publication June 15, 2006; first published online in STEM CELLS EXPRESS July 6, 2006. © AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0447

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genitors, in the BM. Furthermore, rolling and migration studies with enriched HSPCs from nicotine-exposed mice suggest that nicotine is likely to alter the BM homing of these cells by inhibiting their ability to roll and migrate across BM endothelial cells (BMECs), possibly due to decreased expression of ␤2 integrin, a molecule that is known to play a role in trafficking of HSPCs [14].

MATERIALS

AND

METHODS

Endothelial and Stromal Cell Lines S17, a stromal cell line, was kindly provided by Dr. Kenneth Dorshkind (Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles) [15]. STR-12, a murine BMEC line was provided by Dr. Masanobu Kobayashi (Hokkaido School of Medicine, Sapporo, Japan) [16]. Both cell lines were cultured in RPMI (Invitrogen, Carlsbad, CA, http://www. invitrogen.com) supplemented with 10% fetal calf serum (FCS).

Exposure to Nicotine Female BALB/c mice (7– 8 weeks) were anesthetized by inhalation with 5% isoflurane, and each mouse was implanted s.c. with a 21-day slow-release nicotine pellet (5 mg/pellet; Innovative Research of America, Sarasota, FL, http://www.innovrsrch. com) as described previously [17, 18]. Based on previous studies [18] that indicate that the steady-state level of nicotine achieved in the blood of nicotine pellet-exposed mice is severalfold lower than that implanted, the dosage of nicotine used in the current study is likely to correlate to physiological levels previously reported in heavy smokers (ranging from 10⫺6 to 10⫺8 M) [19]. Assuming uniform release, mice are likely to be exposed to approximately one-fifth the concentration of nicotine intake from one cigarette in humans [20]. Age- and gendermatched untreated BALB/c mice housed under similar conditions served as controls. Mice were euthanized at the end of 21 days, and the BM and spleen were collected. The care and maintenance of mice during performance of these studies were in accordance with institutional guidelines.

Colony-Forming Unit Assay BM was collected by flushing the femurs of nicotine-exposed (test) and control mice with RPMI and suspended uniformly in the same medium. Spleens of test and control mice were collected, and spleen cells were obtained by mashing the spleens through 70-␮m cell strainers (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) in RPMI. Whole unlysed BM and spleen cell suspensions were centrifuged and resuspended in MyeloCult M5300 medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). To evaluate erythroid colonies (CFU-E and burst-forming unit [BFU]-E), granulopoietic colonies (CFU-granulocyte-macrophage [CFUGM]), and colonies containing multiple lineages of cells (CFUgranulocyte, erythrocyte, monocyte, megakaryocyte [GEMM]), BM and spleen cell suspensions were plated (1 ⫻ 104 cells per milliliter) on methylcellulose media (MethoCult 3434 medium; StemCell Technologies) supplemented with stem cell factor, IL-3, IL-6, and erythropoietin as recommended by the manufacturer. For granulocyte-macrophage (CFU-GM)-, eosinophil (CFU-Eos)-, and B-lymphoid (CFU-B)-specific progenitors, cells (1 ⫻ 104 cells per milliliter, 5 ⫻ 105 cells per milliliter, and

5 ⫻ 104 cells per milliliter, respectively) were cultured in MethoCult 3234 medium (StemCell Technologies) containing GM-CSF (granulocyte macrophage-colony-stimulating factor) (10 ng/ml), IL-5 (50 ng/ml), or IL-7 (10 ng/ml), respectively. Cells were plated and maintained at 37°C in a humidified atmosphere at 5% CO2 for 10 –12 days as recommended by the manufacturer (StemCell Technologies); the colonies were then counted in situ under an inverted microscope and expressed as CFU per number of cells plated. Colonies were identified as described in the Atlas of Human Hematopoietic Colonies published by StemCell Technologies (www.stemcell.com/technical/ 28405 methocult% 2OM.pdf).

LTBMC Assay Freshly isolated BM cells were cultured in six-well plates in MyeloCult M5300 medium (4 ml/well at 106 cells per ml) containing 10⫺6 M hydrocortisone (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 8 weeks. At the end of each week, cultures were gently rocked to loosen nonadherent cells. All of the medium was drawn up into a pipette, and one-half of the original volume (2 ml) was returned to the dish, and the remaining half was used to determine the number of nonadherent cells. The cultures remaining in the dish were replenished by gently adding fresh MyeloCult M5300 medium (with hydrocortisone). Nonadherent cells in the withdrawn culture medium were counted and assayed in MethoCult 3434 medium for multipotential progenitor cells by colony formation (CFU assay described above).

