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Role of interleukin-18 in experimental group B streptococcal arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 6, June 2004, pp 2005–2013
DOI 10.1002/art.20014
© 2004, American College of Rheumatology
Role of Interleukin-18 in
Experimental Group B Streptococcal Arthritis
Luciana Tissi,1 Bradford McRae,2 Tariq Ghayur,2 Christina von Hunolstein,3 Graziella Orefici,3
Francesco Bistoni,1 and Manuela Puliti1
Objective. To assess the role of interleukin-18
(IL-18) in the evolution of septic arthritis induced by
group B streptococci (GBS) in mice.
Methods. CD1 mice were inoculated intravenously with 8 ⴛ 106 colony-forming units (CFU) of type
IV GBS (strain 1/82), and administered intraperitoneally 1 hour before infection with anti–IL-18 monoclonal
antibodies (0.25 mg/mouse). In a subsequent set of experiments, mice infected with a suboptimal arthritogenic dose
of GBS (4 ⴛ 106 CFU/mouse) were administered different
doses of recombinant IL-18 for 4 days, starting 1 hour
after infection. Mortality, evolution of arthritis, bacterial
clearance, joint histopathology, and cytokine production
were examined in infected mice that did or did not receive
treatment with anti–IL-18 antibodies or IL-18.
Results. IL-18 was produced during GBS infection. Neutralization of IL-18 resulted in a decrease in
mortality rates, and in the incidence and severity of
arthritis. Amelioration of arthritis was accompanied by
a dramatic reduction in local IL-1␤, IL-6, macrophage
inflammatory protein 1␣ (MIP-1␣) and MIP-2 production, and reduced bacterial burden. Administration of
exogenous IL-18 resulted in increased mortality rates
and increased incidence and severity of GBS arthritis,
concomitant with a higher number of GBS and increased levels of IL-6, IL-1␤, MIP-1␤, and MIP-2
production in the joints.
Conclusion. The present study indicated some
involvement of IL-18 in the pathogenesis of GBSinduced arthritis. The role of IL-18 in joint pathology is
shown by a regulatory effect on inflammatory mediator
levels and local cell influx. Thus, IL-18 should be
regarded as a potential therapeutic target in GBS
infection and arthritis.
Group B streptococci (GBS) are a leading cause
of life-threatening infections in neonates and infants (1).
Invasive neonatal GBS infection has either an early
(usually the first 24 hours after birth) or late (7 days after
birth) onset. Common manifestations of GBS disease in
neonates include pneumonia, septicemia, meningitis,
bacteremia, and bone or joint infections (1). Invasive
disease caused by GBS has also been recognized in
adults (2,3).
Septic arthritis is one of the clinical manifestations of late-onset GBS infection in neonates (1) and
requires prolonged antibiotic treatment to ensure an
uncomplicated outcome. In adults, GBS septic arthritis
is often associated with age and severe underlying
diseases (4–7). GBS arthritis is usually hematogenously
acquired, and the most frequently affected joints are the
hip, ankle, and wrist (1). In our mouse model of
hematogenously induced GBS arthritis, mice inoculated
with the reference serotype IV GBS strain manifested
clinical signs of arthritis characterized by early onset,
with evolution from an acute exudative synovitis to
permanent lesions with irreversible joint damage and/or
ankylosis (8).
This laboratory mouse model offers outstanding
potential for GBS arthritis, in that bacteremia persists
for ⬎10 days after GBS infection and localization of
articular lesions is similar to that in humans. We initially
demonstrated that induction of GBS arthritis depends
on the viability and number of microorganisms injected
(8), the presence and amount of bacterial capsule, and
Supported by a grant (2001061479-003) from the Ministero
dell’Università e della Ricerca Scientifica 2001–2002, Italy.
1
Luciana Tissi, PhD, Francesco Bistoni, MD, Manuela Puliti,
PhD: University of Perugia, Perugia, Italy; 2Bradford McRae, PhD,
Tariq Ghayur, PhD: Abbott Bioresearch Center, Worcester, Massachusetts; 3Christina von Hunolstein, PhD, Graziella Orefici, MD:
Istituto Superiore di Sanità, Rome, Italy.
Address correspondence and reprint requests to Luciana
Tissi, PhD, Department of Experimental Medicine and Biochemical
Sciences, Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail: tissi@unipg.it.
Submitted for publication October 7, 2003; accepted in revised form February 23, 2004.
