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TRANSPLANTATION
Reduced stem cell mobilization in mice receiving antibiotic modulation of the
intestinal flora: involvement of endotoxins as cofactors in mobilization
Gerjo A. Velders, Ronald van Os, Henny Hagoort, Perry Verzaal, Henri F. L. Guiot, Ivan J. D. Lindley, Roel Willemze,
Ghislain Opdenakker, and Willem E. Fibbe
Since endotoxins are potent inducers of
stem cell mobilization, we hypothesized
that their presence in the gut may play a
role in cytokine-induced mobilization. To
address this possibility we added ciprofloxacin and polymyxin B to the drinking
water of Balb/c mice mobilized with either
interleukin-8 (IL-8), granulocyte colonystimulating factor (G-CSF), or flt3 ligand
(FL). The yield of colony-forming units
(CFUs) was significantly reduced in all
mice treated with these antibiotics when
compared with controls (IL-8: 192 ⴞ 61 vs
290 ⴞ 64, P < .05; G-CSF: 1925 ⴞ 1216
vs 3371 ⴞ 1214, P < .05; FL: 562 ⴞ 213 vs
1068 ⴞ 528, P < .05). Treatment with cip-
rofloxacin eliminated only aerobic Gramnegative bacteria from the feces without
effect on mobilization. Polymyxin B treatment did not result in decontamination
but significantly reduced the number of
mobilized hematopoietic progenitor cells
(HPCs) most likely due to the endotoxin
binding capacity of polymyxin B. More
than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of
the anaerobic flora by metronidazol led to
a significantly reduced number of mobilized HPCs when compared with controls
(IL-8: 55 ⴞ 66 vs 538 ⴞ 216, P < .05).
Germ-free OF1 mice showed a significantly reduced mobilization compared
with their wild-type controls (IL-8 controls: 378 ⴞ 182, IL-8 germ free: 157 ⴞ 53,
P < .05). Finally, we performed reconstitution experiments adding Escherichia coli–
derived endotoxins to the drinking water
of decontaminated mice. This resulted in
partial restoration of the IL-8–induced mobilization (67 ⴞ 28 vs 190 ⴞ 98.1, P <.01).
Our results indicate that endotoxins serve
as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the
yield of cytokine-induced mobilization.
(Blood. 2004;103:340-346)
© 2004 by The American Society of Hematology
Introduction
To reduce the risk of infections in neutropenic patients a
combination of protective isolation and gut decontamination by
oral administration of antibiotics is often used.1-4 Since it has
been shown that selective elimination of the major potentially
pathogenic microorganisms from the digestive tract is as effective
or even superior to total decontamination in preventing infections in neutropenic patients,5-8 selective gut decontamination9
or partial antibiotic decontamination10 mainly directed against
aerobic Gram-negative rods and fungi is usually applied. The
various antibiotics used for selective decontamination can be
divided into 2 main groups, those that are absorbed11-13 after oral
administration (eg, cotrimoxazole, quinolones) and those that
are not absorbed2-4 after oral administration (eg, polymyxin,
colistin, aminoglycosides).
Mobilized hematopoietic progenitor cells (HPCs) are increasingly used for hematopoietic recovery instead of bone marrow to
allow rapid hematopoietic recovery after autologous and allogeneic
transplantation.14-18 An increasing number of different growth
factors and chemokines such as granulocyte colony-stimulating
factor (G-CSF),19,20 granulocyte-macrophage colony-stimulating
factor (GM-CSF),19,21 stem cell factor (SCF),22 flt3 ligand (FL),23
interleukin-1 (IL-1),24 IL-3,25 IL-8,26 and thrombopoietin27 when
either administered alone or in combination28 are capable of
mobilizing peripheral blood progenitor cells. In the clinical setting,
the growth factor G-CSF has become the standard for autologous as
well as allogeneic peripheral blood stem cell transplantation.29-31
Although patients receiving selective gut decontamination or
systemic antibiotic treatment while concurrently being treated with
G-CSF are able to mobilize peripheral blood progenitor cells, little
is known of the effect of antibiotics on the efficacy of mobilization.
