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Infection and Immunity, June 2005, p. 3745-3748, Vol. 73, No. 6
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.6.3745-3748.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Division of Molecular Immunology, Institute for Genetic Medicine,1 Department of Microbiology, School of Medicine, Hokkaido University, Sapporo, Japan2
Received 16 November 2004/ Accepted 1 February 2005
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ABSTRACT |
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) levels, muscular necrosis, and death compared with infection of C57BL/6 mice. Anti-TNF- monoclonal antibody treatment decreased mortality and muscular necrosis in the infected IL-6-deficient mice. These results suggest that IL-6 plays a crucial protective role via suppression of TNF- production in S. pyogenes infection. |
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Female C57BL/6 (IL-6+/+) and IL-6-deficient (IL-6–/–) mice, aged 8 weeks, were used. IL-6+/+ mice were purchased from SLC (Hamamatsu, Shizuoka, Japan), and IL-6–/– mice on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, Maine). Mice were infected in the right thigh muscle with 0.1 ml of a solution containing 5 x 108 CFU of viable S. pyogenes cells in phosphate-buffered saline. The S. pyogenes strain used was a clinical isolate from a patient with pharyngitis in Hokkaido University Hospital, Sapporo, Japan. The bacteria were type M12/T12. M and T typing was kindly performed by A. Tamaru at the Osaka Prefectural Institution of Public Health, Osaka, Japan. The strain was stored as stock cultures at –80°C in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.); when needed, cultures from frozen stocks were grown in 200 ml of Todd-Hewitt broth and incubated for 10 h at 37°C, and the bacteria were harvested at the start of the stationary phase. The right thigh muscles of infected animals were excised at 24 h postinfection (p.i.), cut up with scissors, and homogenized in a tissue homogenizer (type NS-310E, PHYSOOTRON) (6). Serial 10-fold dilutions of the organ suspensions were placed on blood agar plates and incubated at 37°C for 24 h; then the number of colonies was counted.
Levels of tumor necrosis factor- (TNF-) and IL-6 were determined by use of sandwich enzyme-linked immunosorbent assays (ELISA) as described previously (9). For investigation of the role of endogenous TNF- in S. pyogenes infection, we performed an experiment in which an anti-TNF- monoclonal antibody (MAb) was administered. A hybridoma cell line secreting a MAb against mouse TNF- (MP6-XT22.11; rat immunoglobulin G1) (9) was used. Mice were given a single intravenous injection of the MAb 2 h before infection. Normal rat globulin (NRG) was injected as a control for the MAb. NRG was prepared as described previously (6). IL-6–/– mice were given a single intravenous injection of 1 µg of recombinant mouse IL-6 (rmIL-6) (Genzyme) simultaneously with infection. Phosphate-buffered saline was injected as a control for rmIL-6.
First, we observed the kinetics of endogenous IL-6 when mice were inoculated intramuscularly with S. pyogenes. IL-6 could be detected in serum from 2 h until 48 h after infection in IL-6+/+ mice infected with S. pyogenes. Peak serum IL-6 titers were observed at 8 h p.i. (Fig. 1a). IL-6 was not detected in the sera of IL-6–/– mice infected with S. pyogenes (data not shown). Because IL-6 was produced in infected IL-6+/+ mice, IL-6 might be involved in S. pyogenes infection. To clarify the role of IL-6, we compared IL-6–/– and IL-6+/+ mice in this study. All of the IL-6+/+ mice infected with 5 x 108 CFU of viable S. pyogenes survived until day 10 p.i. (Fig. 1b). On the other hand, the mortality rate of infected IL-6–/– mice was 70% on day 10 p.i. (Fig. 1b). Muscular necrosis was observed in 20% of infected IL-6–/– mice but not in infected IL-6+/+ mice (data not shown). Our previous study showed that the numbers of S. pyogenes bacteria in the thigh muscle peaked at 24 h after infection (6). Therefore, the bacterial numbers in the muscle tissues of infected IL-6–/– and IL-6+/+ mice were determined at 24 h after infection in this study (Fig. 1c). The numbers of bacteria in infected muscles of IL-6–/– mice were significantly larger than those in IL-6+/+ mice at 24 h p.i (Fig. 1c). To investigate pathological changes in the muscles of the infected mice, we performed a histological study. Infected IL-6–/– mice showed necrosis of muscles and less infiltration of leukocytes; infected IL-6+/+ mice showed only infiltration of leukocytes into the muscles (Fig. 1d). These data indicate that IL-6 deficiency rendered mice susceptible to intramuscular infection of S. pyogenes and to muscular necrosis and death. These appearances of infected IL-6–/– mice are similar to symptoms of human STSS. To further investigate the role of IL-6 produced in muscular infection with S. pyogenes, we investigated whether administration of rmIL-6 to the IL-6–/– mice infected with S. pyogenes improved the outcome of streptococcal diseases or not. Administration of 1 µg of rmIL-6 completely inhibited death and muscular necrosis (data not shown).
