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*
Centre d’Immunologie de Marseille-Luminy, Marseille, France;
Département d’Anatomie, Université de Montréal, Québec, Canada;
Programa de Investigación en Enfermedades Tropicales, Universidad Nacional, Heredia, Costa Rica; and
§
Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, IL-6, IL-1ß, granulocyte-macrophage CSF
(GM-CSF)3, and the
granulocyte CSF (G-CSF) in host resistance against bacterial infections
2, 4, 5, 6, 7 . Particularly during Brucella infection, IFN-
and IL-2 seem to control the growth of both avirulent or virulent
Brucella abortus strains in the murine J-774 macrophages,
whereas IL-1, IL-4, IL-6, TNF-, and GM-CSF do not have a
consistent effect 8 . In IFN--activated peritoneal macrophages,
attenuated strain 19 is eliminated, whereas virulent strain 2308 can
replicate slower than in untreated macrophages and cannot be eliminated
9 .
Several transcription factors have been identified as playing a role in
regulating the macrophage activation program. Among them, NF-IL-6 and
NF-
B are expressed at a high level in LPS-induced macrophages 10, 11 , Stat-1 is involved in the transcriptional activation of
IFN--induced genes 12 , and IFN regulatory factor-1,
expressed during macrophage differentiation 13 , controls the
production of nitric oxide (NO) 14 . NF-IL-6, also known as
liver-enriched transcriptional activator protein, -1 acid
glycoprotein/enhancer binding protein, IL-6-dependent DNA binding
protein, CCAAT/enhancer binding protein ß, or NF-myeloid, was
identified as a transcriptional activator of a variety of genes. Its
binding motifs were found in the regulatory regions of genes whose
expression is specifically induced upon macrophage activation 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 .
High levels of NF-IL-6 correlate with the induction of cytokine
expression 26 , suggesting that NF-IL-6 is a regulatory component for
the expression of macrophage-specific cytokines. Upon activation of
NF-IL-6(-/-) macrophages by IFN- and LPS, induction of
transcription of TNF-, IL-6, IL-1ß, GM-CSF, macrophage CSF, IL-10,
and IL-12 was comparable to that observed in normal mice 6 .
Strikingly, no induction of G-CSF expression was observed in
NF-IL-6(-/-) macrophages, and this defect was restricted to
macrophages and fibroblasts 6 . Moreover, NF-IL-6(-/-) mice
displayed a high susceptibility to Salmonella and
Listeria infections, suggesting that NF-IL-6 plays a role in
controlling intracellular parasite proliferation by an as yet unknown
mechanism 6 . The generation of NO and reactive nitrogen intermediates
is known to play an essential role in bacterial killing. The production
of NO by NO synthase, induced by LPS or cytokines known to be dependent
upon the expression of IRF-1 14 , was not hampered in activated
NF-IL-6(-/-) macrophages 6 . This finding is perhaps surprising
because NO was necessary for Listeria killing in wild-type
macrophages 6 . This result suggests that the effect of NF-IL-6 is
independent of NO or may control the expression of factors required in
normal activated macrophages for bactericidal activity.
To further investigate the mechanism involved in the control of
infection by NF-IL-6, phagocytosis, endocytosis, phagosome-endosome
interactions, and replication rates of the attenuated B.
abortus vaccine strain 19 were studied in NF-IL-6(+/+) and
NF-IL-6(-/-) macrophages. We first demonstrate that the attenuated
vaccine strain 19 could replicate in IFN--activated NF-IL-6(-/-)
macrophages. We also report that NF-IL-6(-/-) macrophages are
defective in endocytosis and that membrane fusion between endosomes and
Brucella-containing phagosomes is inhibited. The addition of
G-CSF, whose expression is controlled by NF-IL-6, was sufficient to
rescue both endocytosis and endosome/phagosome fusion. Therefore,
NF-IL-6 is an important factor for host defence against infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Virulent smooth B. abortus strain 2308 was provided
by J.-M. Verger (Institut National de la Recherche Agronomique,
Nouzilly, France), and attenuated smooth B.
abortus strain 19 was obtained from Professional Biological
(Denver, CO). Murine recombinant G-CSF, IL-6, GM-CSF, TNF-, and
IFN- were from R&D Systems (Abingdon, U.K.). Escherichia
coli LPS serotype 026:B6, horseradish peroxidase (HRP), and
latex beads were from Sigma (Saint Quentin Fallavier, France).