Endotoxins are ubiquitously present and constitute an important
component of the normal intestinal flora and are known to induce
HPC mobilization.32-34 We therefore hypothesized that low levels
of endotoxins passing through the intestinal mucosa into the blood
may contribute to the levels of circulating HPCs. Here, we used a
mouse model to study the effects of selective antibiotic decontamination on cytokine-induced stem cell mobilization. We found that
the ability to mobilize in response to IL-8, G-CSF, or FL was
significantly reduced in animals receiving endotoxin binding
antibiotics or antibiotics that modulate the anaerobic flora. The
mobilizing capacity was partially restored after adding endotoxins
to the drinking water of decontaminated mice. These observations
indicate that endotoxins are involved as cofactors in cytokineinduced hematopoietic stem cell mobilization.
From the Laboratory of Experimental Hematology and the Laboratory of
Medical Microbiology, Leiden University Medical Center, Leiden, the
Netherlands; Novartis Forschungsinstitut, Vienna, Austria; and the Rega
Institue, Laboratory for Molecular Immunology, Leuven, Belgium.
Society (NKB-RUL 99-2029).
Submitted July 26, 2003; accepted August 14, 2003. Prepublished online as
Blood First Edition Paper, September 11, 2003; DOI 10.1182/blood-200207-2270.
Supported by a grant from the Fund for Scientific Research FWO-Vlaanderen,
the “Geconcerteerde Onderzoeks Acties” (GAO), and by the Dutch Cancer
340
Reprints: Willem E. Fibbe, Department of Hematology, Leiden University
Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands; e-mail:
[email protected].
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.
© 2004 by The American Society of Hematology
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
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BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
ENDOTOXINS AS COFACTORS IN STEM CELL MOBILIZATION
Materials and methods
Animals
Male Balb/c mice between 8 to 12 weeks of age were purchased from
Broekman BV (Someren, the Netherlands). OF1 mice either specific
pathogen free (SPF) or germ free were purchased from Iffa-Credo (Lyon,
France). The OF1 mice were kept at the animal facility of the Rega Institue
of the University of Leuven (Belgium). Upon arrival, the germ-free animals
were transferred to and kept in insulators under strict germ-free conditions.
They were fed ad libitum with mixed complete aliment R-03 (Usine
d’Alimentation Rationelle, Epinay sur Orge, France), which was irradiated
at 45 kGy, and canned sterile water (Sleutels Conserven, Panninge, the
Netherlands), which was reautoclaved after transfer into the sterile drinking
bottles. Feces of these mice were cultured and no aerobic Gram-negative
bacteria were found. All other mice were fed commercially available rodent
chow. Control mice received acidified water ad libitum.
Antibiotic treatment
There were 5 different antibiotic regimens consisting of combinations of 3
different antibiotics administered through the drinking water (Table 1). The
first regimen consisted of 100 mg/L ciprofloxacin (Bayer Nederland BV,
Mijdrecht, the Netherlands) 70 mg/L polymyxin B (Bupha BV, Uitgeest, the
Netherlands), and 20 g/L saccharose. The second regimen contained 125
mg/L ciprofloxacin and 20 g/L saccharose. The third regimen consisted of
100 mg/L polymyxin B and 20 g/L saccharose. The fourth regimen
consisted of 500 mg/L metronidazol (NPBI, Amstelveen, the Netherlands)
and 20 g/L saccharose, and in the fifth regimen the mice received 500 mg/L
metronidazol, 125 mg/L ciprofloxacin, and 20 g/L saccharose. All antibiotics were administered during 1 week before cytokine-induced stem cell
mobilization and were continued throughout the administration of the
different cytokines.
341
Sysmex F800 (TOA Medical Electronics, Kobe, Japan). Manual neutrophil
counts were performed after May-Grünwald-Giemsa (MGG) staining of the
peripheral blood. Blood mononuclear cell (MNC) suspensions were
obtained after Ficoll separation as described previously.24 The bone marrow
cells were removed from the femur by flushing the femur under sterile
conditions with RPMI 1640 containing 500 ␮g/mL penicillin, 250 ␮g/mL
streptomycin, and 2% fetal bovine serum (FBS; GIBCO, Grand Island,
NY), and 6% heparin (400 IU/mL).
FACS analysis
Cells were incubated for 30 minutes at 4°C with anti-CD3e and anti-CD45R/
B220 antibodies both phycoerythrin (PE)–conjugated and anti–GR-1
antibody–conjugated to fluorescein isothiocyanate (FITC; PharMingen, San
Diego, CA.).
