Unité des Rickettsies, Université
de la Méditerranée, CNRS UPRESA 6020, Faculté de
Médecine, 13385 Marseille Cedex 05, France
Received 22 July 1999/Returned for modification 1 September
1999/Accepted 5 October 1999
Endocarditis is the most frequent form of chronic Q fever, an
infectious disease caused by Coxiella burnetii. As this
obligate intracellular bacterium inhabits monocytes and macrophages, we wondered if pathogenesis of Q fever endocarditis is related to defective intracellular killing of C. burnetii by
monocytes. Monocytes from healthy controls eliminated virulent C. burnetii within 3 days. In contrast, monocytes from patients
with ongoing Q fever endocarditis were unable to eliminate
bacteria even after 6 days. In patients who were cured of endocarditis,
the monocyte infection was close to that of control monocytes. This
killing deficiency was not the consequence of generalized functional
impairment, since patient monocytes eliminated avirulent C. burnetii as did control cells. The addition of supernatants of
C. burnetii-stimulated monocytes from patients with ongoing
endocarditis to control monocytes enabled them to support C. burnetii survival, suggesting that some soluble factor is
responsible for bacterial survival. This factor was related to tumor
necrosis factor (TNF): expression of TNF mRNA and TNF release were
increased in response to C. burnetii in patients with
ongoing endocarditis compared to cured patients and healthy controls.
In addition, neutralizing anti-TNF antibodies decreased C. burnetii internalization, an early step of bacterial killing, in
monocytes from patients with ongoing endocarditis but did not affect
delayed steps of intracellular killing. We suggest that Q
fever-associated activation of monocytes allows the survival of
C. burnetii by modulating early phases of microbial killing.
 |
INTRODUCTION |
Q fever is caused by Coxiella
burnetii, an obligate intracellular bacterium inhabiting monocytes
and macrophages (1). The disease exhibits acute and chronic
forms with different courses. Endocarditis is the most frequent
clinical expression of chronic Q fever (23). Q fever
endocarditis usually occurs in patients with valvular disease and/or
alterations in cell-mediated immunity, such as infection by human
immunodeficiency virus, lymphoma, chronic renal failure, or pregnancy
(21). The medical treatment of endocarditis is long, even
when antibiotics are used with chloroquine (24). The
evaluation of its efficiency requires prolonged follow-up because of
the possibility of late relapses (22).
Macrophages exhibit microbicidal activity which involves binding and
phagocytosis of microorganisms and the action of oxidative and
nonoxidative compounds within phagocytic vacuoles. Macrophage-mediated killing of microorganisms also requires T-cell-derived cytokines, including gamma interferon (IFN-
) (25). Hence, patients
with IFN- receptor deficiency exhibit increased occurrences of
mycobacterial infections (2). Conversely, cytokines which
down-modulate microbicidal activity of macrophages favor the survival
of intracellular microorganisms (3). The survival strategy
of C. burnetii should interfere with the intrinsic
microbicidal activity of macrophages and/or its regulation. Patients
with Q fever endocarditis exhibit impaired cell-mediated immunity,
including antigen-driven lymphoproliferation (17) and
IFN- production (14). We recently demonstrated that IFN- induces C. burnetii killing via apoptosis of
infected macrophages (10). The suppression of T-cell
responses to C. burnetii depends on the release of soluble
mediators such as prostaglandins (18) or interleukin-10
(IL-10) (6) by monocytes. Beside their suppressive role,
monocytes from patients with Q fever endocarditis overproduce tumor
necrosis factor (TNF), a proinflammatory cytokine (5). This
may be related to the specific inflammatory syndrome of Q fever
endocarditis, consisting of an increase in circulating TNF without
variations in cytokine antagonists (7).
This study was undertaken to assess the survival of C. burnetii in monocytes from patients with Q fever endocarditis.
Control monocytes eliminated C. burnetii, whereas those of
patients with ongoing Q fever endocarditis did not. This defect was not
intrinsic but was related to an increase in TNF production. TNF was
involved in this defect mainly by upregulating C. burnetii internalization. We suggest that the level of monocyte
activation in Q fever determines the survival of C. burnetii.
| |
MATERIALS AND METHODS |
Patients.
