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-Mediated Control of Coxiella burnetii Survival in Monocytes: The Role of Cell Apoptosis and TNF1
Unité des Rickettsies, Université de la Méditerranée, Centre National de la Recherche Scientifique, Faculté de Médecine, Marseille, France
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Abstract |
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. While C. burnetii multiplied in untreated
monocytes, IFN- reduced bacterial viability after 24 h of
treatment and reached maximum inhibition after 96 h. IFN- also
affected the viability of infected cells. Cell death resulted from
apoptosis; occurring 24 h after the addition of IFN-, it
reached a maximum after 48 h and was followed by necrosis.
Reactive oxygen intermediates were not required for C.
burnetii killing, since monocytes from patients with chronic
granulomatous disease were microbicidal in response to IFN-. The
role of cytokines was also investigated. IFN- elicited a moderate
release of IL-1ß in infected monocytes. Moreover, the IL-1 receptor
antagonist did not affect C. burnetii survival,
suggesting that IL-1ß was not involved in the bacterial killing
induced by IFN-. TNF was involved in IFN--induced killing of
C. burnetii and cell death. IFN- induced mRNA
expression and sustained secretion of TNF. Neutralizing Abs to TNF as
well as Abs directed against TNF receptors I and II,
significantly prevented IFN--dependent killing of C.
burnetii and cell death. These results suggest that IFN-
promotes the killing of C. burnetii in monocytes through
an apoptotic mechanism mediated in part by TNF. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis are down-modulated in Q fever endocarditis 5, 6 . The
production of large amounts of IL-10 by mononuclear cells has been
related to disease outcome 7 . Antibiotic treatment of chronic Q fever
is long lasting and does not protect against late relapses 8 . The
relative inefficiency of antibiotics results from the inhibition of
their bactericidal activity by the low pH of phagosomal vacuoles in
which C. burnetii multiplies. The combination of chloroquine
and antibiotics reduces the duration of treatment 9 . Hence, the
up-regulation of macrophage microbicidal activity seems to be critical
in optimizing the efficiency of antibiotic treatment.
As chronic Q fever is associated with defective IFN- production, the
association of antibiotics with this cytokine may be relevant to
patient treatment. The role of IFN- in macrophage activation is
clearly established. IFN- increases the cytotoxic activity of
macrophages via the induction of reactive oxygen intermediates
(ROI),3 reactive nitrogen
intermediates (RNI), and cytokines such as TNF 10 . As a result,
activated macrophages show greatly enhanced microbicidal activity for
many intracellular pathogens including Mycobacteria,
Leishmania, Toxoplasma, and
Trypanosoma 11 . IFN- up-regulates the expression of MHC
molecules and thus amplifies the Ag-presenting function of macrophages.
IFN- is also required for the development of the Th1 protective
immune response 11 . Mice in which the IFN receptor (IFN-R) 
-chain
gene has been disrupted display an impaired ability to resist
infections caused by pathogens such as Listeria
monocytogenes or Mycobacteria 12, 13 . The recent
description of IFN-R deficiency in humans also highlights the critical
role of IFN- in the establishment of protective immunity against
intracellular micro-organisms 14 . However, most in vivo and in vitro
experiments based on the administration of cytokines before the
infection procedure do not support the therapeutical application of
IFN-, which must be delivered after infection. The administration of
IFN- to animals helps to eradicate difficult to treat infectious
agents such as M. tuberculosis 15 . In humans, the local
injection of IFN- converts the lepromatous lesions of leprosy into
tuberculoid-like lesions. A combination of IFN- with antimonial
therapy increases clinical efficiency compared with the effect of
treatment with antimony alone in patients with visceral leishmaniasis
16 .
