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HEMATOPOIESIS
From the Division of Vascular Biology, La Jolla
Institute for Molecular Medicine, La Jolla, CA; Institute for Clinical
Immunology, Novosibirsk, Russia; and the National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
MD.
This study demonstrates that in vivo exposure to cigarette smoke
(CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs)
with nicotine, a major constituent of CS, result in inhibition of
hematopoiesis. Nicotine treatment significantly delayed the onset of
hematopoietic foci and reduced their size. Furthermore, the number of
long-term culture-initiating cells (LTC-ICs) within an adherent layer
of LTBMCs was significantly reduced in cultures treated with nicotine.
Although the production of nonadherent mature cells and their
progenitors in nicotine-treated LTBMCs was inhibited, this treatment
failed to influence the proliferation of committed hematopoietic
progenitors when added into methylcellulose cultures. Bone marrow
stromal cells are an integral component of the hematopoietic
microenvironment and play a critical role in the regulation of
hematopoietic stem cell proliferation and self-renewal. Exposure to
nicotine decreased CD44 surface expression on primary bone
marrow-derived fibroblastlike stromal cells and MS-5 stromal cell
line, but not on hematopoietic cells. In addition, mainstream CS
altered the trafficking of hematopoietic stem/progenitor cells
(HSPC) in vivo. Exposure of mice to CS resulted in the
inhibition of HSPC homing into bone marrow. Nicotine and cotinine
treatment resulted in reduction of CD44 surface expression on
lung microvascular endothelial cell line (LEISVO) and bone
marrow-derived (STR-12) endothelial cell line. Nicotine treatment
increased E-selectin expression on LEISVO cells, but not on STR-12
cells. These findings demonstrate that nicotine can modulate
hematopoiesis by affecting the functions of the
hematopoiesis-supportive stromal microenvironment, resulting in the
inhibition of bone marrow seeding by LTC-ICs and interfering with stem
cell homing by targeting microvascular endothelial cells.
(Blood. 2001;98:303-312) Proliferation, differentiation, and self-renewal of
hematopoietic bone marrow cells are strictly regulated and complex
processes. They are controlled by a number of soluble factors,
including cytokines, interleukins, and chemokines, as well as by
extracellular matrix (ECM) and adhesion molecules. The cellular
compartment of the bone marrow is represented by a heterogeneous
population of mature cells, including hematopoietic progenitor cells at
different stages of differentiation and stromal cells. Stromal cells
are an integral part of the regulatory network within the bone marrow microenvironment and play a critical role in hematopoiesis. Stromal cells elaborate various positive and negative regulators as well as ECM
and adhesion molecules that contribute to the formation of "stem cell
niches." These stem cell niches regulate proliferation, differentiation, and self-renewal of hematopoietic stem
cells.1-4 Stromal cells are also a major component of the
adherent layer of long-term bone marrow cultures (LTBMCs). Several bone
marrow-derived stromal cell lines substituting primary stromal cells
in in vitro cultures have been described.5-7 These cell
lines, as well as primary stromal cells, give rise to stem cell
regulatory signals via production of cytokines, ECM, and expression of
adhesion molecules.
Stromal cells are sensitive to extrinsic regulatory factors. For
example, steroid hormones and cytokines8-11 can alter the production of ECM and cytokines by stromal cells. The expression pattern of adhesion molecules and their ligand-binding activity can be
influenced by exposure to different chemicals and cytokines. In
addition, platelet-derived growth factor (PDGF), fibroblast growth
factor (FGF), and macrophage colony-stimulating factor (M-CSF)
regulate stromal cell function by an autocrine
mechanism.12-14 The function of stromal cells is also
dramatically altered in a number of hematologic
disorders.15-19 These observations imply that the
hematopoietic microenvironment, as a target for extrinsic factors, may
have a significant effect on hematopoiesis during both normal and
pathologic conditions.