The cells were then washed with PBS containing 0.8 g/L albumin, and
incubated at 4°C with Cy-chrome–conjugated anti–Ly-5 antibody (Ly-5–
cyanin-5 (Cy 5); PharMingen) for 30 minutes. After washing, the cells were
resuspended in PBS containing 0.8 g/L albumin, and fluorescence intensity
was analyzed using fluorescence-activated cell sorting (FACS; Becton
Dickinson, Mountain View, CA). The GR-1 strong positive, CD3⫺,
B220-PE–negative cells within the total population of Ly5-Cy5–positive
cells were then regarded as mature neutrophils.43
Progenitor cell assays
Colony forming units–granulocyte macrophage (CFU-GMs) were cultured
as described previously.40 Briefly, peripheral blood MNCs were cultured in
3.5-cm dishes containing 5 ⫻ 105 cells per mL in semisolid medium in the
presence of recombinant murine GM-CSF (1.25 ng/mL, kindly provided by
Dr E. Liehl, Novartis Forschungsinstitut). Bone marrow cells were cultured
in concentrations of 1 ⫻ 105 per mL. After 6 days of culture at a fully
humidified atmosphere of 37°C containing 5% CO2, the number of
colonies, defined as an aggregate of more than 20 cells, was scored using an
inverted microscope.
Endotoxin treatment
Endotoxin lipopolysaccharide (LPS) derived from Escherichia coli, serotype 026:B6 (⬎ 10 000 U/ng LPS; Sigma-Aldrich, Zwijndrecht, the
Netherlands) was used. LPS was administered in the drinking water for 1
week at a concentration of 33 ␮g/mL.
Cytokines
Recombinant human granulocyte colony-stimulating factor (rhu-G-CSF,
filgrastim [Neupogen]; a kind gift from Amgen BV, Breda, the Netherlands)
and FL (a kind gift from Immunex, Seattle, WA) were diluted to the desired
concentration in endotoxin-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and administered as an intraperitoneal injection. FL contained 25 pg endotoxin per milligram of protein.
Recombinant human IL-8 (a kind gift from the Novartis Forschungsinstitut,
Vienna, Austria) was purified from E coli expressing a synthetic gene.42 The
concentration of endotoxin was less than 0.05 U/mL as determined by the
Limulus polyphemus amoebocyte lysate assay.
Preparation of cell suspensions
Mice were killed by CO2 asphyxiation. Peripheral blood was drawn by
cardiac puncture, and white blood cell (WBC) counts were performed on a
Table 1. Characteristics of the different antibiotics used for
selective decontamination of the gut
Effect on
aerobic
bacteria
Effect on
anaerobic
bacteria
Decontamination*
Endotoxin
binding
Ciprofloxacin35
⫹
⫺
⫹
⫺
Polymyxin B36-39
⫾
⫺
⫺
⫹
Metronidazol40-41
⫺
⫹
⫺
⫺
*No aerobic Gram-negative bacteria were present in the feces.
CAFC assay
In vitro determination of hematopoietic progenitor cell frequencies was
performed by limited dilution analysis (LDA) of cobblestone area–forming
cells (CAFCs) in microcultures according to the method previously
described.44,45 Briefly, cells were seeded on a pre-established stromal layer
of the murine preadipocyte cell line, FBMD-1 (kindly provided by Dr R. E.
Ploemacher, Erasmus University, Rotterdam, the Netherlands). At weekly
intervals until day 35 after initiation of the cultures, cobblestone areas were
scored using an inverted microscope. Cobblestone areas are defined as
colonies of immature hematopoietic cells (at least 6 cells per colony)
residing within the pre-established stromal layer. The proportion of
negative wells at each dilution was used in a Poisson-based LDA
calculation to determine the CAFC frequency.44,46
Analysis of mouse feces
Mice were housed in plastic cages on sawdust, both of which were replaced
daily. Fresh fecal pellets were collected in 500 ␮L sterile saline after 1 week
of administration of antibiotics. Then, 10-fold serial dilutions were made,
the lower limit being 1:1000. Samples (100 ␮L) of the dilutions were
inoculated on blood agar plates (MacConkey III–agar plates; Oxoid,
Basingstoke, United Kingdom). The 1:1000 dilution was also inoculated
after it had first been incubated for 2 days at 37°C. Of the 1:10 dilution an
extra Sabouraud and aesculine plate were inoculated for elective purposes.