2005
2006
TISSI ET AL
sialic acid in the capsular polysaccharide (9). In this
model, high-level systemic and local production of
interleukin-6 (IL-6) and IL-1␤ and scant production of
tumor necrosis factor ␣ (TNF␣) were observed in response to GBS infection (10). Direct correlation between the severity of arthritis and joint concentrations of
IL-6 and IL-1␤, but not TNF␣, was observed (10).
IL-18 is a novel cytokine that exhibits proinflammatory features and is a member of the IL-1 family of
proteins, originally identified as the interferon-␥
(IFN␥)–inducing factor (11). IL-18 is structurally related
to IL-1␤; both cytokines require IL-1␤–converting enzyme for cleavage of the precursor to release the bioactive molecules for IL-1␤ and IL-18 (12,13). Pro–IL-18
expression has been detected in antigen-presenting cells
such as activated macrophages, Kupffer cells (11), and
dendritic cells (14), as well as articular chondrocytes (15)
and osteoblasts (16). IL-18 induces the production of
proinflammatory cytokines such as TNF␣ and IL-1 (17).
Furthermore, IL-18 stimulates the proliferation of activated T cells and inhibits the formation of osteoclast-like
cells (16).
In several diseases, including rheumatoid arthritis
(RA), IL-18 is considered a proinflammatory cytokine
(18). IL-18 is expressed in human RA synovium, and
enhanced levels of IL-18 have been found in the sera of
RA patients (19). Furthermore, IL-18 plays a role in the
induction of RA synovial fibroblast expression of CXC
chemokines through NF-␬B (20). IL-18 induces chondrocyte proliferation, up-regulates inducible nitric oxide
synthase, stromelysin, IL-6, and cyclooxygenase 2 expression, and increases glycosaminoglycan release (15).
A central role of IL-18 has also been shown in acute
streptococcal cell wall (SCW)–induced joint inflammation, where neutralization of endogenous IL-18 suppresses joint swelling by reducing local TNF␣ and IL-1␤
levels (21).
The aim of the present study was to investigate
the role of IL-18 in our experimental model of GBSinduced septic arthritis. Endogenous IL-18 was neutralized before arthritis induction and the effect on joint
pathology was determined. The impact of exogenous
IL-18 administration on the severity of articular lesions
using a suboptimal arthritogenic dose of GBS was also
examined.
MATERIALS AND METHODS
Mice. Sex-matched, 8-week-old male or female outbred CD1 mice were obtained from Charles River (Calco,
Italy).
Microorganism. Type IV GBS, reference strain 1/82,
were grown overnight at 37°C in Todd-Hewitt broth (Oxoid,
Basingstoke, UK), washed, and diluted in RPMI 1640 medium
(Gibco Life Technologies, Milan, Italy). The inoculum size was
estimated turbidimetrically, and viability counts were performed by plating on tryptic soy agar–5% sheep blood agar
(blood agar), and incubating overnight at 37°C under anaerobic conditions. Mice were inoculated intravenously via the tail
vein with 8 ⫻ 106 or 4 ⫻ 106 GBS in a volume of 0.5 ml.
Control mice were injected in the same manner with 0.5 ml of
RPMI 1640 medium.
Cytokines and antibodies. Recombinant murine IL-18
(6.7 ⫻ 106 units/mg) was purchased from R&D Systems
(Minneapolis, MN) and diluted according to the manufacturer’s recommendations in phosphate buffered saline (PBS)
containing 0.1% bovine serum albumin (BSA). IL-18 was
injected intraperitoneally in a 0.2-ml volume at different doses
(ranging from 0.01 to 0.1 ␮g per mouse) once a day for 4 days
starting 1 hour before GBS infection. As controls, infected
mice received PBS plus BSA according to the same experimental schedule, and uninfected mice received IL-18. Mouse
anti-mouse IL-18 monoclonal antibody (mAb; clone 1C5,
isotype IgG1) was generated at Abbott Bioresearch Center
(Worcester, MA). Normal mouse IgG was purchased from
Sigma (Milan, Italy). The antibodies (0.25 mg/mouse) were
injected intraperitoneally 1 hour before GBS infection. The
dose was chosen on the basis of preliminary titration experiments showing complete neutralization of in vivo IL-18 serum
levels as determined by enzyme-linked immunosorbent assay
(ELISA). Control mice received PBS according to the same
experimental schedule.