Exposure to certain pathophysiologic factors, including cigarette smoke
(CS) and tobacco smoke (TS) and their byproducts, could lead to an
imbalance in hematopoietic homeostasis.20,21 Exposure to
TS results in the reduction of functionally active immunocompetent
cells.22 Recent studies have revealed that the hematopoietic system is one of the targets of TS. A number of constituents of TS that are distributed throughout the bloodstream directly affect target cells. For example, bone marrow-derived macrophages incubated with the gas phase of CS demonstrated a dose-dependent decrease of tissue-type (thrombin-independent) transglutaminase activity, although viability or adherence of these
cells was not affected.23 Moreover, exposure to components of CS such as acrolein and acetaldehyde inhibits proliferation and
migration of fibroblasts,24,25 a part of the bone marrow stromal microenvironment. Furthermore, CS inhibits the production of
fibronectin and collagen by fibroblasts.25-27 These ECM
molecules play an important role in hematopoietic stem/progenitor cells (HSPC) behavior.28 These studies suggest that bone marrow
cells are a target for components of TS, including nicotine.
In the present study, the effects of CS, nicotine, and its metabolite
cotinine on LTBMCs and their influence on HSPC homing were determined.
Our findings suggest that in addition to other deleterious effects of
CS and nicotine on the immune and vascular systems, the development of
hematopoietic tissue in individuals exposed to TS could also be
affected through altered function of the hematopoietic microenvironment.
Endothelial and stromal cell lines
Flow cytometry
HSPC purification BALB/c mice 4 to 8 weeks old were used for all experiments. The contents of femurs and tibias were flushed out from the bone with PBS supplemented with 5% FCS with a needle (21G) attached to a 1-mL syringe. Cells were kept on ice until use.30 HSPC were isolated according to the manufacturer's instructions (StemCell Technology, Vancouver, BC, Canada). Briefly, bone marrow cells were incubated with a cocktail containing lineage-specific antibodies for 15 minutes at 4°C. After washing, bone marrow cells were incubated with antibiotin tetrameric antibody complexes for 15 minutes, followed by incubation with magnetic colloid for 15 minutes. Thereafter, cells were applied on a magnetic column. Recovered cells were collected, washed, and assayed.Methylcellulose assay Bone marrow cells (2 × 104/mL of plating mixture) were mixed gently with semisolid methylcellulose medium supplemented with 10% FCS, 1% BSA, L-glutamine, and 2-mercaptoethanol (StemCell Technology). Corresponding lineage-specific factors, including interleukin (IL)-7 (1 ng/mL), IL-5 (10 ng/mL), granulocyte macrophage colony-stimulating factor (GM-CSF; 10 ng/mL), or M-CSF, were added to induce growth of B-lymphoid (CFU-B), eosinophil (CFU-eos), granulocyte-macrophage, and macrophage colonies, respectively. To induce the growth of early erythroid cell progenitors, IL-3 and erythropoietin (1 U/mL) were added. The conditioned medium from cell line WEHI-3B was used as a source of IL-3 (15% vol/vol). The conditioned medium from cell line L929 was used as a source of M-CSF (15% vol/vol).30-32 The cultures were incubated in a humidified incubator with 5% CO2 at 37°C for 7 to 14 days. Colonies (a group of more than 50 cells) were scored under the microscope.Long-term bone marrow cultures Bone marrow cells (1 × 106 cells/mL) were cultured in 6-well or 24-well plates in DMEM supplemented with 20% horse serum (StemCell Technology) and hydrocortisone (10 6
M; Sigma) at 33°C in a humidified atmosphere at 5% CO2.
Cultures were fed weekly by replacing half of the culture medium.