All plates were incubated at 37°C. After 24 and 48 hours of incubation
various colonies were isolated and identified based on morphology and
Gram staining. Before starting the mobilization schedule, the feces of all
mice were cultured to determine the presence of aerobic Gram-negative
rods. After treatment with ciprofloxacin either alone or in combination with
polymyxin B, all mice were free of aerobic Gram-negative rods (ie,
adequately decontaminated). After treatment with metronidazol or with
polymyxin B added to the drinking water in a concentration of 100 mg/L,
aerobic Gram-negative rods were still cultured from the feces of these mice.
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342
VELDERS et al
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
Serum concentration of endotoxins, IL-6, and TNF-␣
Quantitative measurements of endotoxins were carried out using the
commercially available endotoxin test QCL-1000 (Bio Whittaker, Boehringer Ingelheim Bioproducts, Verviers, France). The detection limit was
0.06 endotoxin units per milliliter of serum (EU/mL). In the serum of the
same group of mice, IL-6 and TNF-␣ were measured using an enzymelinked immunosorbent assay (ELISA) technique (Diaclone, CLB, Amsterdam, the Netherlands). All test were used according to the manufacturer’s
instructions.
Detection of MMP-9 (gelatinase B) activity
Gelatinase B/matrix metalloproteinase 9 (MMP-9) activity in freshly frozen
plasma samples was determined by sodium dodecyl sulfate/polyacrylamide
gel electrophoresis (SDS/PAGE) zymography as described earlier.47
Experimental design
Mice were treated with different regimens of antibiotics, endotoxin, or a
combination of both for 1 week. Before the injection of cytokines, feces of
the mice were collected and cultured. Mobilization was induced in
pretreated or untreated control animals by either a single intraperitoneal
injection of saline or 30 ␮g IL-8, or daily intraperitoneal injections of 5 ␮g
G-CSF for 5 days, or daily injections of 10 ␮g FL for 5 days. At 20 minutes
after the injection with saline or IL-8 or 24 hours after the last injection of
G-CSF or FL, mice were killed by CO2 asphyxiation, peripheral blood was
obtained by cardiac puncture, and femurs were removed. Cell suspensions
were prepared according to the described procedures. The numbers of
CFU-GMs in the peripheral blood and in the femur of the different animals
were determined. An MGG staining of the peripheral blood was performed
as well as FACS analysis of the bone marrow. In a number of animals, a
CAFC assay was performed of peripheral blood and bone marrow cells. The
institutional ethical committees on animal experiments of Leiden University Medical Center and the Rega Institute, Laboratory for Molecular
Immunology, approved the experimental protocol.
Statistical analysis
Differences were evaluated using the Student t test. P values of less than .05
were considered statistically significant. To calculate the CAFC frequency,
a Poisson-based LDA was used. The 95%, 99%, and 99.9% confidential
intervals were calculated, and when the confidential intervals were not
overlapping the P value was considered to be less than 0.05%, 0.01%, and
0.001%, respectively.
Results
Effect of antibiotic treatment on the peripheral blood counts
and on peripheral blood progenitor cell mobilization
To study the effect of selective antibiotic decontamination (ie,
elimination of Gram-negative bacteria) on stem cell mobilization,
decontaminated mice were treated with mobilization-inducing
cytokines. Stem cell mobilization in response to IL-8, G-CSF, or
FL was significantly reduced in mice decontaminated with ciprofloxacin and polymyxin B compared with mice that had not
received antibiotic treatment (saline: 26 ⫾ 8 CFUs/mL vs 71 ⫾ 42
CFUs/mL; IL-8: 290 ⫾ 64 CFUs/mL vs 192 ⫾ 61 CFUs/mL,
P ⬍ .05; G-CSF: 3371 ⫾ 1214 CFUs/mL vs 1925 ⫾ 1216 CFUs/
mL, P ⬍ .05; FL 818 ⫾ 175 CFUs/mL vs 552 ⫾ 149 CFUs/mL,
P ⬍ .05; Figure 1).
We next studied the influence of each of these 2 antibiotics.
