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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3725-3733
HEMATOPOIESIS
"Emergency" granulopoiesis in G-CSF-deficient mice in
response to Candida albicans infection
Sunanda Basu,
George Hodgson,
Hui-Hua Zhang,
Melissa Katz,
Cathy Quilici, and
Ashley R. Dunn
From the Ludwig Institute for Cancer Research, Melbourne Tumor
Biology Branch, PO Royal Melbourne Hospital, Victoria 3050, Australia.
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Abstract |
Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein
believed to play an important role in regulating granulopoiesis both at
steady state and during an "emergency" situation. Generation of
G-CSF and G-CSF receptor-deficient mice by gene targeting has demonstrated unequivocally the importance of G-CSF in the regulation of
baseline granulopoiesis. This study attempted to define the physiologic
role of G-CSF during an emergency situation by challenging a cohort of
wild-type and G-CSF-deficient mice with Candida albicans. Interestingly, after infection, G-CSF-deficient mice developed an
absolute neutrophilia that was observed both in blood and bone marrow.
In addition, 3 days after Candida infection increased numbers
of granulocyte-macrophage (GM) and macrophage (M) progenitors were
observed in the bone marrow of G-CSF-deficient mice. Of the cytokines
surveyed, interleukin (IL)-6 levels in serum were elevated; interestingly, levels of IL-6 were higher and more sustained in G-CSF-deficient mice infected with C albicans than similarly
infected wild-type mice. Despite the higher levels of serum IL-6, this cytokine is dispensable for the observed neutrophilia because candida-infected IL-6-deficient mice, or mice simultaneously deficient in G-CSF and IL-6, developed neutrophilia. Similarly, mice lacking both
G-CSF and GM-CSF developed absolute neutrophilia and had elevated
numbers of GM and M progenitors in the bone marrow; thus, G-CSF and
GM-CSF are dispensable for promoting the emergency response to candidal infection.
(Blood. 2000;95:3725-3733)
© 2000 by The American Society of Hematology.
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Introduction |
Granulopoiesis is the process whereby mature
granulocytes are produced from a small number of pluripotent
hematopoietic stem cells. Despite the fact that a large number of
granulocytes are produced daily to maintain homeostasis and a still
higher number of granulocytes can be produced during an emergency
response to infection, the process is highly regulated. Multiple
cytokines including granulocyte colony-stimulating factor (G-CSF),
granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin
(IL)-3 and IL-6 are thought to play a role in the regulation of
granulopoiesis.1-3 The critical role of G-CSF in
steady-state granulopoiesis has been clearly demonstrated by the
generation of mice in which the G-CSF gene4 and
subsequently G-CSF receptor gene have been disrupted by gene targeting
in embryonic stem cells.5 These mice have 20% to 30% of
circulating neutrophils, compared to wild-type mice. The existence of
both immature and mature forms of neutrophils in mice lacking G-CSF
indicates that neutrophil production can occur in a G-CSF-independent
manner; thus a factor(s) other than G-CSF has the capacity to promote
neutrophil production in the absence of G-CSF.
During an emergency, such as infection with pathogenic organisms, the
response of the host involves a series of inflammatory events, with
macrophages and neutrophils playing an important role in the cellular
phase, followed by an acquired immunity specific to the pathogen. In
such a situation, the hematopoietic system is triggered to meet the
demand for production of appropriate cell types. The production of
different cells is highly regulated, although the mechanism underlying
emergency hematopoiesis remains poorly understood. On the basis of a
large body of evidence it is thought that granulopoiesis during an
emergency situation is regulated primarily by G-CSF; this notion,
however, has so far been inferred rather than defined. Indirect
evidence implicating G-CSF as an emergency granulopoietic factor
includes its ability to stimulate neutrophil production when
administered in pharmacologic doses6 and the coexistence,
in some infective states, of high neutrophil levels and high serum
levels of endogenously produced G-CSF.7
Infection of G-CSF-deficient mice with pathogenic organisms provides a
valuable model to evaluate definitively the role of G-CSF in regulating
neutrophil production during an emergency situation. Different
pathogens evoke a distinct pattern of inflammatory cell responses;
although the mechanisms underlying these differences remain largely
unknown, it is believed to be due to the different sets of cytokines
produced by the host. Because it is known that neutrophils predominate
the first phase of a host's response to Candida
albicans,8 we infected wild-type and G-CSF-deficient mice with the yeast to study regulation of emergency granulopoiesis. Intriguingly G-CSF-deficient mice, like similarly infected wild-type mice, developed profound and sustained neutrophilia, suggesting that
G-CSF is dispensable for mounting an emergency granulopoietic response.
Because serum levels of IL-6 were elevated in G-CSF-deficient mice, we
infected mice simultaneously deficient in G-CSF and IL-6. Furthermore,
to investigate the role of GM-CSF in candida-induced neutrophilia in
G-CSF-deficient mice we also challenged mice simultaneously deficient
in G-CSF and GM-CSF. Like G-CSF-deficient mice, both G-CSF //GM-CSF/ and
G-CSF//IL-6/ mice
developed a similar degree of neutrophilia indicating that in addition
to G-CSF both IL-6 and GM-CSF are dispensable for emergency
granulopoiesis in response to candidal infection.