Nonadherent cells were collected, counted, and used for the CFU
assay.30
Long-term culture-initiating cell assay The assay was performed according to the manufacturer's instructions (StemCell Technology). Briefly, cell populations to be examined were plated in DMEM supplemented with 20% horse serum and hydrocortisone (106 M) in limiting dilution into 96-well
plates containing the stromal cell line S17. Cultures were fed weekly,
and the numbers of wells containing colonies were evaluated after 14 days of culture.33
Bone marrow-derived macrophage cultures Bone marrow-derived macrophages were cultured according to a protocol described previously.34 Bone marrow cells at a concentration of 1 × 106 cells/mL were cultivated in DMEM containing 2 mM glutamine, 0.37% (wt/vol) NaHCO3, 10% (vol/vol) heat-inactivated FCS, and 10% (vol/vol) L929 cell-conditioned medium as a source of M-CSF. Cultures were maintained at 37°C in a 5% CO2 atmosphere for 8 days. Ninety-nine percent of the cells then on the culture dish were phagocytic for latex particles.Primary bone marrow-derived fibroblastlike stromal cell cultures Bone marrow cells (106/mL) were incubated in DMEM supplemented with 20% horse serum (StemCell Technology) and hydrocortisone (106 M; Sigma) at 33°C in a humidified
atmosphere at 5% CO2 for 24 hours. Thereafter, nonadherent
cells were washed out and the remaining cells were further cultured
under the same conditions. Each week, cultures were passaged into the
new culture flasks with fresh media. After 28 days, monolayers
containing stromal cells were used for experiments.
Nicotine and cotinine treatment Nicotine and cotinine (both from Sigma, St Louis, MO; dissolved in ethanol, 101 M stock) were diluted in PBS, sterile
filtered, and used immediately in a light-protected cell culture hood.
A total of 75% to 90% confluent cells were treated with varying
concentrations of nicotine or cotinine (104 M to
108 M) and incubated at 37°C for 5 minutes, 30 minutes,
1 hour, 2 hours, and 4 hours. Thereafter, the cells were detached using Enzyme Free Cell Dissociation Solution (Speciality Media, Phillipsburg, NJ) and assayed for cell-surface molecule expression by flow cytometry. For LTBMC and CFU assays, nicotine or cotinine was diluted in cell
culture medium and sterile filtered before use.
CS exposure, repopulation assay, and spleen CFU assay To determine the effect of CS on hematopoiesis, we placed BALB/c mice (n = 10) in a hermetically closed chamber filled with CS (n = 10 cigarettes). Animals (n = 10) were killed after 21 days of intermittent exposure to CS, and bone marrow cells were harvested, calculated, and assayed for hematopoietic progenitors in methylcellulose cultures. For the assay of marrow-repopulating ability, mice were lethally irradiated with 8 Gy, and after 24 hours reconstituted with 1 × 106 bone marrow cells injected intravenously.30 Animals were killed after 14 days, and bone marrow cells were collected from a femur of each mouse, calculated, and transplanted into lethally irradiated recipients (4 × 104/mouse) to determine the number of CFU in spleens after 12 days (CFUs-12).35 Spleens were fixed in Tellesnicky's solution, and colonies were counted after several hours of fixation.
CS decreases the number of committed progenitors in the bone marrow To determine the effect of CS on hematopoiesis, we exposed BALB/c mice (n = 10) to CS (n = 10 cigarettes) in a hermetically closed chamber. Compared with control animals, exposure to CS had no impact on the total numbers of bone marrow cells (25.8 ± 3.1 × 106/femur in control mice and 22.5 ± 4.7 × 106/femur in CS-exposed mice). However, in mice exposed to CS, the total number of myeloid progenitors in the femur was decreased by 2.1-fold (from 291.5 ± 46.4 in control mice to 135.0 ± 27 in CS-exposed mice; P < .01) (Figure 1).
