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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 863-869
Essential Roles for Granulocyte-Macrophage Colony-Stimulating Factor
(GM-CSF) and G-CSF in the Sustained Hematopoietic Response of
Listeria monocytogenes-Infected Mice
By
Yifan Zhan,
Graham J. Lieschke,
Dianne Grail,
Ashley R. Dunn, and
Christina Cheers
From the Department of Microbiology, the University of Melbourne,
Parkville, Victoria, Australia; and the Melbourne Tumour Biology
Branch, Ludwig Institute for Cancer Research, The Royal Melbourne
Hospital, Victoria, Australia.
 |
ABSTRACT |
The in vivo roles of granulocyte-macrophage colony-stimulating
factor (GM-CSF) and granulocyte (G)-CSF were studied in
factor-deficient gene-targeted knockout mice infected with the
facultative intracellular bacterium Listeria monocytogenes.
Previous results showed that G-CSF / mice had an underlying
selective deficiency in granulopoiesis, but GM-CSF/ mice had
little disturbance in resting hematopoiesis. Nevertheless, in this
study it is revealed that 3 days after intraperitoneal infection with 2 × 105 Listeria, GM-CSF/ mice harbored
50-fold more organisms in their spleen and liver than similarly
infected wild-type mice. This was accompanied by a severe depletion of
bone marrow hematopoietic cells and a deficient inflammatory response
in their peritoneal cavity. Thus, GM-CSF is essential for emergency,
but not resting, hematopoiesis. In contrast, G-CSF/ mice were
markedly susceptible to low doses (2 × 104) of
Listeria intraperitoneally. After infection, the acute (1 day)
granulocyte infiltration to the peritoneal cavity was normal compared
with wild type, but the more prolonged monocyte response was deficient,
reflecting a continued decrease in bone marrow cellularity and
hematopoiesis over 3 days, which was not observed in infected wild-type
mice. It is thus apparent that G-CSF deficiency affects monocytopoiesis
as well as granulopoiesis during infection.
| |
INTRODUCTION |
THE COLONY-STIMULATING factors (CSFs) are
an important group of cytokines discovered for their ability to support
the culture of hematopoietic cells in vitro. In vitro studies indicated
that CSFs support the survival, proliferation, differentiation, and end
cell function of myeloid cells.1 For example,
granulocyte-macrophage colony-stimulating factor (GM-CSF) supports the
in vitro growth of neutrophilic granulocytes and of macrophages from
their common progenitor cells and enhances the functions of
granulocytes,2 macrophages,2 and dendritic
cells,3 whereas G-CSF controls the survival, proliferation,
differentiation, and function of mature neutrophilic granulocytes and
their precursors.4 However, it has been difficult to
confirm an in vivo physiological role for the various CSFs in
hematopoiesis. One definitive approach to studying the in vivo role of
cytokines is the development of gene-targeted knockout
mice.5 In particular, the generation of mice with targeted
disruption of the G-CSF6 or GM-CSF7,8 genes is
aiding the study of the in vivo roles of these two CSFs.
We have previously reported that the G-CSF-deficient mice suffer a
chronic neutropenia associated with a deficiency in granulopoietic precursor cells.6 The G-CSF/ mice were highly
susceptible to intravenous infection with the facultative intracellular
bacterium Listeria monocytogenes. In contrast, although
suffering a characteristic pulmonary disease,8 the
GM-CSF-deficient mice show little disturbance in resting
hematopoiesis, with normal numbers of colony forming cells when their
bone marrow was cultured in any of a variety of hematopoietic growth
factors, and normal numbers of granulocytes and monocytes in the
blood.7 This surprisingly indicated that despite its
potency in vitro, at least for baseline hematopoiesis, GM-CSF was
apparently redundant in vivo.