Twenty patients, consisting of 13 males and 7 females (mean age, 59 years; range, 39 to 79 years), were included in
the study. The diagnosis of endocarditis was based on modified Duke
endocarditis service criteria (11). Patients had
pathological evidence of endocarditis, a positive echocardiogram,
positive blood culture, and high titers of immunoglobulin G (IgG)
directed against C. burnetii. All of these patients were
subjected to valve replacement and medical treatment with doxycycline
and chloroquine. They were divided in two groups: one consisting of
patients with ongoing endocarditis (n = 10)
characterized by high titers of specific IgG (mean, 21,000; range,
1,600 to 120,000) and the other made up of patients recently cured of
the disease (n = 10) and who had low antibody titers
(mean, 600; range, 400 to 800). The first group was treated during the
course of the study, while treatment of the second group had been
stopped at least 3 months before the investigation. Ten healthy
subjects, sex and age matched, were included in the study as controls.
Monocytes and bacteria.
Blood was drawn in
EDTA-anticoagulated tubes, and peripheral blood mononuclear cells were
separated with Ficoll gradients (Eurobio, Les Ulis, France). Cells were
suspended in RPMI 1640 containing 20 mM HEPES (Gibco-BRL, Life
Technologies, Cergy-Pontoise, France), 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U of penicillin per ml, and 100 µg of
streptomycin (Gibco-BRL) per ml. Monocytes were purified by incubating
5 × 105 peripheral blood mononuclear cells in a glass
Labtek chamber/slide (Miles, Naperville, Ill.) for 60 min at 37°C.
Nonadherent cells were removed by washing, and the remaining cells were
designated monocytes because more than 90% of them were
CD14+ and had phagocytic characteristics (5).
Virulent C. burnetii (Nine Mile strain in phase I; ATTC
VR-615) was injected into mice and 10 days later was recovered from spleens and then cultured in mouse L929 fibroblasts maintained in
antibiotic-free minimal essential medium (Gibco-BRL) supplemented with
4% FCS and 2 mM L-glutamine for two passages. Avirulent
variants were obtained by repeated passages of Nine Mile strain in L929 cells (20). After 1 week, L929 cells were sonicated, and the homogenates were centrifuged at 5,000 × g for 10 min. The
bacterial pellet was layered on a 25 to 45% linear Renografin gradient
and spun down. Purified bacteria were then collected, washed, and suspended in serum-free medium before being stored at 
80°C. The concentration of C. burnetii was determined by Gimenez staining.
Infection procedure.
Monocytes were incubated with C. burnetii in phase I (bacterium-to-cell ratio of 200:1) for 24 h in RPMI 1640 containing 10% FCS (10). The cells were
washed to remove free bacteria (corresponding to day 0) and cultured
for 6 days. As controls, monocytes were incubated with avirulent
C. burnetii at a bacterium-to-cell ratio of 100:1 for
24 h. As avirulent bacteria were more efficiently internalized by
monocytes than virulent organisms (8), we incubated monocytes with a lower number of avirulent C. burnetii
organisms to obtain similar amounts of infection. In some experiments,
a 10-µg/ml concentration of goat antibodies (Ab) neutralizing
bioactive TNF or control IgG (R&D Systems, Abingdon, United Kingdom)
was added to monocytes before C. burnetii infection.
Cellular infection was quantified by Gimenez staining. Results were
expressed as an infection index, as follows: (number of bacteria per
infected cell) × (number of infected cells/100) × 100.
The viability of intracellular bacteria was determined as previously
described (10). Briefly, infected monocytes were sonicated, and serial dilutions of homogenates were added to HEL cell monolayers. C. burnetii replication was revealed by indirect
immunofluorescence with rabbit Ab directed against C. burnetii.
Cytokine determination. (i) Immunoassays.
Monocytes (2 × 105 cells/assay) were incubated with C. burnetii (bacterium-to-cell ratio of 200:1) for 24 h.
Supernatants were assayed for the presence of TNF and IL-6. The limits
of detection of the immunoassay kits (Immunotech, Marseille, France)
were 10 and 3 pg per ml, respectively.
(ii) RNA extraction and PCR amplification.
Monocytes (5 × 105 cells/assay) were incubated with C. burnetii (bacterium-to-cell ratio of 200:1) for 3 h. RNA was
extracted by using the guanidium-phenol chloroform method
(5). cDNA was generated by incubating RNA in a reverse
transcriptase mixture. cDNA specimens were amplified in the presence of
primers specific for TNF or IL-6. Amplification products were
quantified with CytoXpress detection kits (BioSource, Fleurus, Belgium)
as previously described (4).
Statistical analysis.
Results were calculated as mean ± standard error (SE). The statistical analysis was conducted by
analysis of variance. Differences were considered significant when
P was <0.05.
| |
RESULTS |
Killing of C. burnetii by monocytes is defective in Q
fever endocarditis.
