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Department of Rheumatology, Göteborg University, Göteborg, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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were significantly increased in
IL-1R-/- vs IL-1R+/+ mice 4 days after the
bacterial inoculation. In conclusion, IL-1R signaling plays a crucial
role in host protection during systemic S. aureus
infection as seen by the fatal outcome of S. aureus
sepsis and arthritis in IL-1R-deficient mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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40%
of adult cases and the common route of bacterial spreading is via the
bloodstream (1). A mouse model has been established in
which staphylococci are i.v. inoculated to trigger sepsis and septic
arthritis (2). The pathogenesis of murine S.
aureus sepsis and septic arthritis involves bacterial as well as
host factors. The expression of certain bacterial virulence factors,
e.g., capsular carbohydrates, adhesins, and regulatory genes, make the
bacteria more resistant to phagocytic functions, and the increased
bacterial survival results in an increased joint inflammation as well
as increased risk of death (3). The host response must
meet the demand of an effective clearance of bacteria.
One of the first steps for the innate immune system in recognizing
staphylococci is via the Toll-like receptors
(TLRs)3
(4), e.g., TLR2 and TLR4, recognizing peptidoglycan and
lipoteichoic acids, respectively (5). The TLRs share an
intracellular (IC) signal pathway with at least two cytokine receptors,
IL-1R (6) and IL-18R (7). The receptor signal
is conducted in sequence via a myeloid differentiation marker
(MyD88), IL-1R-associated kinase, TNFR-associated factor 6, and
TGF--activated kinase, leading to nuclear translocation of NF-
B
(4, 8). Following activation of NF-B several genes with
proinflammatory end products are transcribed. The importance of this IC
signal pathway for protection against staphylococcal infection was
recently shown using MyD88-deficient mice (9).
While IL-18 was previously shown to protect against joint inflammation
but aggravate septicemia during S. aureus arthritis
(10), the importance of IL-1
signaling during septic
arthritis is hitherto unknown. The results from a model of
noninfectious arthritis show that blockade of IL-1 or IL-1R delays
disease onset and ameliorates already established collagen-induced
arthritis (11). Furthermore, IL-1R antagonist-deficient
mice develop spontaneous arthritis resembling rheumatoid arthritis
(12), altogether indicating arthritogenic properties of
IL-1R signaling in inflammatory joint diseases. IL-1R is responsible
for signal transduction of IL-1 (13), while the type
2 receptor is lacking a cytoplasmic tail and is therefore not capable
of signal transmission but functions rather as a decoy receptor
(14).
Using IL-1R-deficient mice and their wild-type controls, we present evidence for the major importance of IL-1R signaling in S. aureus infection in controlling staphylococcal growth and subsequently protecting the host against septic death and septic arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Age- and sex-matched male or female 6- to 15-wk-old C57BL/6 mice, intact or defective with respect to the IL-1R type I gene, were used throughout the study. Procedure of IL-1RI gene disruption has been described in detail elsewhere (15). IL-1R-deficient mice (B6.129S7-Il1r1tm1Imx) as well as their wild-type controls (C57BL/6J) were obtained from The Jackson Laboratory (Bar Harbor, ME). At the time of experiments, IL-1RI-deficient mice had undergone five backcrosses to the C57BL/6 background. Mice were kept and bred at the animal facility of the Göteborg University (Göteborg, Sweden). They were kept under standard conditions of temperature and light and were fed standard laboratory chow and water ad libitum.
S. aureus strain LS-1 was originally isolated from a spontaneously arthritic New Zealand Black/White mouse (16). Before each experiment, bacteria were cultured on blood agar (5% human erythrocytes) for 24 h and then reincubated on blood agar for another 24 h. A bacterial solution was prepared in PBS at a concentration of 5 x 107 or 5 x 106 bacteria/ml. Two hundred microliters of the solution were injected into one of the tail veins on day 0. Viable counts were used to check the concentration of bacteria injected.
Evaluation of arthritis and septicemia
All mice were labeled and monitored individually. Limbs were inspected at regular intervals. Arthritis was defined as visible erythema and/or swelling of at least one joint. To evaluate the severity of arthritis we used a clinical scoring in which macroscopic inspection yields a score of 0–3 for each paw (0, normal; 1, mild swelling and/or erythema; 2, moderate swelling and erythema; 3, marked swelling and occasionally ankylosis), resulting in an arthritic score ranging from 0 to 12 for each mouse. Arthritic index was constructed by a summation of arthritic scores from all mice in the experimental group divided by the number of animals in each group.