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caused deterioration in cases of streptococcal infection (6). Therefore, we investigated serum TNF- levels in infected mice. Serum TNF- titers in infected IL-6–/– mice were significantly higher than those in infected IL-6+/+ mice (Fig. 2a). Administration of 1 µg of rmIL-6 to IL-6–/– mice inhibited elevation of serum TNF- (Fig. 2b). For investigation of the role of endogenous TNF- in IL-6–/– mice, we performed an anti-TNF- MAb administration experiment. The mortality rate and muscular necrosis decreased with anti-TNF- MAb treatment (Table 1). NRG, which was injected as a control for the MAb, had no effect on the mortality rate or muscular necrosis.
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production in streptococcal infection. We investigated whether endogenous IL-6 in streptococcal infection acts directly on immunocompetent cells or not. Titers of TNF- produced by IL-6–/– mouse splenocytes stimulated with heat-killed S. pyogenes were significantly higher than those from IL-6+/+ mouse splenocytes (Fig. 3a). Splenocytes from IL-6–/– mice were incubated with varying doses of rmIL-6 (0.1 ng, 1 ng, 10 ng, 100 ng, and 1,000 ng). The low-dose rmIL-6 (0.1 ng) treatment significantly reduced TNF- levels in IL-6–/– mouse splenocytes stimulated with heat-killed S. pyogenes (Fig. 3b). In addition, TNF- levels tended to be lower with increasing doses of rmIL-6 in splenocytes from IL-6–/– mice (Fig. 3b). Moreover, rmIL-6 also inhibited TNF- production in IL-6+/+ mouse splenocytes (data not shown). This indicates that IL-6 suppresses TNF- production by immunocompetent cells.
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production in SOCS3 gene knockout mice (18). Our present study shows that TNF- production by splenocytes stimulated with heat-killed S. pyogenes was inhibited by IL-6. This indicates that IL-6 regulates TNF- production in immunocompetent cells. Streptococcal infection is sometimes curable by antibiotic therapy. However, S. pyogenes also causes severe streptococcal disease which cannot be controlled by antibiotics. It has been reported that a superantigen is important in inducing STSS (15). In STSS, bacterial cells proliferate dramatically in the host. This indicates that cytokines, which are produced by the stimulation of the superantigen, induce bacterial growth in the host. Therefore, we speculated that an inhibitory mechanism of cytokine production exists in nonsevere streptococcal infection. It is possible that STSS is triggered by a decrease in IL-6 production and that administration of recombinant IL-6 would be effective in the treatment of severe streptococcal infection.
Our present study shows that IL-6 plays an important role in the negative feedback of the immune reaction in the early phase of infectious disease. It has been reported that IL-6 is necessary to regulate excessive cytokine production in some infectious diseases (1, 8, 14). We expect that administration of IL-6 will be effective in treatment against the cytokine storm, which occurs in severe infection and trauma.
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FOOTNOTES |
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) mice after S. pyogenes injection. (c) Numbers of S. pyogenes bacteria at 24 h after infection. Mice were infected intramuscularly in the right hind thigh with 5 x 108 CFU of S. pyogenes. Numbers of viable S. pyogenes cells in the muscles of IL-6+/+ and IL-6–/– mice were determined. Each data point in panels a through c represents the mean ± standard deviation for a group of 10 mice. A double asterisk indicates a significant difference from values for wild-type mice (P < 0.001). (d) Representative muscle histology of IL-6+/+ (left panel) and IL-6–/– (right panel) mice. Muscles were obtained at 48 h after infection, and muscle sections were stained with hematoxylin and eosin. Original magnification, x200.