Colony Formation in Stromal Cell-Supported BM and Spleen Cultures S17 stromal cells were cultured in RPMI containing 5% FCS in 96-well plates until confluent. BM and spleen cells from nicotine pellet-exposed and control mice were suspended in hydrocortisone-supplemented MyeloCult M5300 medium (4 ⫻ 104/ ml) and added to the plates (100 ␮l/well) containing the supportive S17 stromal cell feeder layer. Five serial 1:2 dilutions were performed for each sample, and 12 wells were set up at each dilution. The number of BM or spleen cells added to the feeder layers ranged from 125 to 2,000 cells per well. Cultures were incubated at 37°C for 2 weeks, and the culture medium was replenished with fresh medium (100 ␮l/well) at the end of the first week. The number of wells positive for colonies at each dilution was scored after 14 days in situ under an inverted microscope and expressed as the number of wells with colonies for each dilution.

Enrichment of Murine HSPCs Progenitor cells in the BM were enriched using a kit containing a cocktail of biotinylated monoclonal antibodies (mAbs) against the following murine cells/cell surface antigens: CD5 (Ly-1), erythroid cells (TER119), CD45R (B220), Ly-6G (Gr-1), CD11b (Mac-1), and neutrophils (7-4) (StemSep Mouse Progenitor Enrichment Cocktail, catalog number 13056; StemCell Technologies). HSPCs were isolated by negative selection using anti-biotin anti-dextran tetrameric antibody complexes and purified by magnetic cell separation. Due to the low recovery after enrichment, BM cells recovered from individual mice in the nicotine pellet-treated and control groups were pooled prior to enrichment of HSPCs. After enrichment, 16.27% ⫾ 7.68% of the cells were positive for CD34

Pandit, Sikora, Muralidhar et al. compared with 4.18% ⫾ 1.11% before enrichment as assessed by flow cytometry (FACScan; BD Biosciences, San Jose, CA, http:// www.bdbiosciences.com) using fluorescein isothiocyanate (FITC)labeled anti-mouse CD34 mAbs (eBiosciences, San Diego, http:// www.ebiosciences.com).

Migration Assay This assay was performed using 24-well Transwell clusters equipped with 5-␮m filters (Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences). STR-12 cells were grown to confluency on the filters in RPMI containing 10% FCS. Medium covering the STR-12 cells was replaced with enriched HSPCs (2 ⫻ 105 in 200 ␮l of Dulbecco’s modified Eagle’s medium [DMEM]) isolated from pooled BM of nicotine pellet-exposed or control mice. DMEM containing stromal cellderived factor (SDF)-1 (50 nM), a chemoattractant produced by the BM stroma, was added to the bottom well. The plates were incubated at 37°C and 5% CO2 for 4 hours; the upper wells were then carefully removed, and the cells in the lower wells were collected and counted using a Neubauer Bright-Line hemocytometer (Reichert, Buffalo, NY, http://www.reichert.com). Results were expressed as percentage of migration based on the total number of cells added to the upper well of the Transwell clusters.

In Vitro Laminar Flow Assay Rolling of enriched HSPCs on STR-12 BMECs was assessed in an in vitro parallel plate laminar flow chamber as described in previous studies from this laboratory [21, 22]. Briefly, STR-12 cells were cultured to confluency on cover slips and positioned at the bottom of a parallel plate flow chamber (100-␮m thickness). The chamber was exposed to different flow conditions by perfusing warm medium (RPMI containing 0.75 mM Ca2⫹ and Mg2⫹ and 0.2% human serum albumin) through a constant infusion syringe pump (Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com). A single-cell suspension of enriched HSPCs (5 ⫻ 104 cells per milliliter) isolated from pooled BM of nicotine pellet-exposed or control mice was perfused into the chamber for 5 minutes, and the interaction of the injected cells with the endothelial cell layer was observed using a Leitz Biomed inverted microscope (Wetzlar, Germany). The images were video-recorded for subsequent offline video analysis to manually determine the number of interacting cells. Rolling cells demonstrate multiple discrete interruptions and flow slowly compared with cells that do not roll. Results are expressed as the number of rolling cells per minute at a flow rate of 1 ml/minute.