The severity of septicemia was judged clinically modified from a previous description (17) where coat appearance (0–1), posture or movements (0–1), and temperature (0–1) yielded a severity score of maximally 3 per mouse. Temperature score was obtained when the mice were severely ill and cold as judged by hand.
Weight was regularly checked with 0.1-g accuracy.
Determination of staphylococcal load ex vivo
Bacterial samples from talocrural and radiocarpal joints were obtained using cotton sticks. The bacterial presence was defined if 15 or more CFU per joint were found. Both kidneys were removed aseptically, placed on ice, homogenized, and diluted in 10 ml PBS. Viable counts were done to examine bacterial concentration. To check the staphylococcal load in blood, mice were bled and viable counts were used to titrate the bacterial concentration.
Colonies were tested for catalase (using 18% hydrogen peroxide) and coagulase (using rabbit EDTA-plasma; BD Microbiology Systems, Sparks, MD) reactivity.
Hematological analyses
Mice were bled from the tail into heparinized tubes. Total leukocyte counts were determined in a hematocytometer (Sysmex KX-21; TOA Medical Electronics, Kobe, Japan). Blood smears were prepared and stained by the May-Grunewald-Giemsa method for differential counts.
In vitro analyses of cytokine production, phagocytosis, and IC killing
Spleen cells were passed through a nylon mesh, and erythrocytes were depleted by NH4Cl lysis. Splenocytes were cultured at 2 x 106 cells/ml in Iscove’s medium supplemented with 10% FCS, 5 x 10-5 M 2-ME, 2 mM L-glutamine, and 50 µg/ml gentamicin and incubated with 1 x 107/ml formalin-killed S. aureus LS-1 strain. Supernatants from cell cultures were collected after 24 and 48 h.
Intraperitoneal macrophages were extracted, adjusted to 2 x 106 cells/ml, and incubated in a 24-well plate (Nunc, Roskilde, Denmark) according to a previously detailed procedure (18, 19). Adherent macrophages were incubated with 500 µl of S. aureus at a concentration of 5 x 106 bacteria/ml for 50 min and subsequently washed three times in Iscove’s medium. The IC content of live S. aureus was then checked at three time intervals: 0, 4, and 24 h. The content at the 0-h interval is regarded as a measure of the phagocytosis, and the two later time intervals are regarded as a measure of the efficacy of the IC killing capacity contra-IC growth of bacteria (18).
Cytokine reagents and analyses
Recombinant murine (rm)IL-1 (R&D Systems, Minneapolis, MN)
was reconstituted in accordance with the manufacturer’s instructions,
then further diluted in PBS to desired concentration.
IL-6 was measured using the murine hybridoma cell line B13.29, subclone B9, selected for its IL-6 dependency (20). For the assay, B9 cells were harvested by centrifugation, and after one wash in IL-6-free medium they were seeded at 5 x 103 cells/well into microtiter plates (Nunc) in the presence of serum samples and grown for 68 h before adding [3H]thymidine to study proliferative responses to IL-6. After 4 h, the cultures were harvested onto filters and counted in a beta counter. Mouse IL-6 (Genzyme, Cambridge, MA) was used as a standard. The serum samples and standards were all set up in triplicate.
TNF and IL-1 were measured using Quantikine mouse immunoassays (R&D
Systems). IL-18 was measured by a quantitative test kit for mouse IL-18
(MBL, Nagoya, Japan). All the assays were performed in accordance with
the manufacturer’s instructions.
Statistics
Categorical data were analyzed using Fisher’s exact test, and
unpaired Student t test was used for statistical analyses of
weight changes. All other data were analyzed using the nonparametric
Mann-Whitney U test. A value of p 
0.05
was regarded as statistically significant. Results are presented as
mean ± SEM (n = number of mice).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-1R-deficient mice and their wild-type controls were i.v.
inoculated with 1 x 107 S.
aureus per mouse. The obvious clinical signs of septicemia in
IL-1R knockout mice were seen on the third day after inoculation of
bacteria, when a majority of the IL-1R-deficient mice displayed
septicemia as compared with none of the wild-type mice (Fig. 1
). As expected, the severity of
septicemia differed significantly between groups (1.2 ± 0.2 vs
0 ± 0; p < 0.0001) at the same time point. The
IL-1R-deficient mice started to die at day 4. In a first experiment,
three of 15 IL-1R-/- were dead at day 4 as
compared with none of the controls (0 of 17; NS). The survivors were
severely ill and were sacrificed at day 4 to obtain organ samples. The
weight was checked daily and, interestingly, 24 h after the
inoculation of 1 x 107 S. aureus
the wild-type mice displayed significantly more weight loss as compared
with the IL-1R-deficient animals. This finding was repeated in a second
experiment with the 1 x 107 dose (data not
shown). However, at days 2 and 3 after the inoculation of 1 x
107 S. aureus no differences in weight
loss were recorded between groups, though the general appearance as
measured by septicemia score was highly different at day 3.