Against this background, we undertook to study the response of the
GM-CSF/ mice to the facultative intracellular bacterium L
monocytogenes. This organism provided the original definition of
cell-mediated immunity (CMI) and has since been used extensively to
study the control of CMI.9 In the murine infection, L
monocytogenes survives within macrophages and liver parenchymal
cells. The early inflammatory response is critically important in
natural resistance, and the major gene governing resistance to this
infection in mice determines the time of onset of that
response,10,11 apparently via its effects on the number of
hematopoietic cells in bone marrow and spleen.12 Macrophage
activation to increased bactericidal activity by interferon- is a
key event in acquired cellular resistance to this and other
intracellular bacteria.13 Until recently, it was assumed
that granulocytes played little role in resistance to intracellular
bacteria, but depletion studies using monoclonal antibody (MoAb) to the
Gr1 marker have shown that granulocytes are essential in both the
primary and secondary infection with Listeria.14,15
The granulocytes are believed to lyse infected parenchymal cells, releasing the bacteria and exposing them to killing by activated macrophages/monocytes.16 It is the importance of both
granulocytes and macrophages which makes this infection a suitable
choice to test the role of GM-CSF and G-CSF in emergency hematopoiesis
in knockout mice. Despite the apparent redundancy of GM-CSF in resting hematopoiesis, the study of intraperitoneal Listeria infection in GM-CSF-deficient mice showed that there was a deficiency in the
emergency hematopoietic response to Listeria infection,
although such deficiency was not as striking as that seen in
G-CSF-deficient mice.
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MATERIALS AND METHODS |
Mice and bacteria.
GM-CSF/ and G-CSF/ mice were produced by targeted
disruption of the respective genes in 129 occipitolaeva anterior (OLA) embryonal stem cells which, after selection, were injected into C57BL/6 blastocysts.6,7 Both 129/OLA and
C57BL/6 mice are genetically resistant to Listeria infection
and show quantitatively similar responses to infection. The CSF/
mice and wild-type control mice used in these experiments were
comparably outbred and maintained in the Department of Microbiology,
University of Melbourne. The mice were housed in isolation and fed
sterile pellets and water to maintain their infection-free status.
Listeria monocytogenes were maintained by weekly subculture on
horse blood agar (HBA). Mice 6 to 8 weeks old were infected
intraperitoneally with 2 × 104 or 2 × 105 Listeria from a 24-hour HBA culture and the
dose was checked retrospectively.
Quantitation of infection.
Mice were killed by CO2 narcosis. Spleen and liver were
removed aseptically and individually homogenized in normal saline with
an Ultra Turrax homogenizer (Janke and Kunkel KG, Breisgau, Germany).
The numbers of Listeria in the organs were established by
plating serial 10-fold dilutions of organ homogenates in saline on an
HBA plate and incubating at 37°C for 24 hours.
Cell preparation.
Bone marrow cells were prepared by flushing the tibia with an enriched
Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) with
10% fetal calf serum (DMEM + 10% FCS) using a syringe with a 25-gauge needle. The cells were centrifuged at 800g for 7 minutes, resuspended in DMEM + 10% FCS, and counted. Peritoneal cells were prepared by washing peritoneal cavities with 5 mL DMEM + 10% FCS. Peritoneal cells were pelleted by centrifuge at 800g for 7 minutes and resuspended in 10 mL DMEM + 10% FCS.
MoAbs.
For MoAbs against the granulocyte marker Gr1 (RB6-8C5),17
ammonium sulfate-precipitated -globulin from ascitic fluid was used.
For MoAbs against the macrophage marker F4/80,18 culture supernatants were used. Ammonium sulfate-precipitated -globulin from
rat serum was used as a negative control.
Phenotypic analysis.
Bone marrow cells were washed once in phosphate-buffered saline (PBS,
pH 7.2) with 5% FCS. Cells were then mixed with appropriately diluted
MoAbs on ice for 30 minutes. After washing twice in PBS-5% FCS, cells
were incubated with fluorescein isothiocyanate-labeled sheep anti-rat
Ig (mouse absorbed) (Silenus, Hawthorn, Australia) on ice for 30 minutes. After two washes in PBS, cells were analyzed by FACSort
(Becton Dickinson, San Jose, CA). Cell populations were analyzed and
percentages calculated using the Becton Dickinson Immunocytometry
system "Cell Quest" application. Because the F4/80 marker is
downregulated on macrophage activation,18 it was not a
satisfactory marker of inflammatory macrophages/monocytes in the
peritoneal cavity. Therefore, these cells were assessed
microscopically. Peritoneal cells were spun through an FCS cushion in a
cytocentrifuge onto microscopic slides and stained with Diff-Quick
(Lab-Aids, Narrabeen, New South Wales, Australia). For differential
counts, 500 cells per slide were scored.