Flow Cytometry

Cell surface expression of CD44, CD49d (␣4), CD18 (␤2), and CD62L (L-selectin) on CD34-enriched HSPCs isolated from pooled BM of nicotine pellet-exposed or control mice was determined by flow cytometry as described in our previous studies [8]. For analysis of CD44 and L-selectin expression, FITC-conjugated mAb Pgp-1 [23] and MEL-14 [24] against murine CD44 and L-selectin, respectively, were used. Expression of ␣4 and ␤2 was analyzed by incubating enriched HSPCs (5 ⫻ 105) with antimurine CD49d mAb PS/2 [25] and anti-murine CD18 mAb 2E6 [26], respectively, followed by appropriate FITC-labeled secondary antibodies (BD PharMingen, San Diego, http://www.bdbiosciences. www.StemCells.com

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com/pharmingen). Expression of CXCR4 was analyzed using rabbit polyclonal CXCR4 antibody that is reactive against murine CXCR4 (ProSci Incorporated, Poway, CA, http://www.prosci-inc. com) followed by FITC-conjugated anti-rabbit IgG (Sigma-Aldrich). Appropriate isotype-matched controls were included (all from BD PharMingen). All antibodies were used at a concentration of 4 ␮g/106 cells. Incubations were carried out at 4°C for 30 minutes, and cells were washed with fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline containing 1% bovine serum albumin, 1 mM Ca2⫹-Mg2⫹, and 0.02% NaN3) after each incubation. The expression of cell surface markers was analyzed on a FACScan using CellQuest Pro (Becton, Dickinson and Company, San Diego, http://www.bd.com).

Statistical Analysis

Results are expressed as the mean ⫾ SE. Statistical significance was determined using the Student’s t test.

RESULTS Effect of Nicotine on LTBMCs The BM is the major hematopoietic tissue in adult mice. In a previous study from our laboratory, we showed that treatment of LTBMCs with nicotine in vitro inhibited the formation of cobblestone areas, which represent loci of active hematopoiesis, although the formation of a confluent adherent layer was not significantly affected [8]. In the current study, our goal was to analyze the effect of nicotine on the proliferation and differentiation of HSPCs in a more physiological situation in which mice were subjected to a sustained exposure (21 days) to nicotine in vivo. Freshly isolated BM cultures from nicotine-exposed and control mice were analyzed by the LTBMC assay. There was no significant difference observed in the number of nonadherent cells recovered from BM cultures of nicotine-exposed mice versus control mice (Fig. 1A), suggesting that sustained exposure to nicotine does not have an effect on proliferation of BM cells. Furthermore, consistent with our previous findings [8], formation of a confluent adherent layer was not affected in these mice and was similar to that observed with BM cultures from control mice (data not shown). To determine whether there was a difference in the ability of BM cells from nicotine-exposed and control mice to generate multipotential progenitor cells, nonadherent cells recovered at the end of each week of culture were evaluated for colony formation as described in Materials and Methods (Fig. 1B). Again, there was no difference between nicotine-exposed and control mice in the ability of cells recovered from LTBMCs to undergo colony formation. Although these results are in contrast to our earlier observation, in which nicotine was found to inhibit the number of nonadherent cells in LTBMCs as well as the number of progenitor cells generated in LTBMCs compared with control cultures [8], a major difference between the two studies is that in the previous study BM cells isolated from normal mice were cultured in vitro in the presence of nicotine at a concentration of 10⫺5 M up to 12 weeks, and in the present study mice were exposed to nicotine in vivo, where the serum concentration of nicotine is much lower (approximately 10⫺7 M). Furthermore, nicotine is rapidly metabolized to cotinine in vivo, and our previous studies have shown that exposure to cotinine in vitro does not inhibit nonadherent cells or progenitor cells generated in LTBMCs [8].