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). With this lowered bacterial dose,
signs of septicemia were not evident in any of the groups at day 3.
However, at day 7 after the inoculation of S. aureus, the
IL-1R-deficient mice had lost significantly more weight as compared
with wild-type animals, and three of 12
IL-1R-/- were dead as compared with none of the
controls. The experiment was finished 2 days later at day 9. At this
time point, only 25% (3 of 12) of the IL-1R-/-
mice were alive, whereas no mortality was seen in the wild-type mice
(Fig. 3).
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At day 3 after inoculation of 1 x 107
S. aureus, 36% of the wild-type mice (n =
25) displayed septic arthritis as compared with 68% of IL-1R-deficient
mice (n = 25; p < 0.05). Similarly,
the severity of arthritis at day 3 was more pronounced in
IL-1R-deficient animals as compared with wild-type mice (Fig. 4). Due to severe septicemia in the
IL-1R-deficient group, experiments were finished at days 3–4. To be
able to follow the development of arthritis for a longer time, a
markedly reduced inoculum size (106 per mouse)
was given i.v. to 12 IL-1R-/- mice and 15
IL-1R+/+ control animals. Again, at day 3,
differences were seen between groups, IL-1R-/-
mice displaying somewhat increased frequency of septic arthritis as
compared with IL-1R+/+ mice (42 vs 7%;
p = 0.06). At day 7, 45% (four of nine) of the
IL-1R-deficient mice displayed arthritis as compared with 7% (1 of 15)
of wild-type mice (p < 0.05). The severity of
arthritis was significantly increased in
IL-1R-/- vs IL-1R+/+ mice
at days 3 and 7 also after inoculation of a lower dose of
staphylococci (Fig. 4). The experiment was finished at day 9 after
inoculation of 1 x 106 S. aureus
per mouse, a time point when one of three IL-1R-deficient mice
displayed arthritis as compared with 2 of 15 wild-type animals.
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High staphylococcal load in IL-1R-deficient mice as compared with wild-type mice
The load of S. aureus was checked in blood, kidneys,
and joints after i.v. inoculation of 1 x
107 S. aureus per mouse. While very
few staphylococci were found in blood 4 h after inoculation of
bacteria in both IL-1R-/- and
IL-1R+/+ groups, increasingly more
S. aureus were found in the blood of
IL-1R-/- as compared with wild-type mice 48 and
72 h after bacterial inoculation (Fig. 5A). Indeed, the increase of
S. aureus in the circulation of
IL-1R-/- mice was 100-fold between the time
points 4 and 72 h. Such an increase was absent in wild-type mice.
The bacterial load in kidneys was checked at day 4 after inoculation of
bacteria. IL-1R-deficient mice displayed a higher load (3.1 ±
1.1 x 108 CFU) as compared with wild-type
mice (4.9 ± 1.2 x 107 (NS)). However,
at day 4, three IL-1R-/- mice of 15 had died,
probably the most sick ones carrying the highest bacterial
load.
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B). Furthermore, five of the seven
IL-1R-/- mice with presence of S.
aureus displayed positive culture in two or more of the four
joints checked. The only wild-type mouse with presence of staphylococci
displayed one positive joint.
Because IL-1 is a potent mediator of neutrophil recruitment from
bone marrow into blood (21), we checked whether IL-1R
signaling influences the neutrophil counts in circulation in response
to S. aureus infection. While no difference in neutrophil
counts was recorded between IL-1R intact or IL-1 defect mice before
inoculation of staphylococci, blood of IL-1R-deficient mice displayed
significantly fewer neutrophils 4 and 24 h after the inoculation
of bacteria (Fig. 6).