Although treatment with only polymyxin B did not result in
adequate decontamination of mice, the IL-8– and G-CSF–induced
mobilizations were significantly reduced (IL-8: 500 ⫾ 252
Figure 1. The cytokine-induced mobilization of progenitor cells is decreased by
treatment with ciprofloxacin and polymyxin B. Mice were treated with 100 mg/L
ciprofloxacin, 70 mg/L polymyxin B, and 20 mg/L saccharose added to their drinking
water for 1 week. While the antibiotic treatment was continued, mice were mobilized
with either a single intraperitoneal injection of 30 ␮g IL-8 (n ⫽ 8), or 5 ␮g G-CSF
(n ⫽ 15) or 10 ␮g FL (n ⫽ 9), both for 5 days. At 20 minutes after the IL-8 injection and
24 hours after the last injection with G-CSF or Flt 3 ligand, mice were killed and blood
was harvested. Results are expressed as means ⫾ SD. *P ⬍ .05 compared with the
saline pretreated controls (n ⫽ 3-10). Results are expressed as means ⫾ SD.
CFUs/mL vs 212 ⫾ 115 CFUs/mL, P ⬍ .05; G-CSF: 2557 ⫾ 825
CFUs/mL vs 1475 ⫾ 460 CFUs/mL, P ⬍ .05; Figure 2A).
In contrast, adequate decontamination was observed after
treatment with ciprofloxacin as monotherapy, but without an effect
on the mobilizing capacity (saline: 77 CFUs/mL vs 88 CFUs/mL;
IL-8: 290 ⫾ 173 CFUs/mL vs 364 ⫾ 171 CFUs/mL, P ⫽ .41;
G-CSF: 2525 ⫾ 2352 CFUs/mL vs 1784 ⫾ 1211 CFUs/mL,
P ⫽ .44; Figure 2B). This observation led to the hypothesis that a
decrease in mobilization was due to the endotoxin binding capacity
of polymyxin B and that ciprofloxacin had no effect on mobilization since the anaerobic Gram-negative endotoxins containing flora
were not eliminated.
To study the role of anaerobic Gram-negative flora on mobilization, animals were treated with metronidazol alone or in combination with ciprofloxacin. Both treatment schedules resulted in a
significant decrease of the IL-8–induced mobilization when compared with the control group (saline ⫹ metronidazol: 12 CFUs/mL
vs saline ⫹ metronidazol ⫹ ciprofloxacin: 0 CFUs/mL vs saline
control: 19 CFUs/mL; IL-8 ⫹ metronidazol: 55 ⫾ 66 CFUs/mL vs
IL-8 ⫹ metronidazol ⫹ ciprofloxacin: 98 ⫾ 165 CFUs/mL vs IL-8
control: 538 ⫾ 216 CFUs/mL, P ⬍ .05; Figure 3).
No effect on the WBC count or the absolute number of
neutrophils in blood or bone marrow was seen in animals treated
with various antibiotic regimes compared with untreated controls
(data not shown).
Peripheral blood cell counts and peripheral blood progenitor
cell mobilization in germ-free mice (OF-1)
It was considered that the reduced mobilizing capacity in decontaminated mice was due to a nonspecific effect of the antibiotics used.
Mobilization experiments were therefore performed in germ-free
OF-1 mice not treated with antibiotics. IL-8–induced mobilization
in germ-free mice and in OF-1 mice treated with ciprofloxacin
and polymyxin B was significantly reduced compared with the
control group not receiving antibiotics (IL-8 controls: 378 ⫾ 182
CFUs/mL, IL-8 antibiotics: 225 ⫾ 109 CFUs/mL, P ⬍ .05; IL-8
germ-free: 157 ⫾ 53 CFUs/mL, P ⬍ .05; Figure 4). The peripheral
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BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
ENDOTOXINS AS COFACTORS IN STEM CELL MOBILIZATION
343
Figure 3. IL-8–induced mobilization is significantly reduced after treatment
with metronidazol. Mice were receiving either normal drinking water (n ⫽ 5), water
treated with 500 mg/L metronidazol and 20 g/L saccharose (n ⫽ 5), or water treated
with 500 mg/L metronidazol, 100 mg/L ciprofloxacin, and 20 g/L saccharose (n ⫽ 5)
for 1 week. Mice were mobilized with a single intraperitoneal injection of 30 ␮g IL-8. At
20 minutes after the IL-8 injection, the mice were killed and blood was harvested.
Results are expressed as means ⫾ SD. *P ⬍ .05 compared with the controls (n ⫽ 2).