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Materials and methods |
Mice
Eight- to 10-week-old wild-type, G-CSF-deficient
(G-CSF/),9 G-CSF/GM-CSF-deficient
(G-CSF//GM-CSF/),4
G-CSF/IL-6-deficient
(G-CSF//IL-6/) mice
were used for these studies.
G-CSF//IL-6/ mice
were generated by intercrossing G-CSF-deficient mice with IL-6-deficient mice.10 The mice of mixed C57BL/6 and
129/OLA background were used for all studies. Within an experimental
group mice were age and sex matched. All experiments were conducted according to the guidelines of The National Health and Medical Research
Council of Australia, and the experiment protocols were approved by the
Animal Ethics Committee of Royal Melbourne Hospital Campus (Victoria, Australia).
Candida albicans
Subcultures of Candida albicans (ATCC 18804) were maintained
at  80°C in tryptone broth containing 10% glycerol. Cultures were plated on Sabouraud dextrose agar plates and grown at 37°C for
48 hours. The plates were stored at 4°C for a maximum of 4 weeks.
Before each experiment, a colony was picked from a plate and grown in 5 mL of Sabouraud agar broth (SAB) at 37°C for 24 hours in a shaker.
The cells were centrifuged and the pellet washed twice with
pyrogen-free phosphate-buffered saline (PBS) and resuspended in PBS.
The cell suspension was briefly sonicated, counted in a hemocytometer,
and then adjusted to the desired concentration.
Experimental model
Disseminated candidiasis was produced by tail vein injection of
2.5 × 105 colony-forming units (CFUs) of C
albicans blastoconidia on day 0. Animals were observed for 7 days for altered behavior and morbidity. On days 1, 3, and 7, subgroups
of animals were bled through retro-orbital puncture and then killed by
cervical dislocation. Animals were autopsied aseptically immediately
after death. The right kidney was homogenized in 5 mL PBS. Dilutions of
homogenate were plated on Sabouraud agar dextrose broth and incubated
at 37°C. Colonies were counted after 48 hours. The left kidney was
fixed in Bouin solution and stained with hematoxylin-eosin. For
detection of fungi in tissues, sections were stained with Gomori
methenamine silver.
Peripheral blood and bone marrow analysis
Mice were bled through retro-orbital plexus using EDTA-coated
microhematocrit tubes (Clay Adams, Pasippany, NJ), diluted immediately 1:4 in PBS containing 2 mg/mL EDTA, and analyzed using a Sysmex-K1000 automated counter (Toa Medical Electronics Co, Kobe, Japan). Peripheral blood smears were stained with May-Grünwald-Giemsa, and manual differential cell counts of at least 200 nucleated cells were performed. In some cases, cytospins were prepared from peripheral blood
after lysing the red blood cells (RBC) with RBC lysis buffer (0.16 mol/L ammonium chloride in Tris buffer; pH 7.2) and stained with
May-Grünwald-Giemsa. Bone marrow cells were harvested from the
femur and suspended in RPMI containing 5% fetal calf serum (FCS).
Total bone marrow cells were counted in a hemocytometer using white
blood cell (WBC) dilution buffer (3% glacial acetic acid in PBS).
Differential blood cell counts were performed on cytospin preparations
of bone marrow cells stained with May-Grünwald-Giemsa stain.
Phagocytosis and candidacidal activity of neutrophils
Mice were injected intraperitoneally with thioglycollate and after 4 hours the peritoneum was lavaged with PBS containing 5% FCS.
Neutrophils were further purified on Percoll gradients (using gradients
of 45%, 54%, 63%, and 72%) and centrifuged at 500g for 25 minutes. Polymorphonuclear cells were aspirated from the interphase of
63% and 72% Percoll gradients.
Phagocytosis
Briefly, 2.5 × 106 neutrophils were added to
1 × 106 heat-inactivated candida in Hanks'
balanced salt solution (HBSS) containing 10% mouse serum in a total
volume of 1 mL. The cells were incubated for 15 minutes at 37°C
with shaking and then placed on ice. Trypan blue (1 mL) was added to
each reaction mixture and a wet mount was prepared. Phagocytosed and
extracellular yeast (including adherent but not internalized yeast)
were distinguished and scored on the basis of trypan blue uptake
(internalized yeast being colorless/clear).
Candidacidal assay
Neutrophils (1.5 × 106) were preincubated with
HBSS containing 25% mouse serum at 37°C for 5 minutes before
adding 1.0 × 107 C albicans. Tubes
containing the cells and the yeast were rotated end-over-end for 60 minutes at 37°C. Thereafter, 250 µL of 2.5% sodium deoxycholate
was added and mixed, followed by 4.0 mL methylene blue; the tubes were
then centrifuged at 900g for 15 minutes at 4°C. Wet preps
were made and 300 yeast cells were counted under a microscope
distinguishing the viable cells (unstained) versus dead (stained) cells.