Peroxidase-conjugated murine transferrin (HRP-Tfn) was purchased from
Pierce (Rockford, IL).
Macrophage infection
NF-IL-6(+/+) and NF-IL-6(-/-) mice (C57BL/6 x 129/sv)
were injected i.p. with 2 ml of 10% proteose peptone and killed 5 days
later. Peritoneal exudate cells were harvested by washing the
peritoneal cavity with 5 ml of PBS (pH 7.4), and seeded in 24-well
tissue culture plates in DMEM, 10% FCS, and 2 mM glutamine (cell
culture medium). In all experiments, macrophages were cultured at
37°C in a 5% CO2 atmosphere. Activated macrophages were
obtained in vitro by the addition of 100 ng/ml LPS and 100 U/ml IFN-
for 12 h before infection. Infections of 5 x 104
macrophages were performed in 0.5 ml of DMEM containing 5 x
106 bacteria (multiplicity of infection of 100) obtained
from an overnight culture. After 4 h of incubation at 37°C in
the presence of bacteria, cells were washed five times with DMEM to
remove nonadherent bacteria, and macrophages were further incubated
with DMEM supplemented with 0.25 µg/ml gentamicin. In experiments
using G-CSF, 5 x 104 macrophages were first incubated
for 36 h with 10 ng/ml of G-CSF before strain 19 infection. Strain
19 infection was performed as described above except that G-CSF was
maintained in all solutions.
Fluid phase endocytosis
A total of 106 macrophages were incubated for 5 min at 37°C with 1.5 mg/ml of HRP in DMEM, 10 mM HEPES, and 5 mM D-glucose (pH 7.4). Then, after three washes in PBS at 4°C, HRP was chased for 10, 30, and 60 min at 37°C by incubating the cells in cell culture medium. After two PBS washings, macrophages were lysed with 1% Triton X-100 in PBS. The lysates were then centrifuged in a table-top centrifuge (TLA100; Beckman Instuments, Gagny, France), the supernatants were collected, and their HRP activity was quantified as previously described 27 .
Cytokine effect on HRP uptake
A total of 106 macrophages were harvested from
NF-IL-6(-/-) and NF-IL-6(+/+) mice and then cultured for 36 h in
cell culture medium. Cells were then incubated for 36 h in the
presence or absence of different combinations of murine recombinant
G-CSF, IL-6, GM-CSF, TNF-, and IFN-. Final concentrations in
culture medium were 10 ng/ml for G-CSF, GM-CSF, and TNF- and 1000
U/ml for IL-6 and 20 U/ml for IFN-. An assay for endocytosis using
HRP internalization as previously described was then performed in the
presence of cytokines.
Transferrin uptake
HRP-Tfn was used in all experiments. A total of 106 macrophages were plated in 12-well dishes and incubated for 60 min at 37°C with DMEM and 0.2% BSA to deplete endogenous Tfn. Binding of HRP-Tfn was conducted by incubating NF-IL-6(-/-) and NF-IL-6(+/+) macrophages (in triplicate) with 30 µg/ml HRP-Tfn in DMEM, 0.2% BSA, and 20 mM HEPES (pH 7.4) for 60 min on ice. Unbound HRP-Tfn was removed by several washes with ice-cold PBS. To measure internalization and recycling of HRP-Tfn, the cells were brought to 37°C for 5, 10, and 30 min and then returned to ice, external media were collected, and cells were washed three times with 150 mM NaCl and 10 mM acetic acid (pH 3.5). The pH of the recovered acidic washes were immediately brought to pH 7.4 by the addition of 1 M Tris (pH 8.0) and HRP quantified as described above. This is a measure of surface-bound HRP-Tfn. Then, cells were lysed with PBS Triton X-100 1%, and total cell HRP activity was quantified.