Assay for colony-forming cells (CFCs).
CFC assays were performed as described.19 Triplicate
cultures containing 5 × 104 bone marrow cells in 1 mL
semi-solid agar were placed in 35-mm Petri dishes (Becton Dickinson,
Oxnard, CA). To this was added 0.1 mL of a 1 in 4 dilution of serum
prepared from BALB/c mice which had been injected
intraperitoneally with 5 mg lipopolysaccharide w
(Escherichia coli 011:B4; Difco Laboratories, Detroit, MI) 6 hours earlier. This lipopolysaccharide (LPS) serum was used to support
CFC growth because LPS serum reflects the cocktail of CSFs that are
encountered during infection.20 Cultures were incubated at
37°C for 6 days and colonies containing more than 50 cells were
counted.
Statistics.
The statistical significance of the experimental data was determined by
Student's t-test.
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RESULTS |
Listeria infection in GM-CSF/ mice.
Sex- and age-matched GM-CSF/ and wild-type mice were infected
intraperitoneally with 2 × 105 Listeria
organisms. The intraperitoneal route of infection was chosen because it
allows ready observation of the phagocytic cells drawn to that site.
The rate of infiltration of cells to the peritoneal cavity correlates
strictly with natural genetic resistance in different strains of
mice.10 Mice were killed 1 or 3 days after infection for
bacterial counts in liver and spleen (Fig
1A and B). At day 1 postinfection,
bacterial numbers in the GM-CSF/ mice were similar to the numbers
in wild-type mice, but by day 3 the GM-CSF/ mice showed more than
50-fold exacerbation compared with wild-type mice
(P < .01). The experiments were terminated at this time
because this dose was uniformly lethal for GM-CSF/ mice after 4 or more days, and lethal also for a proportion of wild-type controls.
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| Fig 1.
Bacterial numbers and inflammatory responses in
GM-CSF/ mice. GM-CSF knockout mice ( ) or wild-type mice ( )
were infected intraperitoneally with 2 × 105
Listeria for 1 or 3 days. Bacterial load and peritoneal cells were quantitated. Data represent mean and standard deviation of groups
of five mice. *P < .05 and **P < .01 compared
with wild-type mice. Results were from one of three similar
experiments. (a) Bacterial counts in spleen. (b) Bacterial counts in
liver. (c) Total cells recovered per peritoneal cavity. (d) Neutrophils
per peritoneal cavity. (e) Macrophages and monocytes per peritoneal cavity.
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Numbers of peritoneal cells in uninfected mice did not differ
significantly between GM-CSF/ and wild-type mice (Fig 1C). After
intraperitoneal infection with 2 × 105 Listeria,
the number of cells in the peritoneal cavity of GM-CSF/ mice was
only half that in wild-type mice by 3 days after infection. There was
no significant difference in numbers of neutrophils and macrophages
between wild-type and GM-CSF/ mice, either uninfected or 1-day
infected (Fig 1D and E). However, by 3 days after infection the numbers
of macrophages were significantly lower in GM-CSF/ mice compared
with wild-type mice (Fig 1E). The percentage of neutrophils and
macrophages was not significantly different in the peritoneal cavity of
uninfected or 1-day infected wild-type and GM-CSF/ mice (Table
1). By day 3 there was a significantly higher percentage of typical macrophages in the wild-type mice. It was
of interest that approximately half of the peritoneal cells (53% ± 5%) from GM-CSF/ mice contained visible
Listeria organisms 3 days after infection. Only one quarter
(27% ± 5%) of the cells from wild-type mice contained visible
bacteria at this time and there were considerably fewer bacteria per
cell.