Cytokine levels
Tumor necrosis factor- (TNF-) and IL-10 levels in serum were
determined using an enzyme-linked immunoabsorbent assay (ELISA) (Genzyme Corp, Cambridge, MA and Pharmingen, San Diego, CA) specific for the cytokines under the conditions recommended by the suppliers. Absorbance values read at 450 nm were converted to concentrations (pg/mL) by comparison with the appropriate standard curve.
IL-6 assay
Serum IL-6 was registered indirectly by its capacity to promote
proliferation of the IL-6-dependent mouse hybridoma cell line 7TD1
(obtained from Dr J. Van Snick, LICR, Brussels, Belgium). Test samples
were serially diluted in 96-well flat-bottomed microtiter plates and
incubated for 96 hours with 2000 7TD1 cells/well at 37°C in a
humidified incubator containing 5% CO2 in air. Dilutions of murine IL-6 (a gift from Dr R. J. Simpson, LICR, Melbourne, Australia) were included as a standard. Cell proliferation was measured
using the MTT (3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide [Sigma, St Louis, MO]) colorimetric method. After 96 hours of
incubation, MTT was added to each well (5 mg/mL 1:20 v/v) and the
cultures were incubated for a further 4 hours at 37°C at 5% CO2 in air. After incubation, the culture fluid was removed
and the cells were lysed by adding 200 µL of acidified isopropanol (isopropanol with 0.04 N HCl). The plates were analyzed on a plate reader (Titertek Multiskan MCC 340; EFLAB, Helsinki,
Finland) at a test wavelength of 560 nm and a reference
wavelength of 690 nm. In assays using neutralizing antibody, samples
were mixed with a 1:1 volume of a 1:100 dilution of polyvalent rabbit
antimouse IL-6 antibody for 2 hours before analysis in the 7TD1
bioassay. These assays were performed in parallel with samples treated
with control rabbit serum.
Flow cytometry
Blood was treated with RBC lysis buffer for 5 minutes at 37°C
and then washed once with PBS containing 5% FCS. After lysing RBCs,
WBCs were preincubated with 2.4G2 antibody (Pharmingen) for 10 minutes
at room temperature to block Fc receptors (Fc R III/II) and were
subsequently treated with indicated antibodies at 4°C for 30 minutes in PBS containing 2.5% heat-inactivated FCS. The following
panel of antibodies were used: fluorescein isothiocyanate
(FITC)-conjugated rat antimouse Mac-1 (CD 11b), biotinylated rat
antimouse Gr-1 (Ly-6G), FITC-conjugated rat antimouse c-kit (CD117)
(Pharmingen), and biotinylated rat antimouse c-fms (AFS98, kind gift
from John Hamilton, Melbourne, Australia). Cells stained with
biotinylated antibodies were finally developed with phycoerythrin-streptavidin (Pharmingen). All cells were analyzed on a
FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using
CellQuest software. Positive populations have been gated on the basis
of staining profiles with isotype-matched control antibody.
Statistical analysis
Data are presented as mean ± SD unless otherwise stated. Organ
loads of C albicans are expressed as the logarithm of the
candida count per kidney. Comparisons were made using 2-tailed paired and unpaired Student t tests and the Mann-Whitney signed rank test as appropriate. A P value less than .05 was considered significant.
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Results |
Peripheral blood neutrophilia in candida-infected mice
Morphologic examination of peripheral blood smears of wild-type and
G-CSF-deficient mice revealed that mice of both genotypes developed
significant neutropenia 24 hours after infection with C albicans
(data not shown). However, an absolute neutrophilia was observed in
both G-CSF-deficient and wild-type mice 3 days after infection.
Neutrophilia in G-CSF-deficient mice was most pronounced on day 7 after infection (Figure 1). Due to baseline neutrophil depletion in G-CSF-deficient mice, the fold increase in
neutrophil numbers was significantly higher in G-CSF-deficient mice
than the wild-type mice. The striking neutrophilia in infected G-CSF-deficient mice was somewhat surprising in view of the prevailing view of a pivotal role for G-CSF in emergency granulopoiesis.
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| Fig 1.
Peripheral blood neutrophil counts in wild type and
G-CSF-deficient mice treated with C albicans.
Peripheral blood neutrophil levels in wild-type, G-CSF-deficient
mice at baseline ( ) and 7 days after candida infection ( ),
sampled identically.