Monocytes were incubated with C. burnetii in phase I for 24 h (day 0), and the infection of
monocytes was assessed at days 3 and 6 postinfection (p.i.). An
incubation time of 24 h was required to obtain the infection of
75% of monocytes with one to two bacteria per cell. While this initial
incubation allows the measurement of the early phase of killing, the
infection after 3 and 6 days is an assessment of the delayed phase of
intracellular killing. At day 0, the bacterial phagocytosis in
monocytes from patients with ongoing endocarditis (active patients) was
different from that in monocytes from cured patients or controls (Fig.
1A). The initial infection index was
significantly (P < 0.005) higher in monocytes from
active patients (250 ± 35) than in control monocytes (115 ± 12) or in monocytes from cured patients (136 ± 15). After 3 days,
the number of bacteria was decreased by 75% in control monocytes, and
only 15% of bacteria present at day 0 were found after 6 days (Fig.
1B). The decrease in the bacterial count resulted from an alteration of
C. burnetii viability, as assessed by culturing homogenates
from infected monocytes on HEL cells (105 ± 35 vacuoles per shell
vial at day 0 and 20 ± 9 vacuoles per shell vial at day 6 for
control monocytes). In contrast, the number of intact bacteria remained
constant during the first 3 days p.i. and decreased slightly thereafter
(15% inhibition) in monocytes from active patients. Bacterial
viability was not altered after 3 days of infection (123 ± 38 vacuoles per shell vial), but it was slightly decreased at 6 days p.i.
(95 ± 32 vacuoles per shell vial). In monocytes from cured
patients, the number of bacteria declined by 55% after 3 days and
slowly decreased thereafter (70% inhibition after 6 days). At each
time, the infection index was significantly lower (P < 0.01) in monocytes from cured patients than in monocytes from
active patients. We then wondered if impaired killing of C. burnetii by patient monocytes was specific. Avirulent variants of
C. burnetii were incubated with monocytes according to the procedure described for virulent bacteria. At day 0, the phagocytosis indices in control and patient cells were similar (132 ± 18 and 156 ± 24, respectively). The number of avirulent bacteria was decreased by 70% after 3 days and by 80% after 6 days in monocytes from active patients, cured patients, and healthy controls (Table 1). Our results indicate that ongoing Q
fever endocarditis is associated with specific defective killing of
virulent C. burnetii.
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FIG. 1.
C. burnetii survival in monocytes from
patients with Q fever endocarditis. Monocytes from controls
(n = 10), patients with ongoing Q fever
endocarditis (active patients) (n = 10), or cured
patients (n = 10) were incubated with C. burnetii at a bacterium-to-cell ratio of 200:1. Monocyte infection
was determined at day 0 (A) and at 3 and 6 days p.i. (B) by
Gimenez staining. In panel A, results (means ± SEs) are
expressed as an infection index; in panel B, they are expressed
as a relative infection index compared to values at day 0. *,
P < 0.005; **, P < 0.01 (for the
comparison of active patient values with control values).
|
|
The defect in C. burnetii killing is reproduced by
supernatants from patient monocytes.
We wondered if defective
bacterial killing of monocytes was intrinsic or might be reproduced
by soluble mediators. For that purpose, monocytes from
healthy controls were treated with supernatants obtained by culturing
monocytes from controls, active patients, or cured patients with
C. burnetii for 24 h at 37°C. C. burnetii was then added to treated cells at a bacterium-to-cell ratio of 200:1,
and the infection was assessed for 6 days as described above. The
uptake of C. burnetii was significantly (P < 0.02) higher in cells treated with monocyte supernatants from
active patients than in cells treated with control supernatants (Fig. 2A). While the bacterial number steadily
decreased in monocytes treated with supernatants from controls, it
remained constant after 3 and 6 days in cells treated with supernatants
from active patients (Fig. 2B). When monocytes were pretreated with
supernatants from cured patients, the phagocytosis of C. burnetii (Fig. 2A) and the bacterial number at 3 and 6 day p.i.
(Fig. 2B) were similar to those observed in cells treated with
supernatants from control monocytes. Hence, monocyte supernatants from
patients with ongoing endocarditis were able to induce defective
intracellular killing in monocytes.
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FIG. 2.