Clinical evaluation of arthritis and mortality. Mice
that were injected with GBS and that did or did not receive
treatment with mAb or IL-18 (as described above) were
evaluated for signs of arthritis and mortality. Mortality was
recorded at 24-hour intervals for 30 days. After introduction of
GBS, mice were examined daily by 2 observers (LT and MP)
for 1 month to evaluate the presence of joint inflammation,
and scores for arthritis severity (macroscopic score) were given
as previously described (10,22). Arthritis was defined as visible
erythema and/or swelling of at least 1 joint. Clinical severity of
arthritis was graded on a scale of 0–3 for each paw, according
to changes in erythema and swelling (0 ⫽ no change, 1 ⫽ mild
swelling and/or erythema, 2 ⫽ moderate swelling and erythema, and 3 ⫽ marked swelling, erythema, and/or ankylosis).
Thus, a mouse could have a maximum score of 12. The arthritis
index (mean ⫾ SD) was constructed by dividing the total score
(cumulative value of all paws) by the number of animals used
in each experimental group.
Histologic assessment. Groups of mice that were infected with GBS and that did or did not receive treatment with
anti–IL-18 mAb or IL-18 were examined 7 days after infection
for histopathologic features of arthritis. Mice were euthanized
and arthritic hind paws (1 per mouse) were removed aseptically, fixed in formalin (10% volume/volume) for 24 hours and
then decalcified in trichloroacetic acid (5% v/v) for 7 days,
dehydrated, embedded in paraffin, sectioned at 3–4 ␮m, and
stained with hematoxylin and eosin (H&E). Samples were
examined under blinded conditions. Tibiotarsal, tarsometatarsal, and metatarsophalangeal joints were examined, and a
histologic score was assigned to each joint based on the extent
IL-18 IN GBS ARTHRITIS
of infiltrate (presence of inflammatory cells in the subcutaneous and/or periarticular tissues), exudate (presence of inflammatory cells in the articular cavity), cartilage damage, bone
erosion, and loss of joint architecture. Arthritis severity was
classified as mild (minimal infiltrate), moderate (presence of
infiltrate, minimal exudate, integrity of joint architecture), or
severe (presence of massive infiltrate/exudate, cartilage and
bone erosion, and disrupted joint architecture).
GBS growth in blood, kidneys, and joints. Blood,
kidney, and joint infections in GBS-infected mice that did or
did not receive treatment with anti–IL-18 mAb or IL-18 were
determined by evaluation of colony-forming units (CFU) at
different times after inoculation. Blood samples were obtained
by retroorbital sinus bleeding before the mice were killed.
Ten-fold dilutions were prepared in RPMI 1640 medium, and
0.1 ml of each dilution was plated in triplicate on blood agar
and incubated under anaerobic conditions for 24 hours. The
number of CFU was determined and the results were expressed as the number of CFU per milliliter of blood. Kidneys
were removed aseptically and placed in a tissue homogenizer
with 3 ml of sterile RPMI 1640 medium. All wrist and ankle
joints from each mouse were removed, weighed, and ground in
a mortar in sterile RPMI 1640 medium (1 ml/100 mg joint
weight). After homogenization, all tissue samples were diluted
and plated in triplicate on blood agar, and the results were
expressed as the number of CFU per whole organ or per
milliliter of joint homogenate.
Sample preparation for cytokine assessment. Blood
samples from the different experimental groups were obtained
by retroorbital sinus bleeding at different times after infection
before the mice were killed. Sera were stored at ⫺80°C until
analyzed. Joint tissues were prepared as previously described
(10). Briefly, all wrist and ankle joints from each mouse were
removed and then homogenized in toto in 1 ml/100 mg joint
weight of lysis medium (RPMI 1640 containing 2 mM phenylmethylsulfonyl fluoride and 1 ␮g/ml final concentration of
aprotinin, leupeptin, and pepstatin A). The homogenized
tissues were then centrifuged at 2,000g for 10 minutes, and
supernatants were sterilized using a Millipore filter (0.45 ␮m)
and stored at ⫺80°C until analyzed.
Cytokine assays. IL-18, IL-6, IL-1␤, macrophage inflammatory protein 1␣ (MIP-1␣), MIP-2, IFN␥, and TNF␣
concentrations in the biologic samples were measured with
commercial ELISA kits (IL-6, IL-1␤; Amersham Pharmacia
Biotech, Little Chalfont, UK, and IFN␥, TNF␣, IL-18, MIP1␣, and MIP-2; R&D Systems), according to the manufacturers’ recommendations. Results were expressed as picograms
per milliliter of serum or supernatant from joint homogenates.