In this study we examined the effect of IFN- administered after
infection on the survival of C. burnetii in THP-1 monocytic
cells. We found that the addition of IFN- to infected monocytes
reduced bacterial viability and also elicited the apoptosis of infected
monocytes. IFN--mediated C. burnetii killing and death of
infected monocytes were both dependent on TNF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic leukemia cell line THP-1, HEL fibroblasts, and L929 mouse fibroblasts were obtained from the European Collection of Animal Cell Cultures (Sophia Antipolis, France). Monocytes and L929 cells were cultured at 5 x 105 cells/ml in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEL cells were grown in MEM (Life Technologies) with 10% FCS and 2 mM L-glutamine. One milliliter of suspension containing 5 x 104 HEL cells was seeded on 12-mm round coverslips in shell vials (Sterilin, Felthan, U.K.) and incubated at 37°C in a 5% CO2 atmosphere for 3 days to obtain confluent monolayers.
BALB/c mice were injected i.p. with 108 C. burnetii (Nine Mile strain, American Type Culture Collection VR-615, Manassas, VA). One week later, mice were sacrificed, and their spleens were dilacerated. Spleen homogenates were added to L929 cell monolayers, and cultures were maintained for five passages. Infected L929 cells were then detached with glass beads and sonicated. Sonicates were spun down at 1,000 rpm for 10 min to remove unbroken cells, and supernatants containing bacteria were centrifuged at 10,000 rpm for 10 min. The pellets were suspended in HBSS, and the number of bacteria was assessed by the Gimenez staining procedure 17 . Isolated C. burnetii were aliquoted at 109 organisms/ml and stored at -80°C.
Infection of monocytic cells
Monocytes at 5 x 105/ml were incubated with
C. burnetii at a bacteria to cell ratio of 200:1 for 24
h at 37°C. Cells were then washed by low speed centrifugation (1300
rpm for 5 min) to remove free bacteria; this time was designated 0
h. Infected cells were cultured in the presence or the absence of human
rIFN- (R&D Systems, Abingdon, U.K.) in flat-bottom 96-well plates
for 96 h. In some experiments, neutralizing anti-TNF
polyclonal Ab, mAbs against TNF-R types I and II, isotypic controls, or
IL-1R antagonist (IL-1 Ra) (R&D Systems) were added to cell cultures
treated or not treated with IFN-.
Cellular infection was quantified by indirect immunofluorescence.
Monocytes (3 x 104 cells) were cytocentrifuged at
1000 rpm for 3 min, fixed with 1% formaldehyde, and permeabilized by
0.1 mg/ml lysophosphatidylcholine (Sigma, St. Louis, MO). Cells were
then incubated with rabbit anti-C. burnetii serum at a
1/250 dilution in PBS containing 0,1% BSA (Sigma) for 30 min.
FITC-conjugated F(ab')2 anti-rabbit Ab (Immunotech,
Marseille, France) was added to monocytes that were counterstained with
Evans blue. Results were expressed as an infection index, which is the
product of the mean number of bacteria per infected cell and the
percentage of infected cells x 100. As the number of THP-1 cells
steadily increased by 4.8 ± 0.6-fold in the absence of IFN-
during the experiment, the effect of cell dilution on the infection was
assessed by relating the infection index to the number of cells as
follows: [(number of cells after n hours/number of cells at
0 h) x (infection index after n hours/infection index
at 0 h)] x 100.
Bacterial viability
Infected monocytes were submitted to brief sonication, and homogenates were used to infect monolayers of HEL cells in shell vials as previously described 18 . After 10 days at 37°C, culture medium was removed, and cells were fixed with methanol. C. burnetii were revealed by indirect immunofluorescence as described above. Results were expressed as the number of fluorescent vacuoles per shell vial.
Apoptosis determination
Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA fragmentation occurring during apoptosis was detected in individual cells by in situ DNA end extension using terminal deoxynucleotidyl transferase as indicated by the manufacturer (Apoptag Plus, Oncor, Appligene, Illkirch, France). Nucleotides added by transferase were digoxigenin labeled. FITC-conjugated anti-digoxigenin Ab was applied to detect incorporated nucleotides, permitting visualization of apoptotic cells under UV light. This procedure was performed directly on slides after cytocentrifugation of monocytes.
Annexin V binding assay Monocytes at 106 cells/assay were suspended in a buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 5 mM CaCl2) containing FITC-conjugated annexin V (Boehringer Mannheim, Meylan, France) and propidium iodide (Sigma) at 1 µg/ml for 15 min. Cells were then analyzed by flow cytometry with an EPICS XL cytometer (Coulter, Hialeah, FL).