Effect of monocyte supernatants on C. burnetii survival. Control monocytes were pretreated with
supernatants of monocytes from healthy individuals (n = 3), active patients (n = 3) or cured patients
(n = 3) which had been stimulated by C. burnetii (bacterium-to-cell ratio of 200:1). The cells were then
infected with virulent bacteria as described for Fig. 1. Monocyte
infection was determined at day 0 (A) and at 3 and 6 days p.i. (B) by
Gimenez staining. In panel A, results (means ± SEs) are expressed
as an infection index; in panel B, they are expressed as a relative
infection index compared to values at day 0. *, P < 0.02; **, P < 0.01 (for the comparison of
active patient values with control values).
|
|
C. burnetii stimulates cytokine overproduction in
patient monocytes.
We previously found that patients with Q fever
endocarditis exhibit an increase in spontaneous TNF production by
monocytes (5). We investigated C. burnetii-stimulated production of TNF in monocytes from controls
and patients. The expression of cytokine transcripts was studied by
using a quantitative method (Table 2). In
the absence of C. burnetii, the amounts of TNF mRNA were significantly higher in active patients than in controls (P < 0.05). After 3 h of stimulation with C. burnetii at a bacterium-to-cell ratio of 200:1, TNF transcripts
dramatically increased, but their levels were significantly higher in
active patients than in controls (P < 0.01). In cured
patients, the amounts of TNF transcripts, spontaneously produced or
induced by C. burnetii, were near the control values.
Cytokine secretion was assessed in supernatants from monocytes
stimulated with C. burnetii for 24 h (Table 2). Monocytes from active patients spontaneously released more TNF than
those from controls (P < 0.01). When monocytes were
stimulated with C. burnetii, the levels of secreted TNF were
higher in active patients than in controls (P < 0.04).
The TNF release by monocytes stimulated or not stimulated by
C. burnetii returned to normal values in patients who
had recovered from the disease. The increase in C. burnetii-stimulated TNF production was specific, since the amounts
of IL-6 transcripts were similar in unstimulated monocytes from active
patients (142 ± 31 copies per ng of RNA) and controls (175 ± 27 copies per ng of RNA). Active patients and controls expressed
IL-6 transcripts in response to C. burnetii in a similar way
(437 ± 25 and 518 ± 50 copies per ng of RNA, respectively). No difference was observed between spontaneous and C. burnetii-induced releases of IL-6 in active patients and controls
(142 ± 31 versus 175 ± 27 pg/ml and 437 ± 25 versus
518 ± 50 pg/ml, respectively). Taken together, our data indicate
that defective elimination of C. burnetii in patients with
active endocarditis is associated with a specific increase in TNF
production.
TNF is involved in defective killing of C. burnetii by
patient monocytes.
The role of TNF in impaired killing of C. burnetii was assessed by using neutralizing anti-TNF Ab, which
completely inhibited C. burnetii-stimulated TNF secretion at
10 µg/ml (data not shown). First, monocyte supernatants from
active patients were pretreated with anti-TNF Ab (or goat IgG) and then
added to control monocytes before C. burnetii infection. The
increase in bacterial phagocytosis and bacterial number at 3 and 6 days
p.i. was prevented (data not shown). Second, anti-TNF Ab or control
IgG was added to patient monocytes before the infection with C. burnetii. In active patients, 10 µg of anti-TNF Ab per ml
decreased the uptake of C. burnetii by 36% ± 5%
(Fig. 3A). The blocking Ab did not affect
the bacterial number at 3 and 6 days p.i. in active patients or in
cured patients (Fig. 3B). These results indicate that TNF in
involved in defective killing of C. burnetii during Q fever
endocarditis by acting mainly on bacterial uptake.
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FIG. 3.
Effect of anti-TNF Ab on C. burnetii killing.
Monocytes from active (n = 5) and cured (n = 5) patients were incubated with C. burnetii in the
presence of anti-TNF neutralizing Ab or control IgG (10 µg/ml) at day
0 (A), and monocyte infection was monitored for 3 and 6 days (B) at
37°C. Monocyte infection was determined as described for Fig. 1. In
panel A, results (means ± SEs) are expressed as an infection
index; in panel B, they are expressed as a relative infection index
compared to values at day 0. *, P < 0.02 (for the
comparison of values in the presence of anti-TNF Ab with values in the
presence of control IgG).