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Sustained Nicotine Exposure and Hematopoiesis

Figure 2. In vivo exposure to nicotine induces the generation of CFU in the spleen but not in the BM. Freshly isolated BM and spleen cells from nicotine-exposed (test) and control mice were grown in MethoCult 3434 medium supplemented with stem cell factor, IL-3, IL-6, and erythropoietin. Colonies comprising of colony-forming unit-erythroid, burst-forming unit-erythroid, colony-forming unit-granulocyte-macrophage, and colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte were identified as described in the Atlas of Human Hematopoietic Colonies published by StemCell Technologies and the results are expressed as mean CFU ⫾ SE. Combined data of three experiments (n ⫽ 3–5 mice per group per experiment) are shown. *p ⬍ .05. Abbreviations: BM, bone marrow; CFU, colony-forming unit; SPL, spleen. Figure 1. Sustained exposure to nicotine does not affect long-term bone marrow cultures (LTBMCs). Long-term cultures of freshly isolated bone marrow cells from nicotine-exposed and control mice were maintained up to 8 weeks at 37°C and 5% CO2 in MyeloCult M3500 medium containing 10⫺6 M hydrocortisone with weekly feedings. (A): Nonadherent cells withdrawn from the culture medium during weekly feedings were counted and expressed as mean ⫾ SE. (B): Nonadherent cells harvested from LTBMCs each week were plated at a concentration of 104 cells per milliliter in MethoCult 3434 medium supplemented with stem cell factor, IL-3, IL-6, and erythropoietin and assayed for colony formation. The number of colonies in the cultures was manually counted between 10 and 12 days and expressed as mean ⫾ SE. Combined data of three experiments (n ⫽ 3–5 mice per group per experiment) are shown. Abbreviation: CFU, colony-forming unit.

Effect of Nicotine on Hematopoiesis in the BM and Spleen Single-cell suspensions of the BM and spleen obtained from nicotine-exposed and control mice were assayed for the presence of multipotential as well as lineage-specific progenitor cells by the CFU assay. Although the total numbers of multipotential progenitor cells in BM cultures from test and control mice were similar, a significantly greater number of multipotential progenitors, surprisingly, were present in the spleen of test mice (p ⬍ .05) compared with control mice (Fig. 2). There was no difference in BM CFU-B and CFU-GM between control and test mice; however, there was a significant (p ⬍ .05) decrease in CFU-Eos in the BM of nicotine-exposed mice compared with control mice (Fig. 3). More interestingly, there was a significant increase (p ⬍ .05) in the number of CFU-B, CFU-GM, and CFU-Eos in the spleen of nicotine-exposed mice compared with control mice. Overall, these data suggest that, while sustained exposure to nicotine has only a modest effect on BM hematopoiesis, affecting primarily the generation of eosinophil-specific progenitor

cells but not the proliferation of BM cells (in LTBMCs) or the generation of multipotential progenitor cells, it has a profound effect on the spleen, inducing the generation of not only total progenitor cells but also lineage-specific progenitors.

Effect of Stromal Cell Microenvironment on Colony Formation by Nicotine-Exposed BM and Spleen SDFs are known to support the proliferation and generation of HSPCs [14]. The effect of a well-established stromal cell layer on the ability of BM and spleen cells to undergo colony formation was evaluated (Fig. 4). With BM cultures, we failed to observe any difference between control and nicotine pelletexposed mice with respect to the number of wells with colonies, whereas with the spleen (at all cell concentrations tested), a significantly larger number of wells with colonies were observed with nicotine-exposed versus control cells. These data are consistent with findings described above (Fig. 2) and suggest that the presence of a supportive stromal cell microenvironment does not appear to alter the effect of sustained nicotine exposure on the hematopoietic potential of the BM and spleen.

Exposure to Nicotine Inhibits Enriched HSPC Rolling and Migration Across BMECs In Vitro Because sustained exposure to nicotine augmented hematopoietic activity in the spleen and not in the BM, we postulated that this might be due to the inability of HSPCs to be retained in the BM microenvironment in response to exposure to SDFs and to their concomitant ability to preferentially traffic and interact with ECs in extramedullary organs, including the spleen. Furthermore, several studies have demonstrated that initial rolling followed by activation-dependent firm adhesion of HSPCs to the BMECs are important cellular events that precede chemo-