Next, to determine the direct effect of nicotine on colony-forming
ability of committed hematopoietic progenitors, we cultured freshly
isolated bone marrow cells in methylcellulose semisolid cultures
supplemented with lineage-specific factors with varying concentrations
of nicotine and its metabolite cotinine. Recent studies have
demonstrated the nonspecific and high levels of intracellular uptake of
nicotine by fibroblasts.36 Because the intracellular uptake of nicotine is likely to vary depending upon the extent of
exposure,36 we tested a wide range of concentrations of
nicotine and cotinine on colony-forming ability of hematopoietic
progenitor cells. Treatment of cultures with nicotine and cotinine at
concentrations of 10
Nicotine inhibits the formation of an adherent layer in LTBMCs We next determined whether nicotine or cotinine might inhibit hematopoiesis by affecting the hematopoietic microenvironment. To examine this, we added physiologic concentrations of nicotine or cotinine (105 M to 108 M) to LTBMCs from
the first day of culture. Treatment of the cultures with nicotine
resulted in inhibition of adherent-layer formation in a dose-dependent
manner (Figure 3). Although the formation
of a confluent adherent layer was not significantly affected in LTBMCs
treated with nicotine or cotinine, the formation of loci of active
hematopoiesis in LTBMCs, "cobblestone areas," in cultures treated
with nicotine (105 to 107 M) was inhibited
(Figure 3). These areas were found to be smaller in cultures treated
with nicotine than in control cultures or those treated with cotinine
(data not shown). In support of this finding, we observed that neither
nicotine nor cotinine affected the adhesion of fibroblast precursors
(CFU-F) to culture dishes (data not shown).
Nicotine inhibits nonadherent cell production in LTBMCs Next, the influence of nicotine and cotinine on hematopoiesis in LTBMCs was examined. Nonadherent cells from LTBMCs were harvested weekly during feedings and counted. No differences in the nonadherent cell numbers were observed in cultures treated with 104
to 106 M nicotine compared with controls after 1 week in
culture (P > .1). At weeks 2 and 3, the numbers of
nonadherent cells in short-term cultures treated with varying
concentrations of nicotine were significantly lower than in controls
(P < .01). The inhibition of nonadherent cell production
was dose dependent (data not shown). Long-term administration of
105 M nicotine resulted in significant inhibition of
nonadherent cell production during the entire period of culture (Figure
4A). Interestingly, addition of cotinine
at concentrations from 105 to 108 M did not
affect hematopoiesis in LTBMCs (data not shown). Because the population
of nonadherent cells in LTBMCs is heterogeneous and contains
hematopoietic progenitors and mature cells, it is conceivable that the
decreased number of total cells in LTBMCs could be due to the low
number of progenitor cells generated in LTBMCs. Therefore, cells
harvested during feedings were assayed for a number of myeloid
progenitors. Although cotinine failed to affect the production of
myeloid progenitors in LTBMCs (data not shown), treatment with nicotine
resulted in a significant reduction in the numbers of myeloid
progenitors (Figure 4B). This observation could be explained by low
numbers of long-term culture-initiating cells (LTC-ICs) in
nicotine-treated cultures. To examine this, we harvested adherent
layers of LTBMCs treated with nicotine or cotinine and assayed them for
LTC-ICs. We found that the numbers of LTC-ICs in nicotine-treated
cultures were 2.5-fold lower than in control cultures or cultures
treated with cotinine (Figure 4C).
We further investigated whether nicotine affects LTBMCs in steady
state. Cultures were initially set up in the absence of nicotine for 3 weeks. After 5 weeks, we observed a well-formed adherent layer with
cobblestone areas. Before the addition of nicotine (10 These studies suggest that nicotine may affect the formation of hematopoietic "niches" within the adherent layer of LTBMCs, and this in turn leads to a reduced number of HSPC seeded. Effect of nicotine on adhesion molecule expression by stromal cells We next examined whether nicotine affects the expression of adhesion molecules by stromal cells because several of these cell-surface receptors, including CD44, 4,  2, 7, and VCAM, may
participate in the formation of hematopoietic niches.3,4
We incubated freshly isolated bone marrow cells with varying
concentrations of nicotine and cotinine and then evaluated cell-surface
expression of CD44, 4, 2, 7, and VCAM by flow cytometry. We
observed that within 4 gated populations of bone marrow cells, only a
small population (18%) of large and granulated cells (population
number 3) demonstrated a decrease in the intensity of CD44 expression after treatment with nicotine, but not cotinine. Interestingly, the
expression levels of 4, 2, 7, and VCAM were not affected by
either nicotine or cotinine (Table 1).