Electron microscopy
Macrophages seeded in 3-cm Petri dishes in culture medium were fed with latex beads for 30 min at 37°C in normal culture medium or infected with B. abortus for 60 min at 37°C. After internalization, cells were washed thoroughly three times with cold PBS for 5 min. At the end of each experiment, cells were fixed in the dishes with 1% glutaraldehyde, postfixed in OsO4, and processed for flat embedding in Epon 812 resin as described previously 28 . Pieces of the flat embedded cell monolayer were mounted on top of a bloc of Epon and thin sections were made. Cells displaying the nucleus, indicating that sections were cut neither at the top nor at the base of the cell, were analyzed by electron microscopy.
In vivo fusion assay
Macrophages were harvested from NF-IL-6(-/-) and NF-IL-6(+/+) mice and then cultured for 36 h at 37°C in the presence or absence of 10 ng/ml murine recombinant G-CSF. Cells were then infected with Brucella S19. To visualize the fusion of Brucella-containing phagosomes with endosomes at 12 h postinfection, infected macrophages were incubated for 30 min at 37°C of DMEM containing BSA and 16-nm gold particles (OD520; 10). Cells were washed five times with DMEM and once with PBS and fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer for 30 min at 4°C, processed, and analyzed by electron microscopy as above. For the quantitative analysis of the in vivo fusion assay, intermixing of the BSA-gold and Brucella-containing phagosomes was recorded for each combination of incubations performed. The presence of a single gold particle in a Brucella-containing phagosome was considered to be a sign of fusion between the gold-labeled endosomes and the phagosomes. All experiments and time points were done twice, and at least 100 phagosomes per time point were recorded; extreme care was taken to avoid serial sections.
Immunofluorescence and confocal microscopy
The intracellular brucellae and the lysosomal marker cathepsin D were analyzed by confocal microscopy after immunofluorescence labeling as described by Pizarro-Cerdá et al. 29 . Cells were extensively washed to remove nonadherent bacteria prior fixation for 15 min with 3% paraformaldehyde in PBS, washed once in PBS, and incubated for 10 min with PBS and 50 mM NH4Cl to quench free aldehyde groups. For detection of intracellular bacteria, cells were further permeabilized with 0.05% saponin (Sigma) and incubated for 30 min with a serum (used at 1/5000 dilution in PBS, 10% horse serum) from a B. abortus-infected goat and revealed with donkey FITC-conjugated anti-goat IgG Abs (Immunotech, Marseille, France). Cathepsin D was revealed with rabbit anti-cathepsin D Abs (a gift from Dr. S. Kornfeld, St. Louis, MO) revealed with donkey Texas red-conjugated anti-rabbit IgG Abs (Immunotech). Samples mounted in Mowiol were observed under a Leica TCS 4DA confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany).
Statistical analysis
Results are expressed as the mean ± SEM except in cases in which the SE was <10% of each point value. Confidence intervals were calculated to compare the differences of a measured outcome between groups 30 . Time series were analyzed by analysis of variance (ANOVA) 31 .
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peritoneal macrophages were isolated from transgenic mice and
activated and infected with attenuated B. abortus strain 19
or virulent B. abortus strain 2308. As expected, B.
abortus strain 19 could not replicate in NF-IL-6(+/+) macrophages
compared with strain 2308 (Fig. 1
), thus
confirming that in contrast with pathogenic Brucella
strains, B. abortus strain 19 cannot survive in wild-type
murine-activated macrophages 9 . Indeed, at 72 h postinfection,
most of bacteria from strain 19 underwent degradation process, showing
bacteria debris colocalizing with the lysosomal marker cathepsin D
(Fig. 2, A and B),
whereas bacteria from strain 2308 replicated actively in the macrophage
perinuclear region (data not shown) as already observed in B.