Figure 2. Treatment with oral polymyxin B reduces cytokine-induced stem cell
mobilization, while treatment with ciprofloxacin has no effect on IL-8– or
G-CSF–induced mobilization. (A) Mice were treated with 100 mg/L polymyxin B and
20 mg/L saccharose added to their drinking water for 1 week. Mice were then
mobilized with either a single intraperitoneal injection of 30 ␮g IL-8 (n ⫽ 13) or 5 ␮g
G-CSF (n ⫽ 15) per day for 5 days. At 20 minutes after the IL-8 injection and 24 hours
after the injection with G-CSF, mice were killed and blood was harvested. Results are
expressed as means ⫾ SD. *P ⬍ .05 compared with the controls (n ⫽ 6-8). (B) Mice
were treated with 125 mg/L ciprofloxacin and 20 mg/L saccharose added to their
drinking water for 1 week. The control mice received normal drinking water. Antibiotic
treatment was continued when mice were mobilized with either a single injection of 30
␮g IL-8 (controls: n ⫽ 6, treatment group: n ⫽ 9) or 5 ␮g G-CSF (controls: n ⫽ 4,
treatment group: n ⫽ 10) per day for 5 days. At 20 minutes after the IL-8 injection and
24 hours after the injection with G-CSF, the mice were killed and blood was
harvested. Results are expressed as means ⫾ SD.
blood counts between the 3 groups of mice were not different (data
not shown).
Serum concentration of endotoxins, IL-6, TNF-␣, and MMP-9
It was considered that enhancement of mobilization by endotoxins
would be mediated by the induction of proinflammatory cytokines
(ie, IL-6 and TNF). In all serum samples of the 4 different groups of
mice (control mice, mice treated with endotoxins only, mice treated
with ciprofloxacin and metronidazole, and mice treated with
ciprofloxacin, metronidazole, and endotoxins) the level of endotoxins measured was below the detection level of 0.06 EU/mL (data
not shown). Similarly, all serum levels of IL-6 and TNF-␣ were
below the detection limit. Previously, we demonstrated that IL-8–
induced mobilization coincides with an increase in proteolytic
neutrophil-derived enzymes, in particular MMP-9. MMP-9 levels
as determined by zymography showed a similar increase after
administration of IL-8 in all 4 groups.
Effect of treatment with LPS on the mobilization of HPCs in
control and partially decontaminated mice
To confirm that the reduced mobilizing capacity in decontaminated
mice was due to elimination or inactivation of endotoxin, we
performed endotoxin reconstitution experiments in decontaminated
mice. The IL-8–induced mobilization in mice, decontaminated by
treatment with ciprofloxacin and metronidazol, was significantly
increased when LPS was added to the drinking water in comparison
with decontaminated controls (67 ⫾ 28 CFUs/mL vs 190 ⫾ 98
CFUs/mL, P ⬍ .002; Figure 5), indicating that addition of endotoxin partially restored mobilization.
Administration of drinking water containing endotoxins had no
effect on circulating steady-state levels of HPCs (endotoxins:
9 ⫾ 6 CFUs/mL vs controls: 26 ⫾ 16 CFUs/mL). However, the
IL-8–induced mobilizing capacity in mice receiving LPS was also
significantly increased when compared with the controls (459 ⫾ 135
CFUs/mL vs 320 ⫾ 66 CFUs/mL, P ⬍ .02; Figure 5). These
results indicate a relation between the IL-8–induced mobilization
and the concentration of endotoxins in the gastrointestinal tract (ie,
a higher concentration of endotoxin leads to an increased mobilizing capacity).
Figure 4. In germ-free mice the IL-8–induced mobilization is significantly
reduced. There were 3 groups of OF1 mice mobilized with a single intraperitoneal
injection of 30 ␮g IL-8 or saline: normal controls (saline: n ⫽ 3, IL-8: n ⫽ 7), mice that
had been treated with ciprofloxacin and polymyxin B for 1 week (saline: n ⫽ 3, IL-8:
n ⫽ 7), and germ-free OF1 mice (saline: n ⫽ 3, IL-8: n ⫽ 7) not treated with
antibiotics. At 20 minutes after the IL-8 injection, the mice were killed and blood was
harvested. Results are expressed as means ⫾ SD. *P ⬍ .05 compared with the controls.
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344
VELDERS et al
Figure 5. The IL-8–induced mobilization in decontaminated mice is restored
after oral administration of endotoxins. Mice were receiving either normal drinking
water (n ⫽ 7); drinking water containing endotoxins (33 mg/L) and saccharose (20
mg/L) (n ⫽ 8); drinking water containing 125 mg/L ciprofloxacin, 500 mg/L metronidazol, and 20 mg/L saccharose (n ⫽ 7); or water containing 125 mg/L ciprofloxacin, 500
mg/L metronidazol, 20 mg/L saccharose, and endotoxins (n ⫽ 7). After one week of
treatment, mice were mobilized with a single intraperitoneal injection of PBS
containing 1% bovine serum albumin or 30 ␮g IL-8. At 20 minutes after the injection,
mice were killed and blood and bone marrow were obtained. Results are expressed
as means ⫾ SD. *P ⬍ .05 compared with the controls.
