Journal of Medical Microbiology (2005), 54, 527531 DOI 10.1099/jmm.0.

45936-0

Reduced interleukin-18 secretion in Brucella

abortus 2308-infected murine peritoneal
macrophages and in spleen cells obtained from
B. abortus 2308-infected mice
Luis Fernandez-Lago,1 Antonio Orduna2 and Nieves Vizcano1
1
Correspondence Departamento de Microbiologa y Genetica, Edificio Departamental, Universidad de Salamanca,
Luis Fernandez-Lago Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
lrlago@usal.es 2
Departamento de Microbiologa, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain

Received 21 October 2004
Accepted 4 February 2005
Th1 immune responses in which gamma interferon (IFN-) production predominates are associated
with protective immunity against intracellular bacteria. Following infection, interleukin-18 (IL-18) may
contribute, in association with IL-12, to optimal IFN- production. In this study, the secretion of IL-18
following intracellular infection with virulent Brucella abortus 2308 in CD-1 cultured peritoneal
macrophages and splenocyte cultures was investigated. The production of IL-18 was reduced in
both CD-1 mouse peritoneal macrophages infected with B. abortus 2308 and splenocyte cultures
obtained from B. abortus 2308-infected mice at 3, 6 and 10 days post-infection (p.i.). In contrast,
splenocyte cultures obtained from B. abortus 2308-infected mice at 3 days p.i. secreted significant
amounts of IFN-. Stimulation of these cells with recombinant IL-18 (rIL-18) and/or rIL-12 did not
significantly increase IFN- secretion at the splenocyte level. These data suggest that once the
infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting
endogenous IFN- production.

INTRODUCTION synthesized as a biologically inactive 24 kDa precursor

protein. Cleavage to an 18 kDa active mature protein,
Brucella abortus is a facultative intracellular bacterium and is
mediated by the interleukin-1 (IL-1)-converting enzyme,
one of the causative agents of brucellosis in animals and
also called caspase-1, is essential for IL-18 to become
humans (Fernandez-Lago et al., 1999). Professional phago-
biologically active (Ghayur et al., 1997). Although IL-18
cytes are the first target of B. abortus invasion and the bacteria
alone does not induce significant IFN- production, it
are able to survive and multiply within these cells (Pizarro-
synergistically enhances IL-12-stimulated IFN- production
Cerda et al., 1998). Resistance to B. abortus depends on the
(Yoshimoto et al., 1998) and promotes cell-mediated im-
effective generation of a T-cell-mediated response (Araya et
munity (Takeda et al., 1998). Besides its IFN- inducing
al., 1989) as well as on an effective activation of macrophages
effect, IL-18 has a direct proinflammatory effect on T and
by gamma interferon (IFN-)-producing CD4 T-lympho-
natural killer cells, enhancing proliferation and cytotoxicity
cytes (Zhan et al., 1993). IFN- is a key mediator in
and stimulating the production of cytokines such as tumour
conferring protection against Brucella spp. infections both
necrosis factor alpha (TNF-), IL-1
, IL-6 and IL-8 (Netea et
in vivo (Zhan & Cheers, 1995) and in vitro (Jiang & Baldwin,
al., 2000).
1993), and an elevated IFN- production, mainly in the early
phase of the infection, could be crucial in their control
Previous studies have demonstrated the role of IL-18 in the
(Pasquali et al., 2002). In consequence, cytokines secreted in
hosts response to infection. Endogenously synthesized IL-18
this initial response, which can affect IFN- secretion, can
has been demonstrated to be required in mice for an adequate
strongly affect the outcome of infection.
host defence against mycobacterial infections (Sugawara et al.,
Interleukin-18 (IL-18) is a proinflammatory cytokine pro- 1999), disseminated candidiasis (Stuyt et al., 2004), pneumo-
duced by several cell types, including activated macrophages, coccal pneumonia (Lauw et al., 2002), and Pseudomonas
dermal keratinocytes, osteoblasts, adrenal cortex cells and aeruginosa (Schultz et al., 2003), Yersinia enterocolitica (Bohn
intestinal epithelial cells (Gerdes et al., 2002), that is et al., 1998), Salmonella typhimurium (Mastroeni et al., 1999),
Leishmania major (Ohkusu et al., 2000) and Listeria mono-
Abbreviations: HK, heat-killed; i.p., intraperitoneal; p.i., post-infection; cytogenes (Neighbors et al., 2001) infections. Recently, it
S-LPS, smooth lipopolysaccharide. has been shown that only a combined administration of