Because these cells might constitute the bone marrow hematopoietic
microenvironment, we treated steady-state LTBMCs with nicotine or
cotinine, harvested cells of the adherent layer, and examined adhesion
molecule expression by FACS. Our findings consistently demonstrated a
significant reduction in the cell-surface expression of CD44 on a
population of large and granulated cells treated with nicotine, but not
cotinine. These cells could be either bone marrow macrophages or bone
marrow fibroblastlike stromal cells, a major cell population forming the bone marrow microenvironment. Therefore, we further developed primary cultures of bone marrow-derived macrophages and treated these
cells with varying concentrations of nicotine and cotinine. This
treatment did not affect the expression patterns of any of the adhesion
molecules we examined. We next established primary cultures of bone
marrow-derived fibroblastlike stromal cells. These cells demonstrated
a significant reduction (2.3-fold) in the intensity of CD44
cell-surface expression after treatment with nicotine, but not
cotinine. However, expression of other adhesion molecules remained
unaffected. Finally, the MS-5 stromal cell line was used to study the
effects of nicotine because it supports the growth of hematopoietic
progenitors in vitro.7 Cell-surface expression levels of
CD44, 4, 2, 7, and VCAM on harvested stromal cell line MS-5
after treatment with nicotine and cotinine were examined by FACS
analysis. Exposure to nicotine resulted in a 3.5-fold decrease in the
intensity of CD44 expression and a 3.5-fold increase in 7-integrin
expression. In contrast, the expression of other adhesion molecules
remained unaffected by this treatment.
Nicotine inhibits HSPC homing and platelet recovery To investigate the effect of nicotine on HSPC homing, we reconstituted lethally irradiated mice with bone marrow cells and exposed them to CS for 5 hours daily for 14 days. The numbers of leukocytes and erythrocytes in peripheral blood of mice exposed to CS were not significantly different from those in the control group. However, the reconstitution of platelets was significantly delayed. One week after transplantation, the number of platelets in the control group was 576 ± 82/mL of blood, whereas in the CS group, it reached only 363 ± 65/mL of blood. By week 2, the number of platelets in the CS group (356 ± 30/mL of blood) was still lower than in the control group (639 ± 82/mL of blood).We next examined whether CS affects the trafficking of HSPC into the
bone marrow. We especially assessed whether hematopoietic precursors
from mice exposed to CS would reconstitute bone marrow with efficiency
comparable to that of hematopoietic precursors from untreated mice. The
number of CFUs-12 in the bone marrow of mice exposed to CS was 6-fold
lower than in the control animals (P < .01) (Figure
5). These findings suggest that CS
significantly affects HSPC homing.
Nicotine decreases CD44 expression on endothelial cells Extravasation of HSPC is a complex process involving a number of adhesion molecules. CD44 adhesion molecule is expressed by both endothelial cells and HSPC and has been shown to play a critical role in HSPC homing.37,38 We therefore examined the effect of nicotine or cotinine treatment on CD44 expression on both endothelial and progenitor cells by flow cytometry. CD44 expression on enriched populations of progenitor cells was not altered by nicotine or cotinine treatment (data not shown). Similarly, murine or human endothelial cells were treated with nicotine and harvested, and the cell-surface expression levels of CD44 along with VCAM,7, 4, and P- and
L-selectins were determined. Treatment of HUVECs and the murine bone
marrow-derived endothelial cell line STR-12 with nicotine or cotinine
resulted in a significant reduction of CD44 expression (Table
2). Treatment of HUVECs with nicotine and
cotinine resulted in 2-fold and 1.8-fold decreases in CD44 expression,
respectively. We also observed a decrease (1.5-fold) in the intensity
of CD44 expression on STR-12 endothelial cells after exposure to
nicotine and cotinine. In contrast, both nicotine and cotinine failed
to alter the surface expression of VCAM, 7, 4, and P- and
L-selectins on HUVECs and STR-12. Interestingly, a comparison of
E-selectin expression on lung endothelial cell line (LEISVO) and STR-12
cells revealed that a 4-hour exposure to nicotine induced E-selectin
expression on LEISVO, but not on STR-12 (Figure
6). However, no significant change in
cell-surface VCAM expression on LEISVO and STR-12 was observed after
exposure to nicotine for varying intervals.