The detection limits of the assays were 25 pg/ml for IL-18, 7
pg/ml for IL-6, 3 pg/ml for IL-1␤, 1.5 pg/ml for MIP-1␣, 1.5
pg/ml for MIP-2, 15 pg/ml for IFN␥, and 5.1 pg/ml for TNF␣.
Statistical analysis. Differences in the arthritis index,
number of CFU, and cytokine concentrations between the
groups of mice were analyzed by Student’s unpaired t-test.
Between-group differences in survival data were analyzed by
the Mann-Whitney U test, and incidence of arthritis and
histologic data were analyzed by the chi-square test. Each
experiment was repeated 3 times. Results are expressed as the
mean ⫾ SD. P values less than 0.05 were considered significant.
2007
Figure 1. Interleukin-18 (IL-18) levels in supernatants from joint
homogenates and sera from mice infected with 8 ⫻ 106 colony-forming
units/mouse of group B streptococcus (GBS) and from uninfected
controls. Supernatants from joint homogenates and sera were collected as described in Materials and Methods. IL-18 levels in the
biologic samples were determined by enzyme-linked immunosorbent
assay. Values are the mean ⫾ SD of 3 separate experiments. In each
experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍
0.01 versus uninfected controls.
RESULTS
IL-18 production during GBS infection. IL-18
production was assessed in sera and supernatants from
the joints at different times after infection with 8 ⫻ 106
CFU/mouse of GBS. As shown in Figure 1, joint levels
of IL-18 were significantly higher (P ⬍ 0.01) in GBSinfected mice compared with naive mice (mean ⫾ SD
60.1 ⫾ 9.8 versus 25.8 ⫾ 4.9 pg/ml of joint homogenate,
day 1 after infection). Maximum values were reached on
day 7 (578 ⫾ 70.2 pg/ml of joint supernatant versus
20.6 ⫾ 5.5 pg/ml in naive animals). Circulating levels of
IL-18 were also augmented during infection and peaked
on day 7, although systemic production was lower and
later than that observed locally.
Effect of IL-18 blockade on clinical course of
arthritis. Clinical signs of joint swelling were observed in
30% of the mice as early as 24 hours after infection with
8 ⫻ 106 CFU of GBS. The incidence of arthritis increased to 70% by day 5, and the maximum prevalence
was observed on day 7 after inoculation, when 80% of
the mice manifested clinical signs of arthritis (Figure
2A). Similarly, the arthritis index progressively increased
and reached maximum value 10 days after GBS introduction (mean ⫾ SD 3.0 ⫾ 0.5) (Figure 2B); most of the
animals had articular lesions in both the hind paws and
fore paws. Forty percent of mice died during the course
of infection (Figure 2C).
Neutralization of endogenous IL-18 was performed by administering anti–IL-18 mAb (0.25 mg/
mouse) 1 hour before infection. Efficacy of anti–IL-18
2008
Figure 2. Effect of endogenous IL-18 neutralization on mortality rates
and on the incidence and severity of arthritis in mice infected with
GBS (8 ⫻ 106 colony-forming units/mouse). Monoclonal antibodies
(0.25 mg/mouse) were injected intraperitoneally 1 hour before GBS
infection. Control mice received phosphate buffered saline (PBS)
according to the same protocol. Ten mice were used in each experimental group. A, Incidence of arthritis (percentage of mice with visible
arthritis). B, Arthritis index (clinical severity of arthritis, evaluated as
described in Materials and Methods). For A and B, values are the
mean ⫾ SD of 3 separate experiments. C, Survival curves. Mortality
was recorded at 24-hour intervals for 30 days. Data represent the
cumulative results of 3 separate experiments. D, Histopathologic
severity of arthritis in joints from hind paw sections, assessed 7 days
after infection. Arthritis was scored as mild, moderate, or severe, as
described in Materials and Methods. For anti–IL-18 treatment, 10
paws and 24 joints were assessed; for PBS treatment 10 paws and 28
joints were assessed. ⴱ ⫽ P ⬍ 0.01 versus controls; F ⫽ P ⬍ 0.05 versus
controls. See Figure 1 for other definitions.