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Figure 4. Effect of stromal cells on colony formation in bone marrow (BM) and spleen cells from nicotine-exposed mice. Freshly isolated cells from the BM and spleen of nicotine-exposed (test) and control mice were cultured in a 96-well plates in limiting dilution on a feeder layer of S17 stromal cells for 2 weeks. Cultures were replenished with fresh media at the end of the first week, and wells containing colonies were scored at the end of 14 days. Results are expressed as the mean ⫾ SE of the number of wells with colonies. Combined data of three experiments (n ⫽ 4 – 6 mice per group per experiment) are shown. *p ⬍ .05. Figure 3. Effect of nicotine on the generation of lineage-specific progenitors in the bone marrow (BM) and spleen. Freshly isolated cells from the BM and spleen of nicotine-exposed (test) and control mice were cultured in MethoCult 3234 media containing IL-7 (10 ng/ml), granulocyte macrophage-colony-stimulating factor (10 ng/ml), or IL-5 (50 ng/ml) to evaluate CFU-B, CFU-GM, and CFU-Eos, respectively. Results are expressed as mean CFU ⫾ SE. Combined data of three experiments (n ⫽ 3–5 mice per group per experiment) are shown. *p ⬍ .05. Abbreviations: CFU, colony-forming unit; CFU-B, colony-forming unit-B lymphocyte; CFU-Eos, colony-forming unit-eosinophil; CFUGM, colony-forming unit-granuloctye-macrophage.

kine (SDF-1)-mediated recruitment/homing of HSPCs to the BM [27–29]. Accordingly, the ability of enriched HSPCs isolated from the BM of nicotine pellet-exposed and control mice to interact with BMECs (STR-12) under conditions of flow was determined. In comparison with those of control mice, HSPCs isolated from nicotine pellet-exposed mice exhibited considerably decreased ability to roll on BMECs (Fig. 5). Furthermore, enriched HSPCs obtained from control mice demonstrated a twofold increase in their ability to migrate across an EC barrier toward SDF-1, whereas enriched HSPCs obtained from nicotine pellet-exposed mice did not migrate in response to SDF-1 (Fig. 6). These data suggest that exposure to nicotine markedly inwww.StemCells.com

hibits the ability of HSPCs to interact with BMECs (rolling and transmigration).

Exposure to Nicotine Decreases ␤2 Expression on HSPCs To determine whether inhibition of rolling and migration of nicotine-exposed BM HSPCs across BMECs was due to alterations in adhesion molecule or SDF-1 receptor expression on these cells, enriched HSPCs from control and nicotine pellet-exposed mice were analyzed for the expression of ␣4, L-selectin, ␤2, and CXCR4 by FACS. Although there was no difference in surface expression of ␣4, L-selectin, and CXCR4 (data not shown) on live-gated CD34-enriched HSPCs between control and nicotine pellet-exposed mice, the expression of ␤2 on these cells from nicotine pellet-exposed mice was considerably decreased (approximately 66%) compared with cells from control mice (Fig. 7).

DISCUSSION The adverse effects of cigarette smoke and nicotine are wellknown. We have previously studied the effect of nicotine on hematopoiesis in vitro [8]. In the current study, we have extended these findings by evaluating the effect of sustained in

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Figure 5. Nicotine exposure inhibits rolling of enriched hematopoietic stem/progenitor cells (HSPCs) on bone marrow endothelial cells. Single-cell suspensions of enriched HSPCs isolated from pooled bone marrow of nicotine-exposed (test) or control mice (5 ⫻ 104 cells per milliliter) (n ⫽ 3–5 mice per group per experiment) were perfused into a parallel plate laminar flow chamber for 5 minutes, and their ability to roll on STR-12-coated cover slips under conditions of flow was evaluated. The assay was run in duplicate, and the results are expressed as mean ⫾ SE of the number of rolling cells per minute. Combined data of two experiments are shown.

Figure 6. Exposure to nicotine inhibits transendothelial migration of enriched hematopoietic stem/progenitor cells (HSPCs) in response to SDF-1. CD34-enriched HSPCs isolated from pooled bone marrow of nicotine-exposed (test) or control mice were suspended in Dulbecco’s modified Eagle’s medium and placed in the upper wells of Transwell clusters containing 5-␮m filters coated with confluent layers of STR-12 cells. Plates were incubated at 37°C. Migration of enriched HSPCs from test and control mice (n ⫽ 3 mice per group per experiment) across the endothelial cell layer in the presence and absence of SDF-1 in the lower well was assessed by counting the cells in the lower well after 4 hours. The assay was set up in duplicate, and the results are expressed as mean ⫾ SE of percentage of migration. Combined data of two experiments are shown. Abbreviation: SDF-1, stromal cell-derived factor-1.