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Changes in cellular composition of bone marrow after candidal
infection
At baseline, both wild-type and G-CSF-deficient mice have similar
numbers of nucleated bone marrow cells. However, G-CSF-deficient mice
have a significantly lower number of neutrophils; the deficiency is
most striking in the mature neutrophil compartment comprising metamyelocytes, "band" and segmented forms. To investigate
whether the absolute neutrophilia following candidal infection was due to mobilization of neutrophils from bone marrow into circulation, or, that there was an increased production of neutrophilic cells, we examined cytospin preparations of bone marrow cells from femurs of
mice challenged with C albicans. Despite the fact that the total number of bone marrow cells after infection in both
G-CSF-deficient and control mice were similar to basal values, the
increase in numbers of early and late neutrophil forms indicated
preferential amplification within the neutrophil lineage. In
G-CSF-deficient mice the observed increase was more marked for
immature than for mature neutrophils. (Figure
2 and Table 1).
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| Fig 2.
Bone marrow cells.
Cytocentrifuge preparation of bone marrow cells at baseline (top panel)
and 7 days after candida challenge (bottom panel) from wild-type (A, C)
and G-CSF-deficient (B, D) mice. The cytospin preparations were
stained with May-Grünwald-Giemsa stain.
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Table 1.
Changes in bone marrow hematopoietic cells in response
to candida infection in wild-type and G-CSF-deficient mice
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Role of GM-CSF and IL-6 in granulopoiesis in candida-infected
G-CSF-deficient mice
To understand the basis for the neutrophilia in candida-infected
G-CSF-deficient mice, we analyzed the serum levels of various cytokines. In wild-type mice, at least, IL-3 and GM-CSF are thought to
play an important role during emergency hematopoiesis.11 However, neither IL-3 nor GM-CSF was detectable in the sera of either
wild-type or G-CSF-deficient mice during the course of candida
infection (data not shown). In addition to GM-CSF and IL-3, IL-6 is
thought to be of importance in mounting a neutrophilic challenge in
response to candida infection.12 In both wild-type and
G-CSF-deficient mice, elevated levels of IL-6 were observed 24 hours
after candida infection (Figure 3).
However, in G-CSF-deficient mice, the level of IL-6 at 24 hours after
candida infection was almost 4-fold higher than that observed in
similarly infected wild-type mice. Moreover, unlike wild-type mice, in
which IL-6 was undetectable in serum 7 days after candida infection,
IL-6 levels remained elevated in G-CSF-deficient mice
infected with C. albicans.
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| Fig 3.
Kinetics of IL-6 production.
Wild-type and G-CSF-deficient mice were challenged with
2.5 × 105 C albicans intravenously. At the
indicated times, 6 mice of each genotype were killed. Serum samples
from each mouse were tested for IL-6 as described in "Materials and
methods." Data are represented as mean ± SD. One of 2 separate
experiments with similar results is shown.
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Although we were unable to detect GM-CSF in the serum of
G-CSF-deficient and wild-type mice infected with candida, it still remained possible that because of its demonstrated action on GM and G
progenitors, GM-CSF contributed to the candida-mediated neutrophilia.
To address this possibility mice simultaneously deficient in G-CSF and
GM-CSF were challenged with candida. Seven days after infection we
observed an increase in neutrophil numbers in peripheral blood; the
fold increase compared to baseline in each of the genotypes was similar
in magnitude to that observed in G-CSF-deficient mice (Figure
4). To investigate whether increased granulopoiesis in G-CSF-deficient mice in response to candida infection was mediated by IL-6, we challenged mice simultaneously lacking G-CSF and IL-6 with candida. At baseline, neutropenia in adult
G-CSF//IL-6/ mice
is comparable to that in G-CSF/ mice (S.B.
and A.R.D., manuscript in preparation); moreover, following candida
infection, the increase in neutrophils in circulation was of a similar
magnitude in G-CSF//IL-6/
and G-CSF/ mice (Figure 4).
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| Fig 4.
Peripheral blood neutrophil counts in wild type,
G-CSF/,
G-CSF//GM-CSF/ and
G-CSF//IL-6/ mice treated
with C albicans.
Peripheral blood neutrophil levels in wild type,
G-CSF/,
G-CSF//GM-CSF/and
G-CSF// IL-6/
mice at baseline (empty bars) and 7 days after candida infection
(shaded bars), sampled identically. Data are represented as mean ± SD. *There was a significant difference in the neutrophil count on day
7 compared to day 0 for each genotype of mice (P < .05).
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In addition to mature neutrophils, on day 7 after infection there were
also immature neutrophils in the peripheral blood (left shift) of mice
of all genotypes. However, this effect was more pronounced in mice
deficient in growth factor compared to wild-type mice (Table
2). G-CSF-deficient mice showed a higher
proportion of myelocytes than that seen in the blood of wild-type mice.
Also, in G-CSF-deficient mice, on day 7 after infection, the mature neutrophil compartment included a greater proportion of
"band-forms" and only a few segmented neutrophils (Figure
5). Similar observations were made in
G-CSF//GM-CSF/ and
G-CSF//IL-6/ mice.