mAb treatment was assessed by measuring IL-18 levels
in serum and joints after antibody injection (days 1, 2, 3,
and 5). Total abrogation of systemic free IL-18 production and a significant (P ⬍ 0.01) decrease in local
cytokine levels were observed at all time points checked
(data not shown). Anti–IL-18 mAb treatment resulted in
a reduced number of animals showing articular lesions
compared with controls, although the differences between the 2 experimental groups were not significant
(Figure 2A). The arthritis index in mice treated with
anti–IL-18 mAb was significantly lower than that in
controls, reaching a maximum value of 1.8 ⫾ 0.4,
compared with 3.0 ⫾ 0.5, on day 10 after infection
(Figure 2B). Differences in the arthritis index between
the 2 experimental groups were still significant at the
end of the observation period (data not shown). There
were significant differences in mortality rates between
mice treated with anti–IL-18 mAb and control mice (P ⫽
0.042). Only 10% of animals treated with anti–IL-18
mAb died, versus 40% of controls (Figure 2C). Irrele-
TISSI ET AL
vant antibodies did not affect mortality rates, or the
incidence or severity of arthritis (data not shown).
Seven days after infection, mice were killed and
the most frequently affected paw (hind paw) was removed for histologic examination. Microscopic analysis
of H&E-stained sections was performed. In control
animals, 57.1% of the examined joints were classified as
severely affected, with massive infiltrate/exudate, cartilage and bone erosion, and loss of joint integrity, and
35.7% were classified as moderately affected; only 7.2%
of the joints were classified as mildly affected (Figure
2D). In contrast, most (58.3%) of the examined joints
from mice treated with anti–IL-18 mAb were classified
as moderately affected, and 33.4% of the joints were
classified as mildly affected; only 8.3% of the joints in
this group were severely affected (Figure 2D).
Quantitative monitoring of bacteremia and bacterial growth in the kidneys and joints of mice that did or
did not receive treatment with anti–IL-18 mAb was
performed. A significantly lower (P ⬍ 0.01) number of
microorganisms was recovered from the joints of mice
treated with anti–IL-18 mAb compared with controls 3
days after infection (2.4 ⫻ 105 ⫾ 0.4 ⫻ 105 versus 8.4 ⫻
106 ⫾ 0.7 ⫻ 106, respectively). Such differences were
also found in subsequent days (data not shown). A
similar trend was observed in GBS growth rates in the
kidneys, while no significant differences were observed
between the experimental groups in the blood (data not
shown).
Effect of IL-18 blockade on cytokine production.
Since IL-6 and IL-1␤ play a major role in the pathogenesis of GBS arthritis (10), the effect of IL-18 blockade on
these cytokine levels was assayed. Based on results from
other experimental models indicating an involvement of
chemokines in the development of arthritis (23–25),
MIP-1␣ (a CC chemokine) and MIP-2 (a CXC chemokine) concentrations were also determined. Plasma and
joint specimens were collected daily from day 0 to day 3,
and then on days 5 and 10 after GBS infection.
As previously described (10) and as shown in
Figure 3, a rapid increase in IL-6 and IL-1␤ production
was observed in the joints and sera of GBS-treated mice.
Sustained levels of both cytokines were still present 10
days after infection. A time-dependent increase in chemokine concentrations was also observed in the joints.
In particular, MIP-2 levels peaked on day 3 after bacterial inoculation (681 ⫾ 98 pg/ml), while the MIP-1␣
concentration was 256 ⫾ 47 pg/ml. In the serum, MIP-1␣
concentrations slightly increased, whereas more sustained levels were reached by MIP-2. IL-18 neutralization resulted in an early (24 hours) decrease in IL-6 joint
IL-18 IN GBS ARTHRITIS
2009
in IL-18–treated animals than in controls, with a marked
worsening of articular lesions. A similar negative effect
of IL-18 administration upon survival was observed. In
fact, with an inoculum size of 4 ⫻ 106 GBS/mouse only
10% of the control mice had died at the end of the
observation period, while mortality rates in mice treated
with 0.05 or 0.1 ␮g/mouse of IL-18 were 40% and 50%,
respectively (Figure 4C). No effects were observed in
terms of mortality rates and the incidence and severity
of arthritis when mice were injected with 0.01 ␮g/mouse
Figure 3. Effect of anti–IL-18 monoclonal antibody (mAb) administration on IL-6, IL-1␤, macrophage inflammatory protein 1␣ (MIP1␣), and MIP-2 production in the sera and joints of mice infected with
GBS (8 ⫻ 106 colony-forming units/mouse). Anti–IL-18 mAb (0.25
mg/mouse) or phosphate buffered saline (PBS) was injected intraperitoneally 1 hour before infection. Uninfected mice (day 0) received PBS
at the time of mAb administration. Blood samples and supernatants
from joint homogenates were collected at the indicated times after
treatment (see Materials and Methods). Levels of IL-6, IL-1␤, MIP1␣, and MIP-2 were determined by enzyme-linked immunosorbent
assay. Values are the mean ⫾ SD of 3 separate experiments. In each
experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍
0.01 versus control mice. See Figure 1 for other definitions.
