Sustained Exposure to Nicotine Leads to Extramedullary Hematopoiesis in the Sple - 《stemcells》干细胞学杂志干细胞之家



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作者：Terlika S. Pandit, Lyudmila Sikora, Girija Muralidhar, Savita P. Rao, P. Sriramarao作者单位：Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California, USA
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      【摘要】
   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 -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 &szlig;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.
      【关键词】 Nicotine Bone marrow Spleen Hematopoiesis stem progenitor cells Extramedullary hematopoiesis2 y: G3 f9 L! s8 F, |+ ?
   INTRODUCTION/ C; q0 [% G  K4 y% c
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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 .: }! U6 {8 j2 z

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 .

Here, we have extended our previous in vitro studies .

MATERIALS AND METHODS
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Endothelial and Stromal Cell Lines, T# N/ K" f. i9 o

S17, a stromal cell line, was kindly provided by Dr. Kenneth Dorshkind (Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles) . Both cell lines were cultured in RPMI (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal calf serum (FCS).. x, l3 z/ O$ a2 g  j# Z
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Exposure to Nicotine
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Female BALB/c mice (7¨C8 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 . Age- and gender-matched 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.
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Colony-Forming Unit Assay
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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-µm 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 ), BM and spleen cell suspensions were plated (1 x 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 x 104 cells per milliliter, 5 x 105 cells per milliliter, and 5 x 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¡ãC in a humidified atmosphere at 5% CO2 for 10¨C12 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_M.pdf).+ D+ N& b4 J4 t7 A) F

LTBMC Assay% b5 ^0 Q0 D+ P5 G) W

Freshly isolated BM cells were cultured in six-well plates in MyeloCult M5300 medium (4 ml/well at 106 cells per ml) containing 10¨C6 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).: y: I+ K8 k" {( ?

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 x 104/ml) and added to the plates (100 µl/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¡ãC for 2 weeks, and the culture medium was replenished with fresh medium (100 µl/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.. w. |3 k; M' M8 i% _

Enrichment of Murine HSPCs2 s- j( n9 q- C) R& ^1 d" k# x& ]
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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% ¡À 7.68% of the cells were positive for CD34 compared with 4.18% ¡À 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).  D3 p9 W& j8 B4 k7 F

Migration Assay" z# G+ j- Y# l% k+ {

This assay was performed using 24-well Transwell clusters equipped with 5-µm 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 x 105 in 200 µl of Dulbecco's modified Eagle's medium ) isolated from pooled BM of nicotine pellet-exposed or control mice. DMEM containing stromal cell-derived factor (SDF)-1 (50 nM), a chemoattractant produced by the BM stroma, was added to the bottom well. The plates were incubated at 37¡ãC 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.
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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 . Briefly, STR-12 cells were cultured to confluency on cover slips and positioned at the bottom of a parallel plate flow chamber (100-µm 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 x 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.2 ^$ Y; W% k% z! B7 r
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Flow Cytometry

Cell surface expression of CD44, CD49d (4), CD18 (ß2), 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 , respectively, followed by appropriate FITC-labeled secondary antibodies (BD PharMingen, San Diego, http://www.bdbiosciences.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 µg/106 cells. Incubations were carried out at 4¡ãC 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).3 o% ^$ d# u2 q
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Statistical Analysis0 x' V4 E% M' k/ E
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Results are expressed as the mean ¡À SE. Statistical significance was determined using the Student's t test.; [4 Z% j* c6 X2 t
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RESULTS  C6 E0 J2 I* e$ R
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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 .
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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¡ãC and 5% CO2 in MyeloCult M3500 medium containing 10¨C6 M hydrocortisone with weekly feedings. (A): Nonadherent cells withdrawn from the culture medium during weekly feedings were counted and expressed as mean ¡À 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 ¡À SE. Combined data of three experiments (n = 3¨C5 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

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 ¡À SE. Combined data of three experiments (n = 3¨C5 mice per group per experiment) are shown. *p   B' E8 g. J+ U. t9 e% ^0 \6 D( l
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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 ¡À SE. Combined data of three experiments (n = 3¨C5 mice per group per experiment) are shown. *p

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.
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Effect of Stromal Cell Microenvironment on Colony Formation by Nicotine-Exposed BM and Spleen0 H: A. S/ r+ r4 x, O! M9 M

SDFs are known to support the proliferation and generation of HSPCs . 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 pellet-exposed 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.
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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 ¡À SE of the number of wells with colonies. Combined data of three experiments (n = 4¨C6 mice per group per experiment) are shown. *p 7 o: h  l2 L: o: P8 n( H- O* V/ x5 v
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Exposure to Nicotine Inhibits Enriched HSPC Rolling and Migration Across BMECs In Vitro
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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 chemokine (SDF-1)-mediated recruitment/homing of HSPCs to the BM . 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 inhibits the ability of HSPCs to interact with BMECs (rolling and transmigration).
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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 x 104 cells per milliliter) (n = 3¨C5 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 ¡À 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-µm filters coated with confluent layers of STR-12 cells. Plates were incubated at 37¡ãC. 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 ¡À SE of percentage of migration. Combined data of two experiments are shown. Abbreviation: SDF-1, stromal cell-derived factor-1.. a/ L5 n; _: i3 q" C4 Z6 K0 t6 s

Exposure to Nicotine Decreases ß2 Expression on HSPCs
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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, ß2, 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 ß2 on these cells from nicotine pellet-exposed mice was considerably decreased (approximately 66%) compared with cells from control mice (Fig. 7).

Figure 7. Sustained exposure to nicotine decreases ß2 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 (ß2) using anti-murine CD18 monoclonal antibody 2E6 (4 µg/106 cells) by fluorescence-activated cell sorting analysis as described in Materials and Methods, and the results are expressed as the mean ¡À SD of mean fluorescence intensity. Combined data of two experiments are shown.) x$ E( v" b8 C  A7 K

DISCUSSION
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The adverse effects of cigarette smoke and nicotine are well-known. We have previously studied the effect of nicotine on hematopoiesis in vitro , 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 . 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 . 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.- b) O' v0 Z$ E7 e4 F' g$ S

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 , it is likely that the inhibition of transmigration of HSPCs from nicotine pellet-exposed mice is due to the nicotine-induced inhibition of ß2 expression on these cells. Disruption of SDF-1-mediated recruitment is likely to result in cell mobilization and may participate in extramedullary infiltration.
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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  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 ß2 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.

DISCLOSURES! Y# n* @$ }" W4 i) H

The authors indicate no potential conflicts of interest.  {; A) f/ L& N0 q& m8 L
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ACKNOWLEDGMENTS

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