In addition to the observed neutrophilia in these mice, there was also
a pronounced monocytosis (data not shown). To further characterize the
immature myeloid cells in the periphery of knockout mice after candidal
infection, we analyzed the cell types in blood from candida-infected
mice by flow cytometry using surface markers specific for granulocytes
and monocytes. At baseline, there were 2 populations of cells, which
stained positive for Gr-1 and Mac-1, but could be distinguished on the
basis of Gr-1 expression. One population of cells was
Mac-1+Gr-1dull and the other population was
Mac-1+Gr-1bright (Figure
6, top panel). The forward and side scatter
profile of Mac-1+Gr-1dull and
Mac-1+ Gr-1bright cells suggested
that they represented monocyte/macrophage and granulocytic cells,
respectively. At baseline, the proportion of
Mac-1+Gr-1bright cells was lower in growth
factor(s)-deficient mice compared to wild-type mice, consistent with
the neutropenic state of these mice. Seven days after candida
infection, there were 3 populations of cells that could be
distinguished based on Mac-1/Gr-1 staining pattern:
Mac-1+Gr-1lo (region R3 on Figure 6, bottom
panel), Mac-1+ Gr-1med (region R1 on Figure 6,
bottom panel) and Mac-1+Gr-1high cells (region
R2 on Figure 6, bottom panel). Consistent with the findings based on
morphologic examination of WBCs, FACS analysis revealed that after
candida infection, the proportion of
Mac-1+Gr-1hi cells (representing mature
neutrophils) was greater in wild-type mice compared to growth
factor-deficient mice (Table 3).
Furthermore, the proportion of Mac-1+ Gr-1lo
cells in circulation of candida-infected growth factor(s)-deficient mice was significantly elevated, consistent with the monocytosis observed in these mice (Table 4).
Interestingly, monocytes in candida-infected growth factor-deficient
mice had higher expression of Mac-1 than monocytes from similarly
infected wild-type mice (Table 4). Expression of Mac-1 is up-regulated
on monocytes and neutrophils on activation.13 The presence
of activated monocytes in candida-infected growth factor-deficient mice
could be due to unresolved candida infection.
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| Fig 5.
Two-color flow cytometric analysis of peripheral blood
mononuclear cells at baseline and 7 days after candida infection.
Peripheral blood mononuclear cells from (A) wild-type, (B)
G-CSF/, (C)
G-CSF//GM-CSF/ ,
and (D) G-CSF//IL-6/
mice were incubated with Mac-1 and Gr-1 antibodies. An increase
in proportion of granulocytic cells (Gr-1 and Mac-1 double positive
cells) is seen in mice of all genotypes after candida infection.
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| Fig 6.
WBCs at baseline and after infection.
Cytocentrifuge preparation of WBCs at baseline (top panel) and 7 days
after candida challenge (bottom panel) from wild-type (WT),
G-CSF-deficient mice. The cytospin preparations were stained with
May-Grünwald-Giemsa stain.
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Table 3.
Frequency of
Mac-1+Gr-1+-double positive cells in the
peripheral blood of mice 7 days after candida infection
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Table 4.
C albicans infection results in higher frequency
of Mac-1+Gr-1lo cells in peripheral blood of
knockout mice compared to wild-type mice
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Because morphologic examination of peripheral blood smears 7 days after candida infection revealed the presence of an increased proportion of immature myeloid cells compared to baseline, we also
stained WBCs with antibodies against c-fms and c-kit. The expression of
these 2 markers is regulated during the differentiation and maturation
of hematopoietic cells.14,15 It has been shown previously
that both c-kit+c-fms and
c-kit+c-fms+ cells have colony-forming capacity
in soft agar assay and represent progenitor cells.15
However, only a small proportion of
c-kitc-fms+ cells has colony-forming
capacity and most of these cells are lineage-committed myeloid cells.
At baseline, in wild-type and growth factor-deficient mice there were
primarily 2 classes of cells detected in circulation on the basis of
c-kit and c-fms staining most cells were
c-kitc-fms representing
lymphocytes and neutrophils; only a small proportion of cells were
c-kitc-fms+ (region R1), representing
monocytic cells (Figure 7). However, on day
6 after candida infection, 3 populations of cells were identified based
on their c-kit/c-fms staining pattern:
c-kitc-fms (region R4),
c-kit+c-fmslo (region R3), and
c-kit+c-fmshi (region R2) (Figure 7). On the
basis of the forward and side scatter profile it appeared that the
c-kit+c-fmslo and
c-kit+c-fmshi populations represented early
granulocytes and monocytes, respectively. The frequency of
c-kit+ c-fmslo cells in circulation
was higher in the growth factor-deficient mice compared to wild-type
mice following candida infection(Table 5).
Moreover, in growth factor-deficient mice, c-kit expression was
higher than that in wild-type mice suggesting that in wild-type mice
these cells are more differentiated (data not shown).
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| Fig 7.
Flow cytometric analysis of cell surface markers on
wild-type and G-CSF-deficient mice.