45936 & 2005 SGM Printed in Great Britain 527

L. Fernandez-Lago, A. Orduna and N. Vizcano
recombinant IL-18 and IL-12 induces protection against B.
abortus 2308 infection in mice, and this effect is possibly due
to an increase in IFN- production in the early phases of
infection (Pasquali et al., 2001, 2002). However, at present
the dynamics of endogenous IL-18 production in response to
a B. abortus infection have not been thoroughly investigated.
The aim of the present study was to investigate the capacity of
B. abortus 2308 to stimulate the secretion of IL-18 by cultured
CD-1 peritoneal macrophages and splenocyte cultures, and
to evaluate the role of this cytokine in IFN- production in
response to B. abortus 2308 infection.
METHODS
Mice. Female outbred CD-1 mice (Charles River, Barcelona, Spain)
were purchased at 8 weeks of age and kept for 1 week before use. The
animals were housed in microisolator cages in Horsefall units, and were
cared for in accordance with standard guidelines.
Bacteria and infection of animals. Virulent B. abortus 2308, used as
the challenge strain, was cultured on tryptic soy agar (TSA; BBL)
enriched with 0.3 % (w/v) yeast extract (YE; Difco) for 72 h at 37 8C
under a 5 % (v/v) CO2 atmosphere in air. To infect the mice, the bacteria
were suspended in sterile PBS (pH 7.4) and adjusted turbidimetrically to
the desired concentration. The exact dose was established retrospec-
tively by viable cell counting. Mice were infected by the intraperitoneal
(i.p.) route with 0.2 ml PBS containing 5 3 105 c.f.u. of the B. abortus
2308 virulent strain. Control groups received 0.2 ml PBS. At the
indicated time in each experiment, five mice per group were killed by
cervical dislocation.
Reagents. B. abortus 2308 smooth lipopolysaccharide (S-LPS) and
culture supernatant extracts were obtained as described previously
(Vizcano et al., 1991; Joubier-Maurin et al., 2001). The S-LPS con-
tained, as a percentage of dry weight, 1.5 % protein and 1.1 % 2-keto-3-
deoxyoctulosonic acid. S-LPS from Escherichia coli O157 was obtained
from Sigma. Recombinant mouse IL-18 and IL-12 were obtained from
Pharmingen.
Isolation and infection of CD-1 mouse peritoneal macrophages.
Macrophages were isolated as described previously (Fernandez-Lago et
al., 1999), with some modifications. Briefly, CD-1 mice were inoculated
i.p. with 0.3 ml incomplete Freunds adjuvant (Sigma). Three days later,
the mice were killed and peritoneal cells were harvested by washing with
cold RPMI (Gibco) with 10 % (v/v) fetal calf serum (FCS; Gibco). The
peritoneal cells thus collected were washed twice by centrifugation at
1200 g for 5 min, and resuspended in RPMI, 20 mM HEPES (Life
Technologies) supplemented with 2 mM L-glutamine (Life Tech-
nologies), 10 5 M
-mercaptoethanol and 10 % (v/v) FCS. The cell
suspensions were adjusted to 106 cells ml 1 , seeded on 24-well plates
(1 ml per well), and macrophages were allowed to adhere for 90 min at
37 8C under a 5 % (v/v) CO2 atmosphere. Non-adhering cells were
washed off and adhering cells were washed three times with 1 ml of the
same medium. The number and viability of the macrophages obtained
by this procedure were determined by morphological examination and
trypan blue exclusion (Fernandez-Lago et al., 1999). For macrophage
infection, bacteria from overnight cultures were opsonized at 37 8C for
1 h in PBS containing a subagglutinating dilution (1 : 200) of the
monoclonal antibody BmE10-5, which reacts specifically with the
S-LPS of Brucella spp. (Vizcano et al., 1991). Opsonized B. abortus
2308 cells were then centrifuged, washed and diluted in RPMI
supplemented as described above. For infection, macrophage cells were
incubated with 1 ml of the opsonized bacterial suspension at a m.o.i.