concentrations. IL-6 levels in the joints of mice treated
with anti–IL-18 mAb remained significantly lower (P ⬍
0.01) than those in the joints of control mice at all time
points assessed. In addition, anti–IL-18 treatment resulted in lower levels of IL-1␤, MIP-1␣, and MIP-2
production. However, in this case, the phenomenon was
evident starting 2 days after mAb administration, and at
a later time point (day 10), all cytokine levels in treated
and control mice were comparable. In the first days
(days 0–3) after infection, anti–IL-18 treatment resulted
in a strong decrease in systemic levels of IL-6 and MIP-2,
but not IL-1␤. Irrelevant antibodies did not affect cytokine production (data not shown).
Effect of IL-18 administration on mortality and
arthritis. To further define the role of IL-18 in GBSinduced articular pathology, mice were injected with a
suboptimal arthritogenic dose of GBS (4 ⫻ 106 CFU/
mouse) and treated with different doses of IL-18 (ranging from 0.01 to 0.1 ␮g/mouse) for 4 days, starting 1 hour
before infection. Control infected mice received PBS
plus BSA (vehicle) following the same treatment schedule. As shown in Figures 4A and B, IL-18 treatment
dramatically influenced the clinical course of GBS arthritis. The frequency of arthritis was more pronounced
Figure 4. Effect of exogenous IL-18 administration on survival rates
and on the incidence and severity of GBS arthritis in CD1 mice
infected intravenously with GBS (4 ⫻ 106 colony-forming units/
mouse). Murine recombinant IL-18 (0.1 or 0.05 ␮g/mouse) or phosphate buffered saline plus 0.1% bovine serum albumin (vehicle) was
administered intraperitoneally for 4 days starting 1 hour before
infection. Ten mice were used in each experimental group. A, Incidence of arthritis (percentage of mice with visible arthritis). B,
Arthritis index (clinical severity of arthritis, evaluated as described in
Materials and Methods). C, Survival curves. Mortality was recorded at
24-hour intervals for 30 days. Data represent cumulative results of 3
separate experiments. For A and B, values are the mean ⫾ SD of 3
separate experiments. ⴱ ⫽ P ⬍ 0.01; F ⫽ P ⬍ 0.05 versus vehicletreated mice. See Figure 1 for definitions.
2010
Figure 5. Effect of exogenous IL-18 administration on IL-6, IL-1␤,
macrophage inflammatory protein 1␣ (MIP-1␣), and MIP-2 production in the sera and joints of mice infected with GBS (4 ⫻ 106
colony-forming units/mouse). IL-18 (0.05 ␮g/mouse) or phosphate
buffered saline plus 0.1% bovine serum albumin (vehicle) was injected
intraperitoneally for 4 days starting 1 hour before infection. Control
uninfected mice (day 0) received vehicle according to the same
protocol. Blood samples and supernatants from joint homogenates
were collected at the indicated times after treatment (see Materials
and Methods). Levels of IL-6, IL-1␤, MIP-1␣, and MIP-2 were
determined by enzyme-linked immunosorbent assay. Values are the
mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per
group were killed at each time point. ⴱ ⫽ P ⬍ 0.01 versus vehicletreated mice. See Figure 1 for other definitions.
of IL-18 (data not shown). Histologic findings confirmed
the clinical observations (data not shown).