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Table 1.
Percentage of Cells in the Peritoneal Cavity and Bone
Marrow of GM-CSF/ Mice and Wild-Type Mice Before and After
Intraperitoneal Infection
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Hematopoietic response of GM-CSF/ mice to infection.
The explanation for this impairment in defense against infection was
sought by analysis of the bone marrow progenitors. Consistent with the
known data,7 the total numbers of bone marrow cells and the
numbers of CFCs recovered from the tibia of uninfected GM-CSF/
mice were similar to those from wild-type mice (Fig 2A and
B). After intraperitoneal infection with 2 × 105 Listeria organisms, bone marrow cellularity
showed a steady depletion in both GM-CSF/ and wild-type mice. By
day 3, numbers in GM-CSF/ mice were significantly lower than in
wild-type mice (Fig 2A). The numbers of CFCs recovered from the tibia
also showed a decline which was significantly greater in the
GM-CSF/ mice than wild-type mice (Fig 2B).
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| Fig 2.
Number of bone marrow cells in wild-type and
GM-CSF/ mice following Listeria infection. GM-CSF/
mice () and wild-type mice () were either uninfected or infected
intraperitoneally with 2 × 105 Listeria for 1 or
3 days. Bone marrow cells were flushed from the tibia of individual
mice. Data represent the mean and SD of individual tibia of five mice
in each group. *P < .05 and **P < .01 compared with
wild-type mice which were infected with the same dose of
Listeria. Results were from one of three similar experiments.
(a) Total cells recovered per tibia. (b) Colony forming cells per
tibia. (c) Gr1+ cells per tibia. (d) F4/80+
cells per tibia.
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Phenotypic analysis of bone marrow cells by FACS showed no deficit in
Gr1+ or F4/80+ cells in uninfected
GM-CSF/ mice compared with wild-type (Fig 2C and D). By day 3, total numbers of Gr1+ cells in GM-CSF/ mice were only
half those in wild-type mice, whereas no differences in the total
numbers of F4/80+ cells were noted. The percentages of
Gr1+ and F4/80+ cells in the bone marrow were
not consistently different between the two types of mice (Table 1). The
slight excess of granulocytes in GM-CSF/ mice was not observed in
other experiments.
It should be noted that all the above results involved challenge with a
relatively high dose of Listeria (2 × 105).
When mice were injected intraperitoneally with 2 × 104
Listeria organisms, a dose which resulted in marked
exacerbation in GM-CSF/ mice (below), in four out of seven
experiments there was no exacerbation of infection in the GM-CSF/
mice. In those experiments in which no exacerbation was observed,
similar numbers of bone marrow cells, CFCs, and peritoneal exudate
cells were found in the two mouse strains 3 days postinfection. On the
other hand, in the three experiments where exacerbation was observed there were deficiencies in the bone marrow cells and peritoneal exudate
cells similar to those described above for higher doses of
Listeria (results not shown).
Listeria infection in GM-CSF/ mice.
In contrast to GM-CSF/ mice, G-CSF/ mice have reduced
numbers of granulocytes and hematopoietic cells under resting
conditions.6 Our previous study showed that susceptibility
of G-CSF/ to intravenously injected Listeria was
associated with a deficient blood inflammatory response to
infection.6 To allow comparison with intraperitoneally infected GM-CSF/ mice (above), GM-CSF/ and wild-type
controls were infected intraperitoneally in the present study with 2 × 104 Listeria organisms. The low dose (compared
with 2 × 105 for most of the GM-CSF/ experiments)
was necessitated by the marked susceptibility of the G-CSF/ mice
to infection.6 Groups of mice were killed 1 or 3 days
later. Consistent with the previous study of intravenous infection, at
1 day after infection, bacterial counts in liver and spleen of
G-CSF/ mice were similar to counts in wild-type mice, indicating
no impairment of the initial ability of resident macrophages to capture
the organisms. However, by 3 days postinfection, spleens and livers of
G-CSF/ mice harbored at least 100 times more Listeria
than wild-type mice (Fig 3A and B).