The effect of treatment with antibiotics on the number of
progenitor cells in the femur
In Balb/c, OF-1, and germ-free OF-1 mice, the number of
colony-forming units per femur was not influenced by any treatment (data not shown). In Balb/c mice the effect of the ciprofloxacin/
polymyxin B treatment on the number of neutrophils in the bone
marrow was established. The number of neutrophils as determined
by FACS analysis was not changed compared with the control
group (data not shown).
CAFC assay of peripheral blood and bone marrow
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
with untreated controls. The numbers of not only committed
progenitor cells but also the more primitive progenitor cells were
decreased as shown by the CAFC assay; although the differences
for CAFC day 28 were not statistically significant. Next we
determined the contribution of each of the 2 components of this
mobilization-inhibiting antibiotic combination. Following treatment with ciprofloxacin, which is absorbable after oral administration,35 no aerobic Gram-negative bacteria were cultured from the
feces of mice, while mobilization in response to IL-8 and G-CSF
was not reduced. In contrast, treatment with polymyxin B resulted
in a significant inhibition of mobilization without eliminating the
aerobic Gram-negative flora.36 These apparently conflicting results
can be explained by the endotoxin binding capacity of polymyxin
B.37,38 The lack of inhibition of mobilization observed in mice
treated with only ciprofloxacin can be explained by residual
endotoxins derived from the anaerobic Gram-negative flora that
constitute more than 90% of the bacterial flora of the intestines.39,40
In order to eliminate both the aerobic and the anaerobic
Gram-negative flora we then used a combination of ciprofloxacin
and metronidazol.41 Indeed, a significant reduction in the number
of the mobilized progenitor cells was observed. Moreover, treatment with only metronidazol also resulted in a significantly
reduced mobilization. The significantly lower mobilization observed in mice treated with either polymyxin B or metronidazol
was not due to a reduction in the size of the progenitor cell pool,
since the number of CFUs in the bone marrow was comparable
with the control mice. In addition we observed no sign of
diminished colony growth following addition of the different
antibiotics to the culture medium at concentrations that exceeded
the expected serum concentrations (data not shown). These data
indicate that endotoxins derived from anaerobic Gram-negative
bacteria play a major role in cytokine-induced stem cell
mobilization.
Finally, to eliminate possible direct effects of antibiotics on
mobilization we studied stem cell mobilization in germ-free mice
not receiving antibiotic treatment. Germ-free OF-1 mice not treated
with antibiotics showed a significantly reduced mobilization compared with control mice that were not decontaminated. The effects
seen on mobilization in the germ-free mice were similar to the
To determine the effect of decontamination on committed progenitor cells and cells with repopulating ability, the number of early
(day 7) and late (day 28) CAFCs was assessed in peripheral blood
of mice decontaminated with ciprofloxacin and polymyxin B and
mobilized with a single injection of 30 ␮g IL-8. The numbers of
circulating early CAFCs were significantly reduced in IL-8–
mobilized mice decontaminated with ciprofloxacin and polymyxin
B compared with controls not treated with antibiotics (P ⬍ .05).
Similarly, the numbers of late CAFCs were also reduced. However,
the absolute numbers in peripheral blood of late CAFCs were low
and the difference did not reach statistical significance (P ⬎ .05;
Figure 6).
Discussion
In the present study we addressed the role of endotoxins in
cytokine-induced stem cell mobilization. First we studied the effect
of selective antibiotic decontamination, resulting in the elimination
of the Gram-negative aerobic intestinal flora, on IL-8–, G-CSF–,
and FL-induced mobilization. In mice decontaminated with a
combination of ciprofloxacin and polymyxin B we observed a
significant reduction in the mobilizing capacity when compared
Figure 6. The numbers of CAFCs in peripheral blood are decreased after
pretreatment with ciprofloxacin and polymyxin B. Balb/c mice were treated with
100 mg/L ciprofloxacin, 70 mg/L polymyxin B, and 20 mg/L saccharose added to their
drinking water. The antibiotic treatment was continued and mice were mobilized with
a single intraperitoneal injection of 30 ␮g IL-8. At 20 minutes after the IL-8 injection,
mice were killed and blood was harvested. Results are expressed as means ⫾ SD.