Tobacco use, especially smoking, has been associated with an increased risk of cancer and pulmonary and cardiovascular diseases. Exposure to TS is associated with altered immune responses, including lymphocyte proliferation and neutrophil and macrophage functions, leading to tobacco-related immunodeficiency. Moreover, TS may result in imbalance of the hematopoietic system, such as changes in the erythrocyte-leukocyte ratio and the composition of mature leukocytes in the peripheral blood.39 Most of the undesirable effects of TS have been associated with nicotine. For example, exposure of various types of cells to nicotine results in a decrease in DNA synthesis, inhibition of cell proliferation, alteration of cytokine and ECM molecule production, and changes in adhesion molecule expression.40-47 Although these studies indicate the negative effects of nicotine on various tissues, the potential deleterious effects of TS or nicotine on bone marrow hematopoiesis and HSPC trafficking have not been investigated. We demonstrate for the first time that exposure to nicotine or CS results in inhibition of hematopoiesis in LTBMCs as well as homing of HSPC into the bone marrow. The results of our study indicate that the numbers of hematopoietic
progenitors in the bone marrow of mice exposed to CS are significantly
lower than those observed in control mice. To examine the cellular
mechanisms involved in this phenomenon, we treated LTBMCs with varying
concentrations of nicotine and cotinine. The concentration of nicotine
used in the present study is comparable to the physiologic
concentration of nicotine observed in the serum of
smokers.48 Nicotine either specifically binds to
cell-surface receptors49,50 or undergoes nonspecific
uptake, leading to high levels of intracellular buildup.36
Therefore, the concentration of nicotine required to functionally
modulate cellular responses is likely to vary depending on the cell
type, cell-surface or intracellular binding, tissue of origin, and
duration of exposure. We have therefore examined the effect of nicotine
exposure on hematopoiesis in LTBMCs at physiologic concentrations
varying from 10 the adherent layer and affected the formation of cobblestone areas. As a consequence of this, we observed decreased numbers of hematopoietic progenitors and mature cells in nicotine-treated cultures. Interestingly, nicotine treatment of LTBMCs in steady state did not affect cobblestone areas. These findings support the idea that nicotine affects initial stromal cell-stem cell interactions and HSPC seeding of the stromal layer. This results in a decreased number of stem cells that could colonize the adherent layer and produce progeny. In turn, this process could lead to secondary immunosuppression in smokers. We anticipated that stromal cells are the targets for intracellular
uptake of nicotine. The interaction between HSPC and stromal cells is
dependent upon the engagement of several cell-surface adhesion
molecules.51-56 Therefore, we further postulated that stromal cells treated with nicotine may fail to support HSPC by changing the profile of adhesion molecules expressed by stromal cells.
CD44 is one of the adhesion molecules that plays a critical role in
normal hematopoiesis, and anti-CD44 mAbs significantly affect both the
formation of cobblestone areas within the adherent layer of LTBMCs and
the production of mature cells.37,57 Furthermore, we have
previously demonstrated that CD44 contributes to the adhesion of
myeloid progenitor cells to the bone marrow stromal cell line MS-5.58 Here we show that the expression level of CD44
adhesion molecule was 3.5-fold down-regulated on bone marrow-derived
stromal cell line MS-5 after exposure to nicotine, but not cotinine,
which could reflect events taking place in the primary stroma. Indeed, exposure of primary bone marrow-derived fibroblastlike stromal cells,
but not bone marrow macrophages, to nicotine resulted in a 2.3-fold
reduction of cell-surface CD44. Because CD44 is required for
interactions between stromal cells and HSPC, the nicotine-induced decrease of CD44 expression could lead to a decreased number of HSPC
seeded on the stroma, and consequently, reduced hematopoietic activity.