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. However,
the addition of rmIL-1 at 1 ng/ml to the cultures significantly
increased phagocytosis by wild-type macrophages as compared with
IL-1R-deficient macrophages (3973 ± 673 S. aureus per
105 cells vs 2257 ± 110; p
< 0.05). Expression of IL-1R did not affect the IC killing
ability of macrophages. IL-1R signaling increases production of TNF and IL-6 in vitro and controls production of IL-18 in vivo in response to S. aureus
Next we analyzed the importance of IL-1R signaling for production
of cytokines in vivo in response to S. aureus arthritis and
in vitro to formalin-killed S. aureus. While TNF and IL-6
are produced in response to S. aureus, the role of IL-1R
signaling for production of these cytokines during staphylococcal
infection has not been previously analyzed. The serum concentration of
these two cytokines was assayed in IL-1R intact/defect mice 4 h,
24 h, and 4 days after inoculation of S. aureus. The
importance of IL-1R signaling for the in vivo production of IL-6 was
seen already at 4 h after bacterial inoculation. Later,
differences in IL-6 production due to functional IL-1R were diminished
and overcome (Table I). In contrast,
IL-1R deficiency did not affect TNF production during staphylococcal
infection (Table I). In vitro, IL-6 was severely decreased in absence
of IL-1R signaling (Table II). Spleen
cells, defect with respect to IL-1R, stimulated with formalin-killed
S. aureus cell walls produced significantly less TNF and
IL-6 as compared with spleen cells with an intact IL-1R (Table II). The
production of IL-1 was not influenced by the deficiency of IL-1R as
measured by serum concentration at 4 or 24 h after bacterial
inoculation or, in vitro, in response to S. aureus cell
walls. However, 4 days after inoculation of S. aureus the
serum concentration of IL-1 was significantly increased in
IL-1R-deficient mice as compared with wild-type mice (Table I).
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). IL-18 was not detected in vitro in response to
formalin-killed S. aureus. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signals into cells, develop severe septic arthritis and septicemia and
eventually die in response to i.v. inoculation with S.
aureus. One of the obvious reasons for the severe clinical outcome
of septic arthritis in IL-1R-deficient mice, as compared with IL-1R
intact controls, is the accumulation and proliferation of live
staphylococci in various tissues. Such a defective control of S.
aureus growth in vivo could be due to, e.g., a nonoptimal
recruitment of neutrophils and a diminished phagocytosis. Furthermore,
we show a major importance of the IL-1R signaling pathway in
controlling production of proinflammatory cytokines such as TNF, IL-6,
IL-1, and IL-18 in response to S. aureus.
Weight loss the first 24 h after inoculation of staphylococci
depends on IL-1R signaling. This is clearly shown in response to both
doses of staphylococci used in the present study. While IL-1 exerts
directly a down-regulatory effect on food intake (22),
indirect effects of IL-1R signaling are also possible. Indeed, TNF is
also involved in regulation of weight (23) and, as shown
in this study, its production is regulated by IL-1R expression. It is
well known that IL-6 production is triggered by IL-1 (24).
Both in vitro and in vivo, the IL-6 levels in response to S.
aureus were highly decreased in the absence of IL-1R signaling.
IL-6 induces fever (24) and was recently shown to be an
important mediator in controlling body weight (25).
At 48 h after inoculation of bacteria the numbers of circulating
staphylococci were clearly increased in IL-1R-deficient mice as
compared with wild-type animals. Recruitment of neutrophils as well as
uptake of bacteria by phagocytes are two potential ways by which IL-1R
signaling mediates bactericidal effects in vivo. In addition, it has
previously been shown that IL-1 at optimal concentration also
increases IC killing of S. aureus (26). Except
the direct effects of IL-1 on phagocytes, IL-1R signaling might be
important also indirectly by inducing production of IL-18. In this
respect, IL-18 was recently shown to be important for both recruitment
and activation of the respiratory burst of neutrophils
(27). Somewhat surprisingly, the neutrophil counts were
decreased in IL-1R-/- mice and only marginally
increased in IL-1R+/+ mice in response to
S. aureus infection. This is probably due to an early
neutrophil extravasation to the infected organs. Possibly, neutrophil
counts are increased in IL-1R-/- mice at days
3–4 when these animals display severe bacteremia (28).
While wild-type mice displayed only minor changes in number of
circulating bacteria at 4 h or 2 or 3 days postinoculation, there
was a 100-fold increase of staphylococci in the blood of
IL-1R-deficient animals 3 days after bacterial inoculation as compared
with the 4-h time point.
The severe bacteremia in IL-1R-deficient mice is present in parallel with obvious clinical signs of septicemia. The severe condition of IL-1R knockout mice might be caused directly via staphylococcal products or indirectly via host mediators, e.g., cytokines, highly induced by high staphylococcal load. IL-18 was previously shown to be a detrimental mediator in S. aureus infection concerning development of septicemia (10). The high serum levels of IL-18 found in IL-1R-deficient mice as compared with wild-type controls could thus contribute to the severe septicemia of IL-1R-/- mice.