abortus-infected HeLa cells 29 . Strikingly, Fig. 1 also shows
that the replication rate of strain 19 in NF-IL-6(-/-) macrophages
was similar to that of the pathogenic B. abortus strain 2308
in both NF-IL-6(+/+) and NF-IL-6(-/-) macrophages. At 48 h,
strain 2308 and strain 19 replicated actively within NF-IL-6(-/-)
macrophages without disrupting infected macrophages, with the number of
macrophages remaining constant (5 x 104 cells). After
48 h, bacteria started to induce the disruption of macrophage
membranes leading to the release of free bacteria in the external
medium containing antibiotics. At 72 h, 104
NF-IL-6(-/-) macrophages infected with strain 2308 or strain 19
remained attached to plastic. Fig. 2, C and D,
shows bacteria from strain 19 replicating in the perinuclear region of
NF-IL-6(-/-) macrophages devoid of cathepsin D. These results show
that in NF-IL-6(-/-) macrophages, the attenuated-Brucella
strain 19 behaves as the pathogenic strain 2308 in NF-IL-6(+/+)
macrophages. It also suggests that NF-IL-6 plays a critical role in the
killing of Brucella as previously observed for
Listeria and Salmonella 6 by a hitherto
uncharacterized mechanism. We then investigated the endocytic and
phagocytic properties of transgenic macrophages upon
Brucella infection.
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After a 5-min internalization of HRP, less than half of the HRP
activity was detected in NF-IL-6(-/-) compared with NF-IL-6(+/+)
macrophages (Fig. 3A), showing
that NF-IL-6(-/-) macrophages exhibited an important defect in
endocytosis. External media were collected at each chase time point,
and HRP activity was quantified. About 30% of total HRP activity,
measured within NF-IL-6(-/-) and NF-IL-6(+/+) macrophages after a
5-min internalization, were recovered in the extracellular medium at a
60-min chase, indicating that the proportion of recycled HRP was the
same in both types of macrophages (Fig. 3B). Transferrin
receptor-mediated endocytosis was also markedly impaired in
NF-IL-6(-/-) macrophages but not in transferrin recycling (Fig. 4). Indeed, after 15 min, only 30% of
HRP-Tfn was internalized in NF-IL-6(-/-) macrophages (Fig. 4B), compared with 80% in NF-IL-6(+/+) macrophages (Fig. 4A). However, as observed for fluid phase endocytosis (Fig. 3), recycling rates of HRP-Tfn to external media were very similar in
both NF-IL-6(-/-) and NF-IL-6(+/+) macrophages.
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To analyze the influence of cytokines exogenously added,
NF-IL-6(-/-) and NF-IL-6(+/+) macrophages were grown in the presence
of G-CSF, IL-6, GM-CSF, IFN-, or TNF-, and their ability to
internalize HRP was studied (Fig. 5). The
addition of G-CSF alone was sufficient to almost completely rescue HRP
internalization in NF-IL-6(-/-) macrophages and was more efficient
than any of the combinations of two or three of the other cytokines
tested (Fig. 5). In addition, we observed that IL-6 alone was able to
enhance endocytosis both in NF-IL-6(+/+) and NF-IL-6(-/-)
macrophages, but the effect was much lower than that of G-CSF in
NF-IL-6(-/-) macrophages (Fig. 5). It has been shown that IL-6
enhanced endocytosis in endothelial cells 32, 33 . In macrophages, it
is also known that cytokines cooperate for the modulation of
endocytosis and phagocytosis 34 . Th2 cytokines such as IL-6 and IL-4
seem to increase mannose-receptor endocytosis, whereas Th1 cytokines
such as IFN- have an opposit effect 34 . Although there may be a
modulation effect on endocytosis in NF-IL-6(+/+) and NF-IL-6(-/-)
macrophages by IFN-, GM-CSF, and TNF-, these effects are not
significant according our statistical analysis. Altogether, these
results show that G-CSF plays an important role in the control of
endocytosis in macrophages.