Transmission electron microscopy. Monocytes were fixed for 60 min on a rotating agitator in cacodylate buffer (0.1 M; pH 7.2) containing 1% glutaraldehyde and then incubated overnight at 4°C in fresh cacodylate solution. They were incubated for 1 h at 4°C in 1% osmium tetroxide, then stained with 1% uranyl acetate before being dehydrated in acetone and embedded in Epon.
Cytokine determination
RNA extraction and PCR amplification. Total RNA (5 x 106 monocytes/assay) was extracted by the guanidium thiocyanate-phenol method. To synthesize cDNA, RNA (1 µg) was incubated with reverse transcriptase mixture (Superscript, Life Technologies) for 1 h. The reaction was stopped by heat inactivation (10 min, 95°C). Negative control experiments were performed by omitting RNA from cDNA synthesis. cDNA was used as a template in a mixture containing Taq polymerase and specific primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), TNF, and IL-1ß as previously described 19 . The mixtures were subjected to 24 (G3PDH, TNF) or 26 (IL-1ß) cycles of denaturation, annealing (60°C for G3PDH and IL-1ß, 65°C for TNF), and extension. PCR products were electrophoresed in 2% agarose gel containing ethidium bromide. Gels were exposed to UV, and photographs were taken with an imager (Oncor). The sizes of PCR products were determined using DNA m.w. markers (Boehringer Mannheim).
Immunoassays. Supernatants from monocytes were collected and stored at -80°C before use. TNF and IL-1ß were measured by ELISA (BioAdvance, PerSeptive Diagnostics, Emerainville, France) based on a coated well mAb to the cytokine and a peroxidase-conjugated Ab. The detection limit was 1 pg/ml for TNF and IL-1ß.
TNF bioassay. TNF biological activity was determined by cytotoxic assay assessed by crystal violet staining 20 . Briefly, L929 fibroblasts were seeded at 5 x 104 cells/well into flat-bottom 96-well plates in 100 µl of RPMI 1640 supplemented with 10% FCS, L-glutamine, and antibiotics. Fifty microliters of monocyte supernatants were incubated with L929 cell monolayers in the presence of 50 µl of actinomycin D (Sigma) at 4 µg/ml for 18 h at 37°C. Recombinant TNF (R&D Systems) was included in each assay as a positive control. Crystal violet (Sigma; 0.5% in 20% methanol) was added to L929 cells for 10 min at 37°C. After washing, cells were solubilized by 1% SDS, and absorbance changes were measured at 492 nm. To ascertain that the lysis of L929 cells was TNF specific, anti-TNF Ab was used to neutralize the effects of monocyte supernatants. Results were expressed as units per milliliter (1 U is the amount of TNF inducing 50% cytotoxicity).
Chronic granulomatous disease (CGD) patients
Two patients (6- and 12-yr-old boys) with X-linked CGD and five
healthy controls were included in the study after obtaining informed
consent. CGD patients suffered from recurrent infectious diseases
caused by Salmonella typhi and Aspergillus
fumigatus. The oxidative response of circulating monocytes, as
assessed by ferricytochrome c reduction and nitro blue
tetrazolium reduction, was not detectable. Monocytes were isolated from
blood by Ficoll gradient and adherence 20 . Adherent monocytes were
incubated with C. burnetii for 24 h. IFN- was then
added according to the procedure described for THP-1 cells. Monocyte
infection was measured by immunofluorescence, and an infection index
was calculated as the product of the mean number of bacteria per
infected monocyte and the percentage of infected cells x 100.