The protective effects mediated by IL-1R signaling with respect to
development of septicemia and septic death are greater as compared with
protection mediated by TNF/lymphotoxin (29). Indeed,
we have previously demonstrated that, while being susceptible to
S. aureus infection, TNF/lymphotoxin
double-mutant mice or TNF single-deficient mice were protected
against septic arthritis. This is not the case for IL-1R-deficient
mice. The increased frequency and severity of septic arthritis in
IL-1R-/- mice, as compared with
IL-1R+/+ controls, is most probably explained by
staphylococci being densely expressed in the joints of the former
group. The high staphylococcal load in joints is probably leading
to recruitment of phagocytic cells and local (i.e., intra-articular)
induction of proinflammatory cytokines. In analogy to IL-1, IL-18 is
known to promote joint inflammation in noninfectious arthritis
(30, 31, 32). As shown by serum analyses, high amounts of
IL-18 are produced in IL-1R-/- mice only a few
days following bacterial inoculation. It is possible that the high
IL-18 production in IL-1R-deficient mice, compared with the controls,
increases the joint manifestations via recruitment of inflammatory
cells and their production of reactive oxygen intermediates.
The regulation of IL-18 production was recently reviewed
(8). Because its promoter region contains a NF-B
recognition sequence that is downstream of IL-1R signaling, it is not
very surprising that IL-18 production is influenced by an IL-1R
knockout situation. However, the impact of IL-1R signaling on IL-18
production has not been previously described. Evidently, IL-18
production is directly or indirectly dependent on a functional IL-1R,
as seen by higher serum levels of this cytokine 24 h after the
inoculation of bacteria in wild-type mice as compared with
IL-1R-deficient ones. The significantly increased IL-1/IL-18 serum
levels at 4 days after inoculation of staphylococci in
IL-1R-/- vs IL-1R+/+ mice
could simply be due to an increased staphylococcal load, in the former
mice, constituting a stronger stimulus for cytokine production.
Alternatively, IL-1R signaling may inhibit excessive production of
IL-1 and IL-18 during prolonged infection/inflammation via a
presently unknown pathway.
The results obtained in this study clearly show high susceptibility in severe infection outcome in response to S. aureus in an IL-1R-deficient condition. The results are corresponding to the susceptibility for S. aureus infection described for mice with a MyD88 deficiency and are more pronounced as compared with the susceptibility effects of a TLR2 or IL-18 deficiency. In conclusion, IL-1R signaling is protective in S. aureus arthritis against septicemia and subsequent septic death as well as against development of septic arthritis, due to critical importance of this molecule in controlling accumulation of S. aureus in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Olof H. Hultgren, Department of Rheumatology, Göteborg University, Guldhedsgatan 10A, S-413 46 Göteborg, Sweden. E-mail address: olof.hultgren{at}immuno.gu.se
3 Abbreviations used in this paper: TLR, Toll-like receptor; IC, intracellular; rm, recombinant murine.
Received for publication January 22, 2002. Accepted for publication March 21, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by interleukin-1 (IL-1) requires an IL-1 receptor-associated protein kinase activity. J. Biol. Chem. 270:16514. and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-/anti-CD4 therapy. J. Immunol. 165:7240.) from human leukocytes. Clin. Exp. Immunol. 66:303.[Medline]
double-mutant mice resist septic arthritis but display increased mortality in response to Staphylococcus aureus. J. Immunol. 161:5937.
-independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis. J. Immunol. 165:6553.This article has been cited by other articles:
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L. S. Miller, E. M. Pietras, L. H. Uricchio, K. Hirano, S. Rao, H. Lin, R. M. O'Connell, Y. Iwakura, A. L. Cheung, G. Cheng, et al. Inflammasome-Mediated Production of IL-1beta Is Required for Neutrophil Recruitment against Staphylococcus aureus In Vivo J. Immunol., November 15, 2007; 179(10): 6933 - 6942. [Abstract] [Full Text] [PDF]
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 C.-L. Ku, H. von Bernuth, C. Picard, S.-Y. Zhang, H.-H. Chang, K. Yang, M. Chrabieh, A. C. Issekutz, C. K. Cunningham, J. Gallin, et al. Selective predisposition to bacterial infections in IRAK-4 deficient children: IRAK-4 dependent TLRs are otherwise redundant in protective immunity J. Exp. Med., October 1, 2007; 204(10): 2407 - 2422. [Abstract] [Full Text] [PDF]
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 M. C. Garcia, I. Wernstedt, A. Berndtsson, M. Enge, M. Bell, O. Hultgren, M. Horn, B. Ahren, S. Enerback, C. Ohlsson, et al. Mature-onset obesity in interleukin-1 receptor I knockout mice. Diabetes, May 1, 2006; 55(5): 1205 - 1213. [Abstract] [Full Text] [PDF]
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