|
|
| |
DISCUSSION |
In this study, we found that monocytes from patients with Q fever
endocarditis were unable to eliminate C. burnetii, in
contrast to control monocytes. This finding is complementary with early observations of immune dysfunction in Q fever, in which monocytes from
patients with endocarditis are involved in the suppression of
T-cell-mediated responses (18). This defect was a
consequence of the activity of Q fever, since it was corrected in
patients who had recovered from infection. It did not result from a
generalized deficiency of bacterial killing, since patient monocytes
eliminated avirulent C. burnetii and Legionella
pneumophila (data not shown). This finding is reminiscent of data
obtained for leprosy, in which monocytes of lepromatous leprosy
patients support the growth of Mycobacterium leprae but
restrict the multiplication of other bacteria susceptible to
intracellular killing (15). The monocyte defect in C. burnetii killing affected early and delayed steps of microbicidal
activity. C. burnetii uptake was significantly higher in
monocytes from active patients than in monocytes from controls and
cured patients. This finding may be related to the finding by
Chaturvedi and Newman that a low growth of Mycobacterium avium or Histoplasma capsulatum is associated with a
low phagocytosis by macrophages in human immunodeficiency
virus-infected patients (9). However, the relationship
between initial and delayed steps of bacterial killing is complex.
Alveolar macrophages, which ingest more mycobacteria than monocytes,
support a less efficient replication (13). The H37Ra and
H37Rv strains of Mycobacterium tuberculosis are similar in
their capacities to enter human macrophages, but they exhibit distinct
survival patterns (27, 28). Our results show that the
delayed step of microbicidal activity against C. burnetii
was altered in monocytes from patients with Q fever endocarditis. This
step of bacterial killing involves oxidative and nonoxidative
mechanisms. The generation of reactive oxygen intermediates is
apparently not required for C. burnetii killing. Indeed,
monocyte production of reactive oxygen intermediates was similar in
patients and healthy controls (data not shown). In addition, we showed
that monocytes from patients with chronic granulomatous disease, which
are known to be unable to produce reactive oxygen intermediates,
eliminated C. burnetii as did control cells (10).
It is likely that nonoxidative mechanisms such as intracellular traffic
and vacuole environment (12) are modified in monocytes
during Q fever endocarditis. The efficiency of chloroquine, which is
known to alkalinize phagosomes, in Q fever treatment (24)
supports this hypothesis.
The defect in C. burnetii killing by patient monocytes might
be related to our experimental procedure, which was based on the
culture of monocytes in the absence of lymphocytes. The coculture of
monocytes with autologous lymphocytes did not restore C. burnetii killing of patient monocytes (data not shown). The defect
in C. burnetii killing was not intrinsic, since it was
reproduced by monocyte supernatants from patients with ongoing Q fever
endocarditis. Thus, soluble mediators may be responsible for the lack
of C. burnetii killing by monocytes. We provided evidence
that TNF is largely involved in this defect. First, monocytes from
patients with ongoing endocarditis, which were not competent to
eliminate C. burnetii, overexpressed TNF transcripts and
secreted high levels of TNF in response to C. burnetii. The
TNF production by monocytes from cured patients, which efficiently
killed bacteria, was similar to that by control monocytes. These
results may be related to the increase in spontaneous production of TNF
by monocytes from patients with ongoing endocarditis (5).
Second, the treatment of monocyte supernatants with anti-TNF Ab
abrogated their ability to promote C. burnetii survival in
monocytes. The effect of TNF on C. burnetii survival is
distinct from the usual role of TNF in containment of intracellular
microorganisms (16). For instance, strains of M. tuberculosis recently characterized as having increased virulence
elicited higher TNF levels than classical mycobacterial strains, but
all of the strains grew in human monocytes at similar rates
(19). However, IFN-, which stimulates TNF expression in
macrophages, causes increased growth of M. tuberculosis in human cells (26). The multiplication of virulent strains of M. tuberculosis in monocytes has been related to the
production of TNF (27). We suggest that the ability of
C. burnetii to stimulate TNF production may serve primarily
to promote pathogenesis rather than protection. On the other hand, TNF
most probably affects the early phase of the C. burnetii
killing process. Indeed, blocking secreted TNF with anti-TNF Ab
decreased bacterial uptake by monocytes of patients with ongoing
endocarditis, but it did not enable monocytes to kill C. burnetii. Thus, it is likely that other factors related to
monocyte activation interfere with delayed phases of C. burnetii killing. IFN- alone is not a good candidate, since its
addition to patient monocytes infected with C. burnetii did
not correct the deficiency in bacterial killing (data not shown).
We found that ongoing Q fever endocarditis is characterized by a
deficiency of intracellular killing of C. burnetii by
monocytes. This defect is largely corrected in cured patients. It is
not intrinsic but involves secreted TNF. The secreted TNF primarily affects C. burnetii uptake but not delayed steps of
intracellular killing. This finding assigns a role in pathogenesis of Q
fever endocarditis to TNF and monocyte activation.
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Abstract
Introduction