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| Fig 3.
Bacterial numbers and inflammatory responses in
G-CSF/ mice. G-CSF knockout mice () or wild-type mice
() were infected intraperitoneally with 2 × 104
Listeria for 1 or 3 days. Bacterial load and peritoneal cells were quantitated. Data represent mean and standard deviation of groups
of five mice. ***P < .001 compared with wild-type mice. Results were from one of three similar experiments. (a) Bacterial counts in spleen. (b) Bacterial counts in liver. (c) Total cells recovered per peritoneal cavity. (d) Neutrophils per peritoneal cavity.
(e) Macrophages and monocytes per peritoneal cavity.
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To test the local inflammatory response to Listeria infection
in G-CSF/ mice, the total cellularity and cell subpopulations in
the peritoneal cavity were monitored after intraperitoneal infection.
Before infection, the numbers of cells in the peritoneal cavity were
similar in G-CSF/ mice and wild-type, and there were very few
granulocytes in either strain (Fig 3C through E). One day
postinfection, cell numbers were variable and differences not
significant, but by day 3 the G-CSF/ mice showed an approximately twofold deficit in macrophages (Fig 3C and E). This reflected the fact
that total cells in the peritoneal cavity of wild-type mice 3 days
postinfection were almost two times the normal number, whereas in
G-CSF/ mice they had actually decreased to half of the normal
number. This was a reproducible finding, despite some mouse-to-mouse
variation. Percentages of granulocytes and macrophages/monocytes in the
peritoneal cavity (Table 2) showed no
selective deficiency in the granulocyte inflammatory response,
accentuating the monocyte/macrophage deficiency. Furthermore,
histological examination of lesions in the infected liver and spleen
revealed no selective deficiency in the cell subsets (data not shown).
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Table 2.
Percentage of Cells in the Peritoneal Cavity and Bone
Marrow of G-CSF/ Mice and Wild-Type Mice Before and After
Intraperitoneal Infection
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Hematopoietic response of GM-CSF/ mice to infection.
The total number of bone marrow cells recovered from the tibia cavity
of G-CSF/ mice and wild-type mice was not significantly different
in uninfected mice (Fig 4A). Twenty-four
hours after intraperitoneal infection with 2 × 104
Listeria, there was a drop in numbers of bone marrow cells
recovered from the tibia, which was not significantly or consistently
different in G-CSF/ and wild-type mice. By 3 days after
infection, cell numbers in the tibia of wild-type mice were steady, but
had decreased still further in G-CSF/ mice, to less than half of
the numbers in uninfected G-CSF/, or in 3-day infected wild-type
mice.
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| Fig 4.
Number of bone marrow cells in wild-type and G-CSF/
mice following Listeria infection. G-CSF/ mice () and
wild-type mice () were either uninfected or infected
intraperitoneally with 2 × 104 Listeria for 1 or
3 days. Bone marrow cells were flushed from the tibia of individual
mice. Data represent the mean and standard deviation of individual
tibia of five mice in each group. *P < .05, **P < .01, and ***P < .001 compared with
wild-type mice which were infected with the same dose of
Listeria. Results were from one of three similar experiments.
(a) Total cells recovered per tibia. (b) Colony forming cells per
tibia. (c) Gr1+ cells per tibia. (d) F4/80+
cells per tibia.
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The potential to produce granulocytes and monocytes/macrophages in the
bone marrow of Listeria infected mice was tested by measuring
CFCs. Even before infection there was a significant deficiency in CFCs
in uninfected G-CSF/ mice compared with wild-type controls (Fig
4B). One day postinfection, the numbers in wild-type mice had increased
over the numbers in uninfected mice, but in G-CSF/ mice they had
declined. By 3 days after infection, numbers of CFCs in wild-type mice
were about 11/2 times normal. However, the numbers of CFCs in
G-CSF/ mice had declined to half the numbers in uninfected mice
and were only  the numbers in wild-type mice.