Statistical analysis
Results are given as the mean ± SE. Statistical analysis was conducted with analysis of variance and regression analysis. Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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impairs C. burnetii intracellular growth in
monocytes
THP-1 cells were infected with C. burnetii at a 200:1
bacteria to cell ratio for 24 h to allow homogeneous infection of
the cell population. Cells were then washed to remove unbound bacteria;
this time was defined as 0 h. The infection index at 0 h was
240 ± 24, corresponding to the infection of 70% of cells with
three or four bacteria per cell. The infection index was then measured
every 24 h from 0 to 96 h and was expressed as a relative
infection index compared with the value at 0 h (Fig. 1
A). This relative infection
index steadily increased from 24 to 72 h and reached a plateau
between 72 and 96 h, which was 2.5 times higher than the initial
infection. Viable bacteria were assessed by measuring C.
burnetii-induced vacuole formation in HEL cells. The number of
vacuoles per shell vial started to increase after 24 h and reached
a maximum value after 72 h (Table I). As the number of cell-associated
bacteria and the number of vacuoles were significantly related
(r = 0.850; p < 0.001), most
additional experiments used the infection index as a measurement of
viable bacteria.
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were added to infected cells at 0 h
(Fig. 1
A). IFN- at 1000 U/ml prevented an increase in the
relative infection index after 24 h of treatment (47 ± 5%
inhibition; p < 0.02). Maximum inhibition (76 ±
10%) was reached after 72 h of treatment
(p < 0.0001). Inhibition remained constant
thereafter. It is noteworthy that IFN--mediated inhibition of
C. burnetii infection mainly resulted from a decrease in the
number of cell-associated bacteria, rather than a decrease in the
number of infected cells (data not shown). When 100 U/ml of IFN- was
used, C. burnetii multiplication was moderately impaired
whatever the time of culture. After 72 h, the relative infection
index (170 ± 30%) was intermediate between that of untreated
cells (270 ± 22%) and that of cells treated by IFN- at 1000
U/ml (66 ± 9%). IFN- at 10 U/ml had no effect on the
infection of THP-1 cells (data not shown). The IFN--stimulated
decrease in C. burnetii infection as detected by indirect
immunofluorescence was the result of a dramatic decrease in the number
of viable bacteria (Table I). The number of vacuoles dropped after
24 h of treatment with IFN- (59 ± 7% inhibition). It
decreased steadily to maximum inhibition after 72 h of treatment
(90 ± 10% inhibition; p < 0.05). In addition,
the presence of IFN- was necessary for a complete effect, since its
withdrawal from culture medium at 24 h reduced the inhibitory
effect of IFN- on the number of viable bacteria (data not shown).
IFN- induces death of infected monocytes
IFN- at 1000 U/ml reduced the viability of infected monocytes
(59 ± 4 vs 92 ± 2%; 36 ± 5% inhibition) as assessed
by blue trypan exclusion after 72 h of incubation (Fig. 1B). Maximum inhibition (67 ± 8%) occurred after
96 h of treatment. The effect of IFN- was selective, since the
viability of control cells, infected cells, and IFN--treated cells
was not modified. It was necessary for continued IFN- to be present
in culture medium for cell death to occur, since its withdrawal after
24 h led to monocyte viability (data not shown). In an attempt to
understand whether the trypan blue staining of IFN--treated cells
resulted from a unique necrotic process or a sequence consisting of
apoptosis followed by necrosis, cell death mechanisms were investigated
by three complementary methods. Apoptosis was studied by
deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.
Control cells (Fig. 2A),
infected cells (Fig. 2B), and IFN--treated cells (Fig. 2C) did not show any staining. In contrast, infected
monocytes treated with IFN- for 48 h displayed staining typical
of apoptotic cells (Fig. 2D). A large proportion of cells
(65 ± 10%) were brightly stained, and a minority (30 ±
8%) were more slightly stained. IFN--induced apoptosis of infected
cells was an early event. Indeed, the binding of annexin V, an early
marker of apoptosis, was found in infected cells treated for 24 h
with IFN- (Fig. 3). A maximum increase
in the binding of annexin V was observed 48 h after the addition
of IFN-. As described above, there was a discrete subset of THP-1
cells that strongly expressed an increase in the binding of annexin V.
The ultrastructural analysis of infected cells treated with IFN- for
48 h showed hallmarks of apoptosis. These consisted of the
condensation of nucleus and cytoplasmic organelles as well as
cytoplasmic vacuolization (compare Fig. 4, A and B). A loss
of cell membrane integrity was observed in infected cells after 72
h of treatment with IFN- (data not shown), probably corresponding to
secondary necrosis. Taken together, these results demonstrated that
IFN- rapidly induced the apoptosis of infected, but not noninfected,
monocytes and then their necrosis.