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Activated macrophages were incubated with latex beads for 30 min
at 37°C or infected with bacteria for 60 min and analyzed by electron
microscopy (Fig. 6). Macrophages from
both NF-IL-6(-/-) and NF-IL-6(+/+) phagocytosed beads and bacteria to
a similar extend. The finding that initial rates of phagocytosis of
both latex beads and B. abortus were similar in both cell
types excluded the possibility that an important alteration of the
internalization process was responsible for increased microbial
recovery in NF-IL-6(-/-) macrophages.
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The endocytosis defect detected by studying HRP uptake in
NF-IL-6(-/-) macrophages was also observed at the ultrastructural
level when we analyzed the internalization of BSA-gold particles (Fig. 7). Endosomes from NF-IL-6(+/+)
macrophages were filled with BSA-gold (Fig. 7A), whereas
those from NF-IL-6(-/-) macrophages contained fewer gold particles
(Fig. 7B). Although lysosomes from both cell types looked
normal (data not shown), the endosome morphology of NF-IL-6(-/-)
macrophages displayed a less organized structure with numerous tubular
extensions (Fig. 7B). The alteration of the morphology of
NF-IL-6(-/-) endosomes prompted us to investigate their capacity to
interact and fuse with Brucella-containing phagosomes. The
transfer of BSA-gold particles from endosomes to
Brucella-containing phagosomes was used to evaluate the
levels of fusion occurring between these organelles. Reduced fusion
levels between Brucella-containing phagosomes and
endosomes from NF-IL-6(-/-) were observed (Fig. 8). About one-third of the total
population of Brucella-containing phagosomes fused with
endosomes in NF-IL-6(-/-) macrophages, compared with 60% in
NF-IL-6(+/+) macrophages. These results demonstrate for the first time
that Brucella-containing phagosomes are able to fuse with
endosomes. Similar fusion events have been observed in phagosomes
containing mycobacteria 35, 36, 37 or Leismania donovani 28 ,
at least at the onset of infection for the protozoan. We then tested
whether the addition G-CSF could restore endosome/phagosome fusion,
because this cytokine was able to circumvent the endocytosis defect in
NF-IL-6(-/-) macrophages. Indeed, fusion between endosomes and
Brucella-containing phagosomes in G-CSF-treated
NF-IL-6(-/-) macrophages was restored and was comparable to that
measured in NF-IL-6(+/+) macrophages (Fig. 8). The morphology of
endosomes was modified upon G-CSF treatment in NF-IL-6(-/-)
macrophages; endosomes displayed a more organized vesicular structure
similar with that of NF-IL-6(+/+) macrophages (Fig. 7C).
Taken together, these findings show that G-CSF added externally is
capable of modulating endocytosis by controlling early steps of
internalization and consequently phagosome/endosome fusion in
NF-IL-6-deficient mice.
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We then tested the effect of G-CSF on the replication rate of
attenuated strain 19 in NF-IL-6(-/-) macrophages (Fig. 9). At 48 h postinfection, the
number of intracellular bacteria within G-CSF-treated NF-IL-6(-/-)
macrophages was less than half than that measured in untreated
NF-IL-6(-/-) macrophages. Later, at 72 h, only a few viable
bacteria were detected (Fig. 9), indicating that strain 19 bacteria
could not stably replicate in NF-IL-6(-/-) macrophages treated with
G-CSF. These CFU measurements were confirmed by confocal microscopy
observations in which bacteria from strain 19 underwent degradation in
NF-IL-6(-/-) macrophages upon G-CSF treatment (Fig. 2
, E
and F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 4). rab4 and rab5, two members
of the rab family of small GTPases, were localized in the early
endosomal network 38 . Overexpression of rab5 or activating mutations
such as the GTPase-deficient rab5Q79L increases the size of the early
endosomes and the rate of endocytosis 39, 40 , whereas
dominant-negative mutants inhibit endocytosis 27 and cause
fragmentation of early endosomes as evidenced by light and electron
microscopy 39 . In parallel, rab4 plays a critical role in the
recycling of the transferrin receptor and fluid phase markers through
the early endosomal compartments 41, 42 . Because upon
Brucella infection, endocytosis but not recycling is
affected in NF-IL-6(-/-) macrophages, rab5 or its effectors, but not
rab4, may be good targets after G-CSF receptor involvement.