Phenotypic analysis of bone marrow cells by FACS showed a striking
deficit in the absolute numbers (Fig 4C) and the percentage (Table 2)
of granulocytes in G-CSF/ mice compared with wild-type mice. In
uninfected mice, almost 50% of bone marrow nucleated cells in
wild-type mice were Gr1+, whereas in G-CSF/ mice only
12% of bone marrow cells were Gr1+. Although the
percentage of Gr1+ cells in bone marrow increased somewhat
during infection of the G-CSF/ mice, absolute numbers remained
low. The initial deficit in the percentage of F4/80+
monocytic cells (Table 2) in the bone marrow of G-CSF/ mice was
less striking (11% compared with 18% in wild-type mice, and not
significantly different). However, the difference in absolute numbers
increased with time because of the decline in total cellularity of the
bone marrow (Fig 4D).
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DISCUSSION |
The initial reports of normal baseline hematopoiesis in
GM-CSF-deficient mice7,8 raised the possibility that,
despite its potency in support of hematopoiesis in vitro, GM-CSF is not mandatory for balanced granulocyte and monocyte/macrophage development in vivo. Our data now show that, with a sufficient infective challenge, an essential hematopoietic role for GM-CSF can be shown in vivo, and
hence GM-CSF is not wholly redundant with respect to hematopoiesis. Although infection with typical experimental challenge doses of Listeria did not reproducibly reveal a hematopoietic impairment in GM-CSF-deficient mice, when higher doses were used, GM-CSF/ mice failed to control the infection. This failure of host defenses was
accompanied by deficiencies in both the granulocyte lineage (in the
marrow) and the monocyte/macrophage lineage (in the peritoneal cavity),
consistent with the in vitro and in vivo effects of GM-CSF on both
these lineages. The fact that the susceptibility of GM-CSF-deficient mice to Listeria was only manifest at high infecting doses
indicates that emergency host defenses are intact, but it is an
inability to sustain host defences or a lack of reserves of
responsiveness that is missing.
Previously in experiments using the intravenous route of infection, we
had shown that G-CSF-deficient mice were susceptible to
Listeria infection.6 In view of the baseline
hematopoietic defect in G-CSF/ mice, this suggested that a
G-CSF-primed bone marrow was necessary for a normal defense against
infection. The deficits observed in the intraperitoneally infected
GM-CSF-deficient mice prompted a reappraisal of the response of
G-CSF/ mice to Listeria infection, using this route of
infection. While confirming the previous observations,6
several new insights emerged. Despite the image of G-CSF as a factor
primarily supporting the development and function of neutrophilic
granulocytes, the failure of hematopoiesis in Listeria-infected
G-CSF-deficient mice clearly involves not only the granulocytic
lineage but also the monocyte/macrophage lineage. Impaired
monocyte/macrophage lineage production is evident in the
blood,6 in the local peritoneal inflammatory response, and
in the bone marrow itself (present studies).
Because of the different basal states of total- and
granulocyte-hematopoiesis in these two CSF-deficient mouse lines, it is difficult to make direct comparisons. The marked baseline
granulopoietic deficit in G-CSF-deficient mice precludes isolated
evaluation of the nature of an emergency granulopoietic response,
whereas in GM-CSF-deficient mice with normal baseline hematopoiesis, a high dose of Listeria unmasks an inability to sustain an
emergency response without GM-CSF. On the other hand, monocytopoiesis
shows little difference in the two strains before infection, and the impairment of monocytopoiesis in the face of infection is of a similar
magnitude in either G-CSF- or GM-CSF-deficient mice.
The importance of G-CSF in listeriosis is consistent with the elevated
serum levels (>1,000 U/mL) observed during infection.20 In contrast, serum GM-CSF levels are low (<50 U/mL), although evidence is accumulating that endogenous mechanisms do not generally use GM-CSF as a freely circulating cytokine (G.J.L., unpublished, 1996). Indeed, more detailed analysis of events after
infection throws light on the mechanisms of action of these two
cytokines during infection. In both GM-CSF/ and G-CSF/
mice, the initial localization of bacteria in the spleen and liver was
the same as in wild-type mice, indicating there was no deficiency in
the ability of resident macrophages to capture the invading
microorganisms. Defects become apparent later.