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effects are associated with the production of TNF, but not
ROI
IFN- elicits cytotoxic and microbicidal functions in
monocytes/macrophages via several mechanisms, including ROI generation
and cytokine synthesis 10 . THP-1 cells released only low levels of
superoxide (
3 nmol/106 cells) after stimulation with a
potent inducer such as PMA. To overcome this difficulty, we used
monocytes from CGD patients, infected with C. burnetii and
then treated with 1000 U/ml IFN-, to assess the role of ROI in the
response of infected cells to IFN- (Fig. 5). In the absence of IFN-, the
infection index of monocytes from controls and CGD patients slowly
diminished within 96 h, but this decrease never exceeded 30%.
After 24 h of IFN- treatment, the monocyte infection index fell
rapidly (63 ± 7 and 65 ± 10% inhibition for controls and
CGD patients, respectively) and then decreased at a lower rate. Thus,
an IFN--mediated decrease in C. burnetii infection does
not require the production of ROI.
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. They were barely expressed in
infected THP-1 cells after 24 h (Fig. 6) and were undetectable after 48 h
(data not shown). The expression of transcripts for TNF was high
24 h after the addition of IFN- to infected cells (Fig. 6) and
remained so 48 h after treatment (data not shown). No transcripts
for IL-1ß in noninfected cells, treated or not with IFN-, were
detectable. In contrast, the transcripts for IL-1ß were expressed in
C. burnetii-infected cells for 24 h. Again, the
addition of IFN- to infected cells up-regulated the expression
levels of cytokine mRNA. Immunoreactive TNF was detectable in
supernatants of infected cells, and its amounts were clearly increased
by IFN- treatment after 24 h (135 ± 30 vs 15 ± 5
pg/ml; Fig. 7). TNF release steadily
increased to reach a maximum value 72 h after the addition of
IFN- (315 ± 45 pg/ml) and slightly decreased thereafter. In
addition, the kinetics of the release of bioactive TNF (Fig. 7, inset)
and immunoreactive TNF were related. IL-1ß was not detected in
supernatants of THP-1 cells cultivated in the presence or the absence
of IFN-. It was only detectable in supernatants of C.
burnetii-infected THP-1 cells (8 ± 3 pg/ml). IL-1ß release
was induced in infected cells treated with IFN- from 50 ± 7
pg/ml after 48 h to 55 ± 10 pg/ml after 96 h. Thus,
IFN- specifically induced a sustained production of TNF, and to a
lesser extent IL-1ß, in infected cells.
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As TNF and IL-1ß secretions were associated with IFN--induced
C. burnetii killing and the apoptosis of THP-1 cells, their
effects on IFN- responses were investigated. The contribution of
IL-1ß to IFN--stimulated responses was assessed by using IL-1Ra.
Infected cell death and C. burnetii killing were not
dependent on IL-1ß since IL-1Ra could not prevent them (Table II) regardless of the concentration used
(10–200 ng/ml; data not shown).
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-stimulated responses was assessed using
two approaches. First, infected cells were incubated with IFN- in
the presence of neutralizing anti-TNF Ab or an isotypic control. Ab
doses were determined as the lowest doses inhibiting L929 cell lysis
(data not shown). In the presence of an isotypic control at 5 µg/ml,
the infection index of IFN--treated cells markedly decreased after
48 h, and cell viability dropped regularly after 48 h (Fig. 8). When anti-TNF Ab at 5 µg/ml was
used, the decrease in infection index observed after 48 h was
partially prevented. The infection index then increased to reach a
maximum value after 96 h (relative infection index with
anti-TNF Ab, 161 ± 25; relative infection index without
anti-TNF Ab, 55 ± 3). It is noteworthy that an anti-TNF
Ab-mediated increase in the infection index reached 50% of the values
of the infection index found in infected cells (compare Fig. 1A and Fig. 8A). The death of infected THP-1
cells treated with IFN- was completely prevented by anti-TNF Ab
(Fig. 8B). Second, the role of TNF in IFN--stimulated
responses was investigated using Ab directed to TNF-RI or TNF-RII
(Table II). Abs specific for TNF-RI (10 µg/ml) or TNF-RII (5 µg/ml)
were equally potent at preventing IFN--induced monocyte death and
monocyte infection. Rescue was similar to that observed with
anti-TNF Ab. The simultaneous addition of anti-TNF-RI and
anti-TNF-RII Abs induced a cell response equivalent to that
elicited by each Ab. These results demonstrated that IFN--induced
C. burnetii killing and IFN--induced cell death were
dependent on TNF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to C.
burnetii-infected THP-1 cells markedly impairs bacterial survival.