Dot blots of peripheral blood leukocytes depicting c-fms and c-kit
staining pattern at baseline (top panel) and 6 days after candida
infection (bottom panel). Region R1 represents cells that are
c-kitc-fms+. On day 6 after infection,
c-kit+ cells were seen in circulation, represented in
regions R2 (c-kit+ c-fmshi), R3
(c-kit+c-fmslo), and R4
(c-kit+c-fms).
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Increased numbers of colony-forming cells in the bone marrow of
candida- infected mice
Using a soft agar assay, we examined whether increased myelopoiesis
in the bone marrow of candida-infected G-CSF-deficient mice was
associated with an increase in the frequency of myeloid progenitor
cells. On day 1 after infection, there was a decrease in the
frequency of myeloid progenitor cells in G-CSF-deficient mice
(data not shown). However, on day 3 after infection, a significant increase in the frequency of myeloid progenitors, GM colony-forming cells (CFCs) and M-CFCs, in particular, was observed in
G-CSF-deficient mice (Figure 8). Except
for M-CFCs, which remained elevated, all other types of myeloid
progenitors returned to basal values by day 7 after infection. A
similar trend was noted in mice simultaneously deficient in G-CSF and
GM-CSF (Figure 8). In wild-type mice the changes in the frequency of
CFCs were less dramatic; only a slight increase in M-CSF
progenitor cells was observed on day 3 after infection, and the
frequency of CFCs, in general, were similar to basal values on day 7 after infection.
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| Fig 8.
Changes in frequency of colony-forming cells in the bone
marrow of wild-type, G-CSF/ -deficient, and
G-CSF//GM-CSF/ mice following
candida infection.
Data are represented as mean ± SD. Data at each time point are from
9 mice per genotype. *P < .05 comparison between mice of
same genotype at baseline and on the study day indicated.
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Candida load in kidneys of infected mice
The initial clearance of candida from blood appeared to be similar
in both G-CSF-deficient and wild-type mice and at 24 hours after
infection no candida could be detected in mice of either genotype (data
not shown). Because kidneys are the most representative organs for the
determination of in vivo growth of candida16 and candida
load in kidney underscores the severity of disseminated candidiasis,16 we determined the load of candida in the
kidneys of infected mice. At 24 hours after infection the load of
candida in kidneys of wild-type, G-CSF-deficient, and G-CSF/GM-CSF
double-deficient mice was similar (data not shown). By day 3, the
candida load had increased, and the numbers of viable organisms in
wild-type and G-CSF-deficient mice was in the same range. However, by
day 7, unlike wild-type mice where the candida load in kidney had declined and the infection appeared to be resolving, in
G-CSF-deficient and G-CSF/GM-CSF-deficient mice the load of candida
was greater than that observed on day 3 after infection (Figure
9A). This trend was confirmed on
microscopic inspection of kidney sections of infected mice stained with
hematoxylin-eosin. Whereas in infected wild-type mice the acute
inflammation appeared to be subsiding, candida-infected
G-CSF-deficient mice showed signs of acute inflammation. Both
suppurative and nonsuppurative granulomas associated with yeast and
necrosis were observed in the knock-out mice. Analysis of kidney
sections stained with Gomori methenamine stain further revealed that
whereas only a few candida (mostly yeast forms) could be seen in the
kidney of wild-type mice, both the mycelial and yeast forms of
candida were seen more commonly in G-CSF-deficient mice (Figure
10). G-CSF-deficient mice also appeared
sick and had a greater loss of body weight than wild-type
mice following infection (Figure 9B). Large numbers of
G-CSF-deficient mice had to be killed during the course of
infection due to acute illness.
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| Fig 9.
Experimental C albicans infection of wild-type
and G-CSF-deficient mice.
(A) Candida load per kidney (expressed as logarithm of the yeast count)
in wild type () and G-CSF-deficient mice (), 3 and 7 days after
experimental infection with C albicans. (B) Body weight
as a percent of pretreatment values after candida challenge. C
albicans (2.5 × 105 CFUs ) was injected through
lateral tail vein of wild-type ( ) and G-CSF-deficient mice ().
Body weights were recorded before and at the time points shown after
challenge. Ten mice of each type were studied. Data are represented as
mean ± SEM. *P < .05 for comparison of G-CSF-deficient
and wild-type mice.
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| Fig 10.
Tissue sections were prepared from kidneys of wild-type,
G-CSF/, and
G-CSF//GM-CSF/ mice 7 days after
infection with C albicans.
Sections were stained with Gomori methenamine silver stain (top panel)
and haematoxylin-eosin stain (bottom panel). (A, D) Wild-type mice show
healing inflammatory lesions in the cortex and few yeast cells. (B-C,
E-F) G-CSF-deficient and G-CSF/GM-CSF-deficient mice, respectively,
show numerous abscesses throughout the section with large aggregates of
fungal yeasts within abscesses. Inset shows polymorphonuclear
neutrophils in an area of dense leukocytic infiltration surrounding
fungal yeasts (marked by arrows).
|
|
Because G-CSF is also known to modulate neutrophil function,
we next investigated whether the neutrophils from G-CSF-deficient mice
were functionally impaired in their capacity to control candida infection. We compared the thioglycollate-elicited peritoneal neutrophils from wild-type and G-CSF-deficient mice for phagocytic and
candidacidal activity. As shown in Table 6,
the phagocytic activity of the neutrophils from both G-CSF-deficient
and wild-type mice was comparable. Although the candidacidal activity
of neutrophils from G-CSF-deficient mice appeared to be somewhat lower
than that from wild-type mice, statistical significance was not
attained.