The biogenesis of phagolysosomes and their ability to degrade invading
microorganisms have been shown to involve a regulated series of
interactions between newly formed phagosomes and endocytic organelles
28, 43, 44, 45 . Particurlarly, endosome/phagosome fusion regulated by
rab5 can be modulated upon Listeria 46 or
Mycobacterium 47 infections. We are currently
investigating the effect of G-CSF on rab5 expression during
Brucella infection. A direct consequence of the effect of
G-CSF on endocytosis and membrane fusion between endosomes and
phagosomes is an increase in bactericidal activity. In NF-IL-6(-/-)
macrophages, NO synthase is not impaired 6 . However, the production
of reactive oxygen intermediates is lower in NF-IL-6(-/-) than in
wild-type macrophages 6 , suggesting that NF-IL-6 may control the
expression of other elements of the respiratory burst. On the other
hand, it is known that G-CSF enhances the respiratory burst in
phagocytes 48 . NO synthase was found to be associated to
intracellular membrane vesicles different from lysosomes and
peroxisomes in murine macrophages 49 . These vesicles could
translocate to Brucella-containing phagosomes in normal
macrophages and could be hampered in NF-IL-6-deficient macrophages due
to the lack of fusion between endosomes and phagosomes. Our hypothesis
is that G-CSF, by completely restoring endosome/phagosome fusion (Fig. 8), could allow elements of the respiratory burst present in endocytic
compartments to reach the Brucella-containing phagosomes and
thus to partially restore the bactericidal activity of the cells (Fig. 9). The functional translocation of elements present in endosomes after
macrophage activation could alter the environment of phagosomes and
potentiate the efficiency of NO. Restored membrane fusion between
attenuated-B. abortus containing phagosomes and endosomes
transfers bacteria from a relatively nonhostile environment in which
strain 19 can replicate to one that contains reducing agents, acid
hydrolases or oxide and NO radicals that are potentiated in activated
macrophages. Under these conditions, bacteria from attenuated strain 19
could be targeted to lysosomes and killed, whereas pathogenic bacteria
could still replicate but to a lesser extent than in resting
macrophages 9 . The bactericidal responses of activated macrophages
are probably based on the complex interactions between several
molecular mechanisms, and more work is needed to understand the
regulation of membrane trafficking during macrophage activation. The
regulation of endosome/phagosome fusion by cytokines is certainly an
important means for host cells to protect themselves against
intracellular pathogen growth. Identification of target genes, other
than G-CSF, which are under the control of NF-IL-6 and whose expression
affects intracellular pathogen replication, is a challenge for the
future.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Pierre Gorvel, Centre d’Immunologie de Marseille-Luminy, case 906, 13288 Marseille, France. E-mail address:
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF, G-CSF, granulocyte CSF; NO, nitric oxide; HRP, horseradish peroxidase; HRP-Tfn, peroxidase-conjugated murine transferrin.
Received for publication July 16, 1998. Accepted for publication December 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
-dependent bacteriostasis of Mycobacterium avium. Infect. Immun. 64:3224.[Abstract]
interferon-activated murine peritoneal macrophages. Infect. Immun. 60:3011.B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90:10193.B and C/EBP family members: a Rel domain-bZIP interaction. Mol. Cell Biol. 13:3964.: tyrosine phosphorylation of a 91-kD DNA binding protein. Science 258:1808.B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J. Biol. Chem. 265:21128. and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730. and interleukin-6 on fluid-phase permeability and ammonia diffusion in CNS-derived endothelial cells. J. Invest. Med. 44:474.[Medline]
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, NF-IL-6(-/-)
macrophages infected with strain 19; •, NF-IL-6(-/-) macrophages
infected with strain 2308; 
, NF-IL-6(+/+) macrophages infected with
strain 19; and O, NF-IL-6(+/+) macrophages infected with strain
2308.
), intracellular Tfn; (
), released Tfn.