vivo exposure of mice to nicotine. Furthermore, the effect of nicotine exposure on BM hematopoiesis in comparison with an extramedullary organ such as the spleen, which has not been previously reported, was determined in the current study. Our studies demonstrate that whereas BM hematopoiesis did not appear to be dramatically altered, exposure to nicotine significantly induced hematopoiesis in the spleen. Furthermore, in contrast to our previous studies, in which in vitro treatment of BM cultures with increasing concentrations of nicotine resulted

Sustained Nicotine Exposure and Hematopoiesis

Figure 7. Sustained exposure to nicotine decreases ␤2 expression on hematopoietic stem/progenitor cells (HSPCs). Enriched HSPCs isolated from pooled bone marrow of nicotine-exposed or control mice (n ⫽ 4 mice per group per experiment) were analyzed for surface expression of CD18 (␤2) using anti-murine CD18 monoclonal antibody 2E6 (4 ␮g/106 cells) by fluorescence-activated cell sorting analysis as described in Materials and Methods, and the results are expressed as the mean ⫾ SD of mean fluorescence intensity. Combined data of two experiments are shown.

in the inhibition of LTBMC [8], sustained in vivo exposure for 21 days to nicotine had no effect on proliferation of BM cells in LTBMC or on the generation of multipotential progenitor cells. It is important to note that there were major differences in the experimental setup of the two studies. For example, in the previous study, BM cells isolated from normal mice were exposed to increasing concentrations of nicotine in in vitro culture rather than in vivo. In the present study, mice were exposed to nicotine s.c. with no subsequent exposure of BM and spleen cells to nicotine once isolated and while in culture. Although the spleen and liver cooperatively contribute to hematopoietic homeostasis along with the BM during ontogeny [30], extramedullary hematopoiesis in adult life is usually associated with pathological conditions. When it occurs, it is seen mainly in tissues active in hemopoiesis in embryonic life, such as the spleen and liver and (as a rare occurrence) in the skin [31], thyroid nodule [32], kidney [33], and even the heart [13]. For instance, previous studies from our laboratory have also demonstrated that constitutive and sustained expression of IL-5 (that is associated with chronic allergic inflammation) results in onset of extramedullary hematopoiesis in the spleen [34]. Likewise, in a recent study, decreased hematopoiesis in BM along with active and increased extramedullary hematopoiesis in the spleen as well as liver was observed in mice with ganciclovir-induced osteoblast deficiency [12]. Thus, it appears that hematopoiesis at extramedullary sites in adult life is often associated with various pathophysiological conditions. Accordingly, the migration of progenitor cells out of the BM and the extramedullary hematopoiesis observed in the spleen due to nicotine exposure as seen in the present study may potentially lead to altered local immune responses in smokers, and these responses in turn may enable the proliferation of progenitor cells at nonhematopoietic sites, leading to various pathological conditions. In support of this, exposure to nicotine in vitro has been reported to cause inhibition of apoptosis in thymocytes and splenocytes of BALB/c mice, causing an imbalance in the immune response and resulting in vascular damage and cancer [7]. Several studies have demonstrated that the increased number of circulating leukocytes,