Levels of IL-10 and TNF- in circulation
Despite developing neutrophilia, G-CSF-deficient mice had
a greater load of candida in their kidneys and had higher morbidity than similarly infected control mice. Earlier reports have suggested that susceptibility of mice to candida can be due to increased production of IL-10, which in turn can suppress macrophage
function.17 On the other hand, TNF- is thought to be
beneficial during candida infection.18 Indeed,
TNF-deficient mice are more susceptible than wild-type mice to candida
infection.19 To investigate whether the increased
susceptibility of G-CSF-deficient mice to candida was due to an
altered pattern of production of IL-10, TNF-, or both, we measured
the circulating levels of IL-10 and TNF- in candida-infected
G-CSF-deficient and wild-type mice. Low levels of IL-10 were detected
in the sera of the infected mice of both genotypes (data not shown).
However, levels of TNF- were significantly elevated in the sera of
candida-infected wild-type and G-CSF-deficient mice compared to basal
levels (Figure 11). Until 48 hours, the levels of TNF- in G-CSF-deficient and wild-type mice were similar. At 72 hours after candida infection, TNF- was undetectable in circulation in wild-type mice; in G-CSF-deficient mice the level of
TNF- remained elevated and was still detectable 7 days after candida
infection.
View larger version (23K):
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| Fig 11.
Kinetics of TNF- production.
Wild-type and G-CSF-deficient mice were challenged with
2.5 × 105 C albicans intravenously. At the
indicated times, 6 mice of each genotype were killed. Serum samples
from each mouse were tested for TNF- activity as described in
"Materials and methods." Data are represented as mean ± SD. One of 2 separate experiments with similar results is
shown.
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Discussion |
The generation of mice lacking growth factors and cytokines has
provided the means of definitively evaluating their normal physiologic
role. Furthermore, experimental infection of factor-deficient mice with
various pathogens provides the means of gauging the role
played by these factors/cytokines in the host's response to infection.
Until recently, granulopoiesis, both at steady state and during
emergency, has been believed to be regulated primarily by G-CSF.
Studies with G-CSF-deficient and G-CSF receptor-deficient mice have
led to the conclusion that although G-CSF plays an important role in
steady-state granulopoiesis, there exist G-CSF-independent pathways for
generation of granulocytes at steady state.4,5 Although
historically G-CSF has been thought to play a critical role in
emergency granulopoiesis, this remains largely
inferred.6,20 An initial attempt to gauge the effect of
G-CSF-deficiency on the host's capacity to mount an emergency
granulopoietic response was evaluated by infecting G-CSF-deficient
mice with Listeria monocytogenes.4 Compared to
similarly infected control mice, G-CSF-deficient mice mounted only a
modest neutrophilia; the load of organisms in spleen and liver was
higher in infected G-CSF-deficient mice.4 This observation
indicated that G-CSF is indispensable for mounting emergency
granulopoietic response to infection with L monocytogenes in
vivo. However, it is known that macrophages are the predominant
cell type involved in host's response to infection with L
monocytogenes.21,22 Earlier studies have demonstrated that neutrophils represent the first and most important line of host
defense against C albicans.16,23,24 Moreover,
neutrophilia has been observed in both humans and mice following
candida infection.25 Because different pathogens evoke
different cellular responses, we chose C albicans as a model
pathogen to study emergency granulopoiesis and to investigate the role
of G-CSF in this process.