Effect of IL-18 administration on GBS growth
and cytokine production. In vivo GBS growth was
assessed in blood, kidneys, and joints of mice treated
with IL-18 (0.05 ␮g/mouse for 4 days) or vehicle. There
were no significant differences in the number of GBS
recovered from the bloodstream between the experimental groups (data not shown). In contrast, higher GBS
titers were observed in the joints and kidneys of IL-18–
treated mice compared with controls from day 2 after
infection on (data not shown). Ten days after injection,
7.6 ⫻ 107 ⫾ 0.2 ⫻ 107 GBS were recovered in the joints
and 1.2 ⫻ 108 ⫾ 0.2 ⫻ 108 in the kidneys of IL-18–
treated mice, compared with 1.0 ⫻ 106 ⫾ 0.3 ⫻ 106 and
9.8 ⫻ 106 ⫾ 1.0 ⫻ 106 in controls, respectively.
Animals infected with GBS and treated with
IL-18 or vehicle were monitored for systemic and local
production of proinflammatory cytokines and chemokines. As shown in Figure 5, administration of a subarthritogenic dose of GBS resulted in moderate local
production of IL-6, IL-1␤, MIP-1␣, and MIP-2. A significantly dramatic increase (P ⬍ 0.01) in cytokine and
chemokine production was evident upon treatment with
TISSI ET AL
IL-18. This effect was not limited to the period of IL-18
administration, since higher levels of all the secreted
proteins examined were still found in the joints of
IL-18–treated animals compared with controls at the
end of the observation period. Systemic production of
IL-6 and MIP-2 was also increased by IL-18 treatment,
while no effect was evident on IL-1␤ and MIP-1␣
secretion. As expected, IL-18 administration resulted in
rapid (within 4 hours after infection) IFN␥ and TNF␣
production, particularly sustained at the systemic level
(Figure 6). Subsequently, IFN␥ levels remained significantly higher (P ⬍ 0.01) in treated mice compared with
controls until day 4 after infection, while no significant
differences between experimental groups were found for
TNF␣ levels. Treatment with IL-18 alone induced only a
weak systemic production of IL-6, MIP-2, IFN␥, and
TNF␣, which was limited to the period of cytokine
administration (data not shown).
Figure 6. Effect of exogenous IL-18 administration on interferon-␥
(IFN␥) and tumor necrosis factor ␣ (TNF␣) in sera and joints of mice
infected with GBS (4 ⫻ 106 colony-forming units/mouse). IL-18 (0.05
␮g/mouse) or phosphate buffered saline plus 0.1% bovine serum
albumin (vehicle) was injected intraperitoneally for 4 days starting 1
hour before infection. Control uninfected mice (day 0) received vehicle
according to the same protocol. Blood samples and supernatants from
joint homogenates were collected at the indicated times after treatment (see Materials and Methods). Levels of IFN␥ and TNF␣ were
determined by enzyme-linked immunosorbent assay. Values are the
mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per
group were killed at each time point. ⴱ ⫽ P ⬍ 0.01; F ⫽ P ⬍ 0.05
versus vehicle-treated mice. See Figure 1 for other definitions.
IL-18 IN GBS ARTHRITIS
DISCUSSION
The present study assessed the role of IL-18 in
murine GBS arthritis by investigating the effect of its
blockade or supplementation in an experimental model
of GBS infection. The murine model of GBS infection
has been beneficial in elucidating bacterial and host
factors responsible for GBS arthritis (8–10,26,27). In
particular, a strong involvement of IL-6 and IL-1␤, but
not TNF␣, in the pathogenesis of GBS arthritis has been
established (10). Significantly high levels of IL-18 were
evident upon GBS infection, particularly in the joints.
Endogenous IL-18 plays an important role in GBS
arthritis, since neutralization resulted in a decrease in
the severity of arthritis. Similarly, improved outcome
was achieved after administration of neutralizing anti–
IL-18 antibodies or IL-18 binding protein in collageninduced arthritis (CIA) (28,29), and in a mouse model of
SCW arthritis (23).
In these experimental models, attenuation of the
disease was associated with a marked reduction of local
proinflammatory cytokines responsible for articular
damage. In the present study, an early marked decrease
in IL-6 and IL-1␤ production was observed upon IL-18
neutralization. Both cytokines are known to contribute
directly to articular damage. In fact, IL-1␤, together with
TNF␣, induces the release of tissue-damaging enzymes
from synovial cells and articular chondrocytes and activates osteoclasts (30,31). IL-6 participates together with
IL-1 in the catabolism of connective tissue components
at inflammation sites (32,33), and activates osteoclasts,
resulting in joint destruction (34). It is likely that a
decrease in IL-6 and IL-1␤ is one of the factors involved
in the amelioration of articular lesions upon anti–IL-18
treatment in our experimental model.