This finding is of interest because it makes IFN- a candidate for
the modulation of microbicidal competence in cells stably infected.
Most initial studies have shown that the pretreatment of macrophages
with IFN- increases microbicidal activity 10, 11 , but that
infected cells are either poorly responsive or refactory to IFN-.
Indeed, macrophages infected with Mycobacteria or
Leishmania are unable to mount a microbicidal response to
IFN- 21 . The model of C. burnetii infection of
monocytes is clearly different. While the addition of IFN- to
infected cells induced a powerful microbicidal activity against
C. burnetii, the pretreatment of THP-1 cells by IFN- was
only bacteriostatic (data not shown). The microbicidal effect of
IFN- was an early event, since C. burnetii viability was
reduced by 70% within 24 h of treatment with IFN-. It was not
restricted to macrophage cell lines, because IFN- also reduced the
viability of C. burnetii in circulating monocytes with a
similar time course (data not shown).
The second important feature of the response of C.
burnetii-infected cells to IFN- was the induction of cell
death. This was associated with an apoptotic process occurring within
the first 48 h of IFN- treatment and a secondary necrosis. Such
a finding suggests that first, C. burnetii infection of
THP-1 cells was necessary but not sufficient for the induction of cell
death, in contrast to bacteria-producing exotoxins or pathogens such as
Shigella, Salmonella 22 , and
Mycobacteria 23, 24 . Second, IFN- per se did not induce
cell death in THP-1 cells. Although the role of IFN- in apoptotic
events has been established in several cell types, its ability to
induce cell death in macrophages is not clear 11 . IFN- can even
prevent the apoptotic death of monocytes cultured in the absence of
growth factors 25 . Third, the apoptosis of THP-1 cells requires both
IFN- and an additional signal provided by C. burnetii
infection. This result is in agreement with previous reports in which
cytokines or ATP are necessary to prime macrophages for the apoptotic
effect of IFN- 26, 27 . The activation of IFN--treated
macrophages with zymosan particles results in an early apoptosis
followed by a secondary necrosis 28 . We tested the hypothesis that
monocyte activation mediated by LPS provides the cosignal for
apoptosis. THP-1 cells were stimulated with LPS from Escherichia
coli, which is known to strongly activate macrophages, and then
were treated with IFN-. Under these conditions, cell death observed
after 72 h did not exceed 10% of the cell population (data not
shown). Hence, a transient activation of THP-1 cells is insufficient in
priming cells for apoptosis. It is likely that a sustained activation
induced by C. burnetii infection is necessary for cell
death. Finally, the time courses of the IFN--induced apoptosis and
the microbicidal effect of IFN- seem to be similar, whereas the
necrosis of infected cells was delayed. This is in line with a previous
report that apoptosis, but not necrosis, controls the infectious
process 29 . Thus, we can hypothesize that apoptosis favors
microbicidal activity against C. burnetii.
The mechanisms of IFN--mediated responses in myeloid cells may
include the generation of ROI and RNI and the production of cytokines.
Intracellular ROI might be involved in IFN--dependent C.
burnetii killing. ROI production restricts the growth and favors
the killing of several intracellular micro-organisms such as
Mycobacteria 30, 31 . Moreover, IFN- is an inducer of
oxygen-dependent macrophage antimicrobial activity 10, 11, 32 . The
treatment of C. burnetii-infected cells with IFN- did not
induce superoxide production (data not shown). More strikingly,
monocytes from patients with CGD were able to kill C.
burnetii in response to IFN-. In addition, ROI triggered cell
death in myeloid cells 33 , but again the lack of ROI generation in
response to C. burnetii and IFN- treatment did not
support the critical role of ROI in the death of THP-1 cells. RNI
generation has been considered an important effector of macrophage
microbicidal activity 34 and may also be involved in their death.