Our observation that candida-infected G-CSF-deficient mice mount a
profound and sustained neutrophilia in G-CSF-deficient mice following
candida infection is intriguing. The observation indicates that in vivo
factors other than G-CSF can promote neutrophil production during an
infection. In addition to peripheral blood neutrophilia there was an
increase in both early and late forms of neutrophils in the bone marrow
of candida-infected G-CSF-deficient mice. The increase was, however,
more marked in neutrophil precursors (promyelocyte and myelocyte)
compared to late forms of neutrophils (metamyelocyte and bands). This
may be due to increased trafficking of mature neutrophils from bone
marrow to sites of infection in the tissues, due to prolonged candida
infection in G-CSF-deficient mice. Alternatively, there are fewer cell
divisions or a greater proportion of cells die between the early to
late transition in neutrophil development. Because the candida-induced
neutrophilia in G-CSF-deficient mice is sustained and is also
accompanied by an increase in both precursor and mature neutrophils in
bone marrow, this clearly indicates that this phenomenon is not merely
due to mobilization of neutrophil "reservoir pool," but involves
neutrophil production per se. Furthermore, our observation that the
increase in neutrophils in peripheral blood of candida-infected growth factor-deficient mice lagged behind the maximum increase of marrow CFUs
seen on day 3, suggests that the increased mature elements observed in
the periphery originated from marrow progenitors. Candida-infected
G-CSF-deficient mice also develop monocytosis, which is more
pronounced than that seen in wild-type mice. The presence of a higher
proportion of immature neutrophils along with monocytosis in the
peripheral blood of candida-infected G-CSF-deficient mice raises the
possibility that less committed progenitor cells (at least bipotential
progenitors) are undergoing amplification to generate neutrophils and
monocytes in candida-infected G-CSF-deficient mice. Our study has
shown the presence of cells coexpressing c-kit and c-fms in blood of
candida-infected mice. These cells have been previously shown to
represent myeloid progenitors with colony-forming capacity.15 Although we have not analyzed blood for the
presence of progenitors, the presence of
c-kit+c-fms+ cells in the blood suggests that
myeloid progenitors are mobilized cells into the periphery after
candida infection. There has been no report published so far regarding
mobilization of hematopoietic progenitor cells in G-CSF-deficient
mice; this phenomenon has, however, been found to be altered in G-CSF
receptor-deficient mice.26 In G-CSF receptor-deficient
mice, mobilization of hematopoietic progenitor cells in response to
cyclophosphamide and IL-8 is impaired; however, Flt-3 ligand-mediated
mobilization is unaffected.26 In addition to its capacity
for mobilization, Flt-3 ligand has the capacity to expand early
progenitor cells. It is therefore possible that the increased
myelopoiesis in the growth factor(s)-deficient mice in response to
candida infection is mediated by Flt-3 ligand. However, because Flt-3
ligand could not be detected in circulation in candida-infected
wild-type or growth factor(s)-deficient mice, it is unlikely that this
effect is being mediated by Flt-3 ligand (data not shown).
On the basis of the data presented here we conclude that both GM-CSF
and IL-6 are dispensable for the observed neutrophilia. Clearly there
exists in vivo a factor(s), other than G-CSF, GM-CSF, and IL-6, that
can promote granulopoiesis. A similar conclusion was drawn by Zhang et
al27 in their study on C/EBP knockout mice. These mice
do not express G-CSF and IL-6 receptors and have a complete blockade in
granulocytic differentiation. However, in this study, the expression of
these receptors on fetal liver hematopoietic cells by retroviral
transduction resulted in only partial restoration of granulopoiesis in
vitro. In the study the authors raised the possibility of existence of
the other C/EBP target genes, possibly cytokine receptors, that are
also important for the block of granulocyte differentiation in
C/EBP-deficient mice.27
Despite developing neutrophilia, G-CSF-deficient mice were found to be
more susceptible to C albicans infection than wild-type mice.
In response to candida infection, the loss in body weight was greater
in G-CSF-deficient mice than wild-type mice. Candida-infected G-CSF-deficient mice also had a greater load of candida in their kidneys and showed more extensive tissue damage. However, this was
unlikely to be due to impaired neutrophil function because phagocytic
and candidacidal activity of neutrophils from G-CSF-deficient mice was
comparable to that of neutrophils from wild-type mice. The prolonged
infection may be due to the basal neutropenic state of G-CSF deficiency
rather than some functional deficiency of neutrophils in the absence of
G-CSF.
Both TNF- and IL-6 levels were elevated in candida-infected
G-CSF-deficient mice as compared to similarly infected wild-type mice.
These cytokines seem to have a dual role during infection with
pathogens. Although the absence of TNF- and IL-6 has been shown to
aggravate candida infection in mice,19,28 high levels of
these cytokines correlate with the fatal outcome following septicemia.29,30 Elevated levels of TNF- and IL-6,
coupled with a higher candida load in the kidney, are probably the key factors responsible for greater loss in body weight and higher mortality in candida-infected G-CSF-deficient mice than similarly infected wild-type mice.
Taken together these results clearly demonstrate that emergency
granulopoiesis, particularly in response to candida infection, can
occur in the absence of G-CSF. Furthermore, GM-CSF and IL-6 are
dispensable for candida-driven neutrophilia in G-CSF-deficient mice.
Our current efforts are focused on identifying factor(s) that account
for residual neutrophils in G-CSF-deficient mice as well as those that
underlie the G-CSF-independent neutrophilia after infection with C
albicans.
| |
Acknowledgments |
We are thankful to T. Helman, B. Morrow, and M. Pitt for assistance in
the animal house; J. Strickland for assistance with the artwork; and
Prof A. W. Burgess for critically reviewing the manuscript.
| |
Footnotes |
Submitted November 29, 1999; accepted February 3, 2000.
Reprints: Sunanda Basu, Ludwig Institute for Cancer
Research, PO Royal Melbourne Hospital, Victoria 3050, Australia; e-mail: sunanda.basu{at}ludwig.edu.au.
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.
| |
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Abstract
Introduction