of 30 : 1 (bacteria : macrophage) for 1 h. After incubation, non-
528
phagocytosed bacteria were washed off. Following this, 1 ml RPMI
supplemented as described above and containing 50 g gentamicin
ml 1 was added to the macrophages to kill any remaining extracellular
bacteria. The cells were then cultured in the same medium with
gentamicin (50 g ml 1 ) at 37 8C under a 5 % (v/v) CO2 atmosphere
in air for 48 h, and culture supernatants were obtained from each well,
filtered through 0.45 m pore-size filters, and kept at 70 8C until used
for cytokine analysis. Supernatants obtained at the same time from
uninfected macrophage cultures were used as negative controls. Unin-
fected macrophage cultures (five wells per sample) were also incubated,
in the same medium with gentamicin (50 g ml 1 ), in the presence of
either purified S-LPS (20 g ml 1 ) or a 1/10 dilution of culture
supernatant obtained from B. abortus 2308, and S-LPS from E. coli
O157 (1 g ml 1 ; Sigma). Macrophage supernatants were also obtained
at 48 h from each well, filtered, and kept at 70 8C until assayed.
Splenocyte cultures. Infected CD-1 mice were killed on days 3, 6 and
10 p.i. The spleens were removed aseptically, and individual cell
suspensions were prepared by gentle teasing through cell strainers
(Becton Dickinson). Erythrocytes were lysed by adding lysis buffer
(0.155 M NH4 Cl, 10 mM KHCO3 , 10 mM EDTA, pH 7.4) to the
pellet for 3 min. The cells were then washed three times in RPMI,
20 mM HEPES supplemented with 2 mM L-glutamine, 10 5 M
-
mercaptoethanol and 10 % (v/v) FCS. Spleen cells (106 ) were cultured in
24-well plates (0.5 ml per well) and stimulated with 0.5 ml heat-killed
(HK) B. abortus 2308 at 107 cells ml 1 . Cultures were incubated at 37 8C
under a 5 % (v/v) CO2 atmosphere in air. Supernatants were harvested
at 48 h, filtered through a 0.45 m pore-size filter, and frozen at 70 8C
for use in cytokine assays. Controls including cultures of splenocytes
obtained from uninfected mice or from infected mice stimulated with
the mitogen concanavalin A (5 g per well; Sigma) were also established.
In some experiments, splenocytes obtained at 3 days p.i. from mice
infected with 5 3 105 c.f.u. B. abortus 2308 were cultured, once
stimulated with 107 HK B. abortus 2308 cells, in the presence of
recombinant IL-18 (10 ng ml 1 ) and/or rIL-12 (10 ng ml 1 ). Super-
natants were also removed after 48 h, filtered, and tested for IFN-
production.
Cytokine ELISA. Cytokine contents in macrophage and splenocyte
supernatants were determined by ELISA, using commercial kits for
IL-18 (biologically active and non-active forms) and IFN- (OptEIA;
Pharmingen). Protocols were as recommended by the manufacturer.
Known concentrations of each cytokine were used to generate standard
curves in each assay and used as references to calculate cytokine
concentrations. Samples were assayed in duplicate and values are
expressed as means  SD. The lowest limit of detection for IL-18 and
IFN- was 30 pg ml 1 .
Statistical methods. All experiments in this study were performed at
least twice. Experimental values are given as means  SD. The data
obtained were analysed by one-way analysis of variance, comparing the
mean values with the Fishers protected LSD (least significant differ-
ences, P , 0.05).
RESULTS AND DISCUSSION
IL-18 secretion is reduced in B. abortus
2308-infected macrophages
To investigate the effect of B. abortus 2308 infection on
macrophage-derived IL-18 secretion, CD-1 mouse elicited-
peritoneal macrophage cultures were used. As shown in Fig.
1, 24 h culture supernatants of uninfected macrophages
constitutively secreted IL-18. A significant (P , 0.05) in-
crease in the secretion of this cytokine was observed when
Journal of Medical Microbiology 54
 Reduced IL-18 secretion by B. abortus