M. tuberculosis-induced apoptosis of murine macrophages is
prevented by nitric oxide inhibitors 24 . IFN--mediated macrophage
microbicidal activity may also result from the stimulation of RNI
production 11 . Nevertheless, nitric oxide-independent killing of
micro-organisms has been reported in IFN--stimulated macrophages
35, 36 . The contribution of RNI to human macrophage function is still
a source of controversy 37 . We did not detect any production of
nitric oxide in THP-1 cells infected by C. burnetii and/or
treated by IFN-. L-arginine inhibitors did not affect
the survival of C. burnetii (data not shown). Indeed, RNI
are not involved in the IFN--mediated control of C.
burnetii infection in THP-1 cells.
Apoptosis of infected THP-1 cells may be due to cytokine secretion.
Indeed, inflammatory cytokines such as TNF or IL-1ß have a
proapoptotic activity in myeloid cells 22 . IFN- elicits the
synthesis of several cytokines, including inflammatory and
immunoregulatory cytokines in macrophages 10, 11 . It seems that
IL-1ß is not responsible for IFN--induced microbicidal activity
against C. burnetii and cell death. The addition of IFN-
to infected cells induced a sustained expression of IL-1ß transcripts
but only a moderate IL-1ß release. In addition, IL-1R antagonist had
no effect on THP-1 cell microbicidal activity or cell death. In
contrast, there is evidence to support the role of TNF in
IFN--mediated C. burnetii killing and cell apoptosis.
IFN- induced sustained expression of TNF mRNA and markedly
up-regulated TNF secretion in infected THP-1 cells. Neutralizing
anti-TNF Ab and Abs directed to TNF-RI and TNF-RII substantially
prevented the reduction in bacterial viability. Thus, TNF may
contribute to IFN--stimulated monocyte microbicidal activity. This
emphasizes the protective role of TNF in infections caused by
intracellular pathogens 38 . The role of TNF in macrophage cell death
is debated in infectious models. The apoptosis of murine macrophages
caused by M. tuberculosis or IFN- is prevented by
anti-TNF Ab 24 . M. avium/intracellulare induced
apoptosis of macrophages that is dependent on TNF 23 . In contrast,
M. tuberculosis prevented the apoptosis of human monocytes
via the secretion of TNF 39 . In C. burnetii-infected
cells, the induction of cell death in response to IFN- was prevented
by anti-TNF Ab and by Abs directed to TNF-R types I and II. This
finding suggests that both receptors for TNF are necessary for the
TNF-related apoptosis of infected cells. TNF-R type I has been
described as mediating most TNF biological functions, including
cytotoxicity 40 , but the latter seems to be in part signaled by TNF-R
type II 41 . The modulation of TNF responsiveness in T cells infected
by HIV required cooperative signaling between TNF-R types I and II
42 . Both TNF receptors are also required for DNA fragmentation 43 .
In addition, TNF had the capacity to synergize with IFN- to exert
antileishmanial activity 44 .
In this report we have shown that IFN- induces the killing of
C. burnetii in infected THP-1 cells and also their cell
death by apoptosis and secondary necrosis. Both events are dependent on
TNF. Thus, the death of monocytes infected by strictly intracellular
pathogens may confer an advantage for the host by denying
micro-organisms their sanctuary site. In addition, as IFN- was fully
active on cells already infected, it may be useful as an adjuvant
treatment of C. burnetii-infected patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Louis Mege, Unité des Rickettsies, Centre National de la Recherche Scientifique, UPRESA 6020, Faculté de Médecine, 27 blvd. J. Moulin, 13385 Marseille Cedex 05, France. E-mail address:
3 Abbreviations used in this paper: ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates; IFN-R, interferon receptor; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; CGD, chronic granulomatous disease; IL-1 Ra, IL-1 receptor antagonist.
Received for publication July 23, 1998. Accepted for publication November 9, 1998.
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