There appears to be a connection between inflammatory cell accumulation and joint destruction in
septic arthritis (35–37). In fact, invading macrophages
and granulocytes produce cytokines and proteolytic enzymes that contribute to cartilage and bone destruction
(38–40). In addition to proinflammatory cytokine production, in our experimental model the extent of articular inflammatory infiltrate and exudate and the number
of microorganisms that reach the joints also dictate the
severity of GBS arthritis (41). Selective recruitment of
activated leukocytes into a site of inflammation is mediated by many factors, including chemokines (42). These
low molecular weight proteins, divided into 2 distinct
groups, CXC and CC, based on the position of the first
2 cysteine amino acid residues (42), are produced by
leukocytes, endothelial cells, chondrocytes, osteoblasts,
2011
and other cell types in response to antigens, microbial
products, and endogenous cytokines, and are detected in
various inflammatory diseases (42,43). As expected,
MIP-1␣ and MIP-2 production was observed during
GBS infection, particularly at the joint level, and treatment with anti–IL-18 antibodies significantly impaired
chemokine production. IL-18 induces CXC chemokine
production from synovial fibroblasts in RA patients (22).
It is likely that, in our experimental model,
blockade of IL-18 acts directly on the production of
MIP-2 (a CXC chemokine), thus lowering polymorphonuclear cell influx into the joints. Since there is evidence
that polymorphonuclear cells from the synovial fluid of
RA patients produce high levels of MIP-1␣ (44), the
observed decrease in MIP-1␣ concentrations in the
joints of mice treated with anti–IL-18 may be due to a
small number of polymorphonuclear cells that are locally recruited. In fact, histopathologic analysis revealed
reduced infiltrate/exudate in the joints of mice treated
with anti–IL-18. It should be noted that GBS persist in
macrophages for up to 24–48 hours (45), and macrophages may carry GBS to different body sites, such as
the joints, thereby disseminating infection. Thus, it is
likely that the small number of GBS recovered from the
joints of mice treated with anti–IL-18 is due to the low
number of locally recruited inflammatory cells.
Administration of exogenous IL-18 to mice injected with GBS led to a worsening of articular lesions,
as observed in a model of CIA (46,47). In the latter case,
IL-18 likely mediated inflammatory arthritis not only by
enhancing Th1 activity, but also by directly inducing the
production of proinflammatory cytokines (IL-6, TNF␣,
IFN␥) from different cell types of the innate immune
system. In our experimental model, infection with a
suboptimal arthritogenic dose resulted in mild articular
lesions in a few animals. Low levels of proinflammatory
cytokines and chemokines, together with a low number
of GBS, were evident in the joints. Upon treatment with
recombinant IL-18, a dramatic increase in both cytokines and chemokines was observed.
IL-18 may exacerbate arthritis by multiple mechanisms. It might directly act on synovial macrophages
and articular chondrocytes. In fact, in vitro experiments
have demonstrated that IL-18 induces the release of
proinflammatory cytokines from macrophages and the
release of matrix metalloproteinases and glycosaminoglycans by articular cartilage, supporting a possible
direct contribution of IL-18 in joint destruction (17).
But, by inducing MIP-2 production, IL-18 might enhance the local influx of polymorphonuclear cells, which,
in turn, secrete MIP-1␣ with consequent recruitment of
2012
TISSI ET AL
mononuclear cells. As stated above, all these inflammatory cells not only contribute to articular damage by
cytokine and proteolytic enzyme production (35), but by
carrying microorganisms (45) to the different body sites,
also augment local bacterial load, thus amplifying the
inflammatory response. Finally, early (4 hours) IL-18–
mediated IFN␥ production may concur to worsen articular lesions, since we previously demonstrated that
administration of IFN␥ close to the time of infection
exerts detrimental effects on arthritis (26).
In conclusion, our results offer direct evidence
for a proinflammatory role of IL-18 in GBS arthritis.
The disease-modifying activity of IL-18 neutralization in
this experimental model of septic arthritis provides a
rationale for clinical studies.
12.
13.
14.
15.
16.
17.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. Eileen Mahoney
Zannetti for dedicated editorial assistance and Alessandro
Braganti, Carla Barabani, and Stefano Temperoni for their
excellent technical assistance in histologic processing and
animal care.
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