100 abortus 2308 to induce the secretion of IL-18 mediated by

Interleukin-18 (pg ml21)

* CD-1 mouse elicited-peritoneal macrophages. As shown in

80
60
40
20
,30 pg ml21
0 in IL-18 synthesis in mouse macrophages is possibly depen-
Uninfected LPS-Ec Ba-infected LPS-Ba
Fig. 1. B. abortus 2308-infected peritoneal macrophages secrete
reduced amounts of IL-18. Cultured peritoneal macrophages were
uninfected, infected with virulent B. abortus 2308 (Ba-infected), or
treated with S-LPS from E. coli (LPS-Ec; 1 g ml 1 ), S-LPS from B.
abortus 2308 (LPS-Ba; 20 g ml 1 ) or a B. abortus 2308 culture
extract (Extract-Ba; 1 : 10 dilution). After 2 days of culture, super-
natants were harvested and assayed for IL-18 by ELISA. Results are
expressed as means  SD (five wells per point). *, Statistically differ-
ent from uninfected macrophage cultures (P , 0.05).
cultured peritoneal macrophages were exposed to S-LPS
from E. coli O157 (1 g ml 1 ) (Fig. 1). In contrast, when
infected with opsonized B. abortus 2308 for 90 min, followed
by removal of extracellular bacteria, supernatants of these
macrophage cultures showed no IL-18 activity (, 30 pg
ml 1 ; Fig. 1). These results demonstrate that B. abortus 2308
infection induces a reduction of IL-18 secretion in mice at the
macrophage level. It has previously been demonstrated that
Brucella culture supernatants contain a protein factor(s) that
is able to inhibit the secretion in LPS-activated human
macrophages of other macrophage-derived cytokines, such
as TNF- (Joubier-Maurin et al., 2001). Accordingly, we also
analysed the capacity of both the culture supernatants (1 : 10
dilution) and the S-LPS (20 g ml 1 ) obtained from B.
Fig. 1, neither B. abortus 2308 culture supernatants nor B.
abortus 2308 S-LPS had a significant effect on IL-18 secretion.
Similar results were obtained upon using different concen-
trations of both preparations (data not shown). These
findings suggest, as happens with wild-type Salmonella
dublin infections (Elhofy & Bost, 1999), that this decrease
Extract-Ba
dent on the intracellular replication of B. abortus 2308,
although the mechanism responsible for this remains to be
elucidated.
IFN- production in B. abortus 2308-infected
splenocyte cultures is not dependent on the
presence of IL-18
In subsequent experiments we analysed the effect that the
reduction in IL-18 secretion, observed in cultured macro-
phages as a result of B. abortus 2308 infection, had on IFN-
secretion in B. abortus 2308-infected mice at the splenocyte
level. As shown in Table 1, a 48 h splenocyte culture super-
natant obtained from uninfected CD-1 mice constitutively
expressed IL-18. A significant decrease (P , 0.05) in the
secretion of this cytokine was observed in 48 h splenocyte
cultures obtained from B. abortus 2308-infected mice on days
3, 6 and 10 p.i., and stimulated in vitro with 107 HK B. abortus
2308 cells. In contrast, a statistically significant (P , 0.05)
increase in IFN- secretion by the splenocyte cultures was
observed on days 3, 6 and 10 p.i. (Table 1). These results show
that the limited secretion of IL-18, as a result of B. abortus
2308 infection, does not significantly affect IFN- produc-
tion, and therefore that IL-18 would not be involved in B.
abortus-induced IFN- secretion at the splenocyte level. To
investigate this further, splenocytes were obtained from B.
abortus 2308-infected mice at 3 days p.i. and, once stimulated