As might be expected in mice suffering no disturbance of resting
hematopoiesis, the numbers of cells reaching the peritoneal cavity of
GM-CSF mice were not deficient at 1 day postinfection, and only became
significant by day 3, when numbers were only half those in wild-type
mice. By this time the neutrophil response had waned, and the
deficiency was reflected particularly in macrophage numbers. It is
difficult to say whether this final failure of the inflammatory system
in the GM-CSF/ mice allowed the increase in bacterial numbers, or
if the failure in hematopoiesis and inflammation is secondary to the
high bacterial load reached by 3 days. The fact that GM-CSF/ mice
given a lower dose of Listeria often showed no exacerbation of
infection and no deficiency in their 3-day hematopoietic or
inflammatory responses favors the latter interpretation. What, then,
exacerbates infection? Although a deficient inflammatory response can
certainly exacerbate infection,11 the peritoneal cells of
GM-CSF/ mice showed functional deficiencies (Y.Z. and C.C.,
manuscript in preparation), in particular a reduced capacity to produce nitric oxide. Nitric oxide is of primary importance in macrophage killing of intracellular bacteria21 and the
deficiency would undoubtedly contribute to the exacerbation of
infection. This result, suggesting a role for GM-CSF in macrophage
activation, is the converse of the activation of peritoneal cell
function observed in transgenic mice overexpressing
GM-CSF.22
It is of interest that at the site of infection in the peritoneal
cavity of G-CSF/ mice there was no significant difference in
numbers of granulocytes compared with wild-type mice. However, by 3 days postinfection, there was a marked deficit in total cellularity and
in the number of monocytes/macrophages in the G-CSF/ mice. Our
earlier observation of the inflammatory response in the blood after
intravenous infection also showed a deficit in the monocytic response.6 However, mature monocytes and macrophages do not carry receptors for G-CSF. This raises the possibility that, after infection, G-CSF contributes to the expansion of stem cell precursors common to all of these hematopoietic cells. G-CSF has been shown in
vitro to act synergistically with interleukin-3 (IL-3), IL-1, and IL-6
in different systems to promote the proliferation or survival of
primitive multipotent progenitor cells.23 In mice whose
bone marrow was depleted with 5-fluorouracil, G-CSF and IL-1 act
synergistically to stimulate multilineage hematologic recovery.24
It is not surprising that a halving of the numbers of CFCs in the bone
marrow of G-CSF/ mice could increase susceptibility to L
monocytogenes infection. Naturally susceptible BALB/c mice have
only half the numbers of bone marrow CFCs compared with resistant C57BL mice, and this forms the basis of the major gene
governing resistance of mice to listeriosis.12 What is more
surprising is the fact that the deficiency of neutrophilic granulocytes
in the G-CSF/ mice is not more profound. This indicates a
redundancy in the action of CSFs or other cytokines controlling
myelopoiesis. It is relevant to note that the CSFs are not the only
cytokines which control myelopoiesis during infection. Depletion of
IL-6 during listeriosis markedly exacerbates infection25
and may act at least in part by controlling granulocyte
production.26 Thus, it would be of interest to test the
effect of further depletion of IL-6 in these mice.
These experiments with gene-targeted knockout mice indicate that
although a particular factor may initially appear redundant for some
expected functions, closer examination is likely to reveal that it does
play a specialized role under some circumstances. In GM-CSF-deficient
mice, the in vivo hematopoietic role of GM-CSF only becomes crucial
under the stress of a high-infecting dose of bacteria.
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FOOTNOTES |
Submitted July 14, 1997;
accepted September 29, 1997.
Supported by the National Health and Medical Research Council of
Australia Project Grant No. 921126.
Address reprint requests to Christina Cheers, PhD,
Department of Microbiology, University of Melbourne, Parkville,
Victoria, 3055, Australia.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance wth 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Sunanda Basu for helpful discussions.
| |
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Abstract
Introduction
Abstract