In addition to the effects on CD44 expression, treatment of MS-5 cells
with nicotine results in a 3.5-fold increase in Homing of HSPC into the bone marrow is a complex process mediated by various adhesion molecules expressed on HSPC and on bone marrow endothelial cells. Because nicotine inhibits the repopulation of irradiated bone marrow by intravenously injected HSPC, we speculated that nicotine may target bone marrow endothelial cells. This, in turn, may result in alteration of HSPC extravasation and homing. We therefore examined the effects of nicotine and its major metabolite cotinine on bone marrow endothelial cells (STR-12) in comparison with lung microvascular endothelial cells (LEISVO). Exposure of STR-12 and LEISVO to nicotine resulted in a decrease of cell-surface CD44 expression. Interestingly, in sharp contrast to its lack of any effect on hematopoiesis and expression of adhesion molecules by bone marrow stromal cells, cotinine was observed to inhibit CD44 expression by HUVECs as well as bone marrow endothelial cells. Previous studies including ours37,38 have demonstrated that blockade of cell-surface CD44 expression on HSPC with anti-CD44 mAb results in the inhibition of HSPC homing into the bone marrow. The molecular basis of this phenomenon is still not well understood. Furthermore, the endothelial cell ligand for CD44 expressed on HSPC is not known. Because CD44-CD44 homotypic interactions could be involved in cell-cell adhesion,64 it is conceivable that CD44 expressed on the endothelium may serve as a ligand for CD44 expressed on HSPC. Therefore, reduction of CD44 expression on endothelial cells would lead to a decreased adhesion of HSPC to the endothelium and, as a consequence, inhibition of HSPC homing. In contrast to the reduced CD44 expression, treatment with nicotine for 4 hours resulted in up-regulation of E-selectin on LEISVO, but not on STR-12 cells, suggesting an organ-specific targeting effect of nicotine. Although previous studies have demonstrated that treatment of HUVECs with nicotine containing CS condensate resulted in increased expression of E-selectin, which is associated with increased monocyte adhesion in vitro,45 the effect of nicotine-induced E-selectin expression on HSPC homing is not known. Our observations suggest that E-selectin induction is restricted to lung endothelium and not bone marrow-derived endothelial cells, and, as such, may not influence HSPC homing to the bone marrow. Up-regulation of E-selectin on endothelial cells and Overall, these studies identify for the first time the differential cellular effects of nicotine, cotinine, and CS exposure in vivo and in vitro and demonstrate that long-term exposure to nicotine affects both hematopoiesis and HSPC homing, whereas cotinine is likely to inhibit only cell trafficking.
Submitted October 3, 2000; accepted March 14, 2001.
Supported by National Institutes of Health grant AI35796 and Tobacco Research Disease Related Program (TRDRP) grant 7RT-0197 to P.S.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: P. Sriramarao, Division of Vascular Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Dr, San Diego, CA 92121; e-mail: rao{at}ljimm.org.
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Top
Abstract
Introduction
) or with 10
) and the fifth week
of culture (
). (A) Nonadherent cells were harvested weekly during
feedings of the cultures (n = 4) and calculated, and numbers were
expressed as mean ± SD. (B) Nonadherent cells harvested
from LTBMCs treated with nicotine were plated at a concentration of
104 cells/mL in methylcellulose cultures (n = 4)
supplemented with GM-CSF (10 ng/mL). After 7 days in culture, the
number of colonies was counted, recalculated for a total number of
cells for each culture, and expressed as mean ± SD. (C)
LTBMCs were cultured with nicotine or cotinine for 3 weeks.
Thereafter, the adherent layer of LTBMCs was harvested, calculated, and
tested for LTC-IC content in a 14-day limiting-dilution assay on the
S17 cell line. Data shown are means ± SD of the LTC-IC content of
2 experiments.