*P ⬍ .05 compared with the controls.
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BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
ENDOTOXINS AS COFACTORS IN STEM CELL MOBILIZATION
inhibition observed in control mice that were decontaminated with
ciprofloxacin and polymyxin B.
To further support the hypothesis that endotoxins in the
gastrointestinal tract serve as cofactors in cytokine-induced peripheral blood stem cell mobilization, we performed endotoxin reconstitution experiments in decontaminated mice. IL-8–induced mobilization was significantly enhanced in decontaminated mice
(metronidazol/ciprofloxacin) receiving drinking water also containing endotoxins. In these animals a partial restoration of mobilization was observed following oral administration of endotoxins.
The mechanisms underlying this effect are presently unknown.
Endotoxins are also known as potent inducers of mobilization
following systemic administration.32,33 Under clinical and experimental conditions the lipopolysaccharide component of the aerobic
Gram-negative bacteria wall damages the endothelial cell48 finally
leading to the synthesis and the release of proinflammatory
mediators (TNF, IL-1, IL-6, and IL-8) from macrophages.49 The
endotoxin concentration in the gut is very likely to be decreased by
the treatment with polymyxin B or metronidazol, resulting in a
diminished production of cytokines by macrophages. We hypothesized that the increase in stem cell mobilization observed after
administration of endotoxin was due to the induction of proinflammatory cytokines that could synergize in the induction of stem cell
mobilization. The serum concentrations of TNF-␣ and IL-6 were
not increased. These data, however, do not rule out the possibility
that these or other cytokines act at a local level in the intestinal tract
rather than acting at a systemic level.
Previous studies in our laboratory have shown that neutrophils
in the peripheral blood are indispensable for IL-8–induced mobilization,50 whereas neutrophils in the bone marrow were suggested to
be important in the G-CSF–induced mobilization.51 The antibiotics
used did not alter the number of neutrophils in the peripheral blood
or the number of neutrophils in the bone marrow. Pruijt et al
showed that IL-8–induced mobilization in rhesus monkeys is
345
dependent on gelatinase B.52 In the present study we found that
gelatinase B levels induced by IL-8, measured by zymography,
were not affected by the treatment with antibiotics (data not
shown). This supports more recent data in mice showing that
gelatinase B levels reflect in vivo activation of neutrophils but are
not essential for HPC mobilization in the mouse.50
Recent evidence indicates that LPS-activated signaling through
toll-like receptor 4 augments chemokine-induced neutrophil migration through modulation of chemokine receptors.53 It is therefore
conceivable that LPS-induced modification of neutrophil migration
is related to alterations in stem cell mobilizing capacity.
The enhancement of the mobilization may be dependent on the
concentration of endotoxins in the gastrointestinal tract. As a
consequence, modification of the endotoxin content or binding of
endotoxins may affect the levels of cytokine-induced mobilization.
Cytokine-induced mobilization was lowest in mice receiving
antibiotic decontamination and highest in control mice receiving
oral endotoxins. These data suggest that cytokine-induced mobilization is enhanced by endotoxins in a dose-dependent way.
In conclusion, our studies indicate that endotoxins may serve as
cofactors in cytokine-induced mobilization of peripheral blood
progenitor cells. Our observations could potentially have implications for the antibiotic treatment of patients undergoing treatment
with rHu-G-CSF to mobilize peripheral blood progenitor cells for
autologous transplantation. Elimination of the anaerobic flora of
the gut or the use of antibiotics that bind endotoxins may impair the
yield of the peripheral blood stem cell mobilization.
Acknowledgments
We thank Ilse Vanaelst, Jan Mertens, Marijke Fröhlich, and Jan
Didden for technical help.
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2004 103: 340-346
doi:10.1182/blood-2002-07-2270 originally published online
September 11, 2003
Reduced stem cell mobilization in mice receiving antibiotic modulation
of the intestinal flora: involvement of endotoxins as cofactors in
mobilization
Gerjo A. Velders, Ronald van Os, Henny Hagoort, Perry Verzaal, Henri F. L. Guiot, Ivan J. D. Lindley,
Roel Willemze, Ghislain Opdenakker and Willem E. Fibbe
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