Table 1. Production of IL-18 and IFN- in stimulated spleen cells from B. abortus 2308-
infected mice
CD-1 mice were i.p.-infected with 5 3 105 c.f.u. B. abortus 2308. Mice, five per group, were killed
at 3, 6 and 10 days after infection. Data are mean values of five animals  SD. An asterisk indicates
that the results are statistically significant (P , 0.05) as compared to those for the uninfected
control group.

Time after infection that Spleen wt Cytokine level (pg ml21 )

splenocytes were obtained (mg)
(days) IL-18 IFN-

Uninfected control 100  17 125  10 , 30

3 164  20* 60  8* 137  20*
6 204  15* 35  3* 120  15*
10 404  47* , 30 130  12*

Splenocytes were cultured in RPMI medium and stimulated with 107 HK B. abortus 2308 cells as
described in Methods. Supernatants were collected at 48 h for cytokine analysis.
Culture supernatants were tested for IL-18 and IFN- by ELISA. The level of sensitivity for both
cytokines was 30 pg ml 1 .

http://jmm.sgmjournals.org 529
L. Fernandez-Lago, A. Orduna and N. Vizcano
Table 2. Effect of IL-18 and IL-12 on IFN- production by
stimulated spleen cells from B. abortus 2308-infected mice
CD-1 mice were i.p.-infected with 5 3 105 c.f.u. B. abortus 2308. Mice,
five per group, were killed at 3 days after infection. Data are mean values
of five animals  SD.
Treatment* IFN- level (pg ml21 )
None 130  26
IL-18 145  60
IL-12 142  33
IL-18 + IL-12 145  50
Concanavalin A 550  125
*Cytokines (10 ng ml 1 ) were added to splenocyte cultures. Concana-
valin A was used at 5 g ml 1 .
Splenocytes were cultured in RPMI medium and stimulated with 107
HK B. abortus 2308 cells as described in Methods. Supernatants were
collected at 48 h and tested for IFN- by ELISA. The level of sensitivity
was 30 pg ml 1 .
with 107 HK B. abortus cells, were treated with rIL-12 (10 ng
ml 1 ) and/or rIL-18 (10 ng ml 1 ) or concanavalin A (5 g
ml 1 ). The levels of IFN- were then measured in the culture
supernatants 48 h after stimulation. As shown in Table 2, the
addition to splenocyte cultures of rIL-12, rIL-18 or both
cytokines simultaneously did not significantly affect IFN-
secretion as compared to untreated cells. A significant
(P , 0.05) increase in IFN- production was only seen in
the concanavalin A-treated splenocyte cultures (Table 2). It
may therefore be concluded that once the infection has been
established, the exogenous addition of IL-12, IL-18 or both
cytokines simultaneously does not contribute to IFN-
production in response to B. abortus 2308 infection. Thus
it is possible that both cytokines are active, inducing a
protective endogenous IFN- production only when phar-
macologically effective levels of them in a biologically active
form are present in the first stages of infection (Pasquali et al.,
2002).
ACKNOWLEDGEMENTS
This work was financed by project G03/204 Red para la Investigacion de
la Brucelosis, Instituto de Salud Carlos III, Ministerio de Sanidad y
Consumo, Spain.
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