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including immature or “younger” peripheral polymorphonuclear neutrophils, which are observed in smokers as well as in animals exposed to cigarette smoke, sequester to the lungs [35–37]. These studies have focused on the effects of cigarette smoke on the release of leukocytes from the BM and their recruitment to the lungs, whereas our study focuses on the effects of nicotine, a major constituent of cigarette smoke, on hematopoiesis in the BM as well as at an extramedullary site. Although only modest effects were observed on BM hematopoiesis, our studies suggest that the exposure to nicotine can result in the spleen’s being able to function as a potential site for the generation of inflammatory leukocytes that could consequently be released and eventually recruit to the lungs, causing lung inflammation. Previous studies have shown that direct cellular contact between HSPC and BMEC monolayers through specific adhesion molecules plays a critical role in trafficking, migration, and possibly proliferation of HSPCs and that BMECs support the proliferation and differentiation of hematopoietic progenitors via production of various cytokines [14]. Stem cell motility, directed movement, and homing are defined by a wide variety of adhesion receptors, including L-selectin [38] and integrins (very late antigen [VLA]-4, VLA-5, and lymphocyte function-associated antigen-1), nonintegrin receptors such as the Ig family, as well as other ligands that mediate cell-to-matrix and cell-to-cell interactions [39]. In the present study, enriched HSPCs isolated from the BM of nicotine-exposed mice exhibited diminished rolling on STR-12 BMECs compared with HSPCs from control mice. Compared with control HSPCs, analysis of adhesion molecule expression on these cells demonstrated considerably lower levels of ␤2— but not ␣4 or L-selectin—that are known to participate in trafficking, accounting for the decreased interactions with BMECs. Previous studies have shown that decreased expression of VLA-4 and ␤2 integrins by HSPCs is associated with mobilization and decreased adhesive interactions with BMECs [40]. Importantly, the ability of nicotine to inhibit ␤1 and ␤2 integrin expression has also been reported for fibroblasts (␤1) [41] as well as neutrophils (␤2) [42]. Although we did not observe any change in ␣4 expression on nicotine pellet-exposed HSPCs, previous studies from our laboratory have demonstrated that ␣4 integrin has multiple adhesive states that can be regulated from less adhesive to more adhesive upon activation [43]. We postulate that nicotine may have a similar effect by altering the functional state of ␣4 on HSPCs such that these cells exhibit decreased interaction with the BMECs although the level of ␣4 expression is not altered. SDF-1, constitutively expressed and produced by BM stromal cells and BMECs, induces chemotaxis of both committed and primitive hematopoietic progenitors via interaction with the CXCR4 receptor [44 – 46]. SDF-1-induced migration of CD34⫹ cells across BM endothelium is also mediated by ␤1 and ␤2 integrins [47]. This chemokine is known to play a central role in the repopulation of the BM by circulating CD34⫹ progenitors via integrin-mediated arrest of CD34⫹ cells on BM endothelium [27,

48]. Furthermore, not only does SDF-1 induce chemotaxis of HSPCs, but it has also been shown to increase adhesive interactions of rolling CD34⫹ progenitors by increasing adhesiveness between integrins and their respective endothelial ligands, which is critical for recruitment of HSPCs to the BM [27]. In the present study, enriched HSPCs from BM of nicotine-exposed mice migrated poorly in response to SDF-1, whereas those from control mice exhibited a near twofold increase in migration. Recent in vitro studies have demonstrated that exposure of total BM cells to SDF-1 in the presence of nicotine (10⫺5 M) inhibited the number of total BM, as well as progenitor, cells that underwent migration [49]. Whereas no difference in CXCR expression was observed on enriched HSPCs from nicotine pellet-exposed mice compared with control mice in our study, a considerable decrease in ␤2 expression was discerned. Given the involvement of ␤2 integrins in SDF-1induced migration of HSPCs across BM endothelium [47], it is likely that the inhibition of transmigration of HSPCs from nicotine pellet-exposed mice is due to the nicotine-induced inhibition of ␤2 expression on these cells. Disruption of SDF-1-mediated recruitment is likely to result in cell mobilization and may participate in extramedullary infiltration. These studies further support our thinking that sustained exposure to nicotine results in adhesion molecule-dependent altered migration and trafficking of progenitor cells. Although recruitment of progenitor cells to the spleen in nicotine-exposed mice is likely to be dependent on altered expression of one or more cell surface adhesion molecules on progenitor as well as vascular endothelial cells, it is also likely that the generation of CFU-F (fibroblast progenitors) in the spleen as previously reported during certain inflammatory conditions [34] may induce the generation of a hematopoietic niche for the migrating BM progenitor cells to seed and proliferate. Overall, whereas in vivo exposure to nicotine did not appear to have an effect on the generation of multipotential progenitor cells, a significant inhibition of lineage-specific progenitors (CFU-Eos) was observed in the BM. However, a more distinct effect was observed on the spleen, where a marked increase in generation of multipotential as well as lineage-specific progenitors was observed. Hematopoiesis in the spleen could potentially be due to decreased interaction (rolling and transendothelial migration) of HSPCs with BMECs due to decreased ␤2 expression facilitating their ability to traffic to other organs, causing altered homing of these cells and leading to their seeding and proliferation at extramedullary sites.

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ACKNOWLEDGMENTS This study was supported by California Tobacco-related Disease Research program Grant 10 RT-0171.

DISCLOSURES The authors indicate no potential conflicts of interest.

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