This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Diao, H.
Right arrow Articles by Kohanawa, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Diao, H.
Right arrow Articles by Kohanawa, M.

 Previous Article  |  Next Article 

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.

Endogenous Interleukin-6 Plays a Crucial Protective Role in Streptococcal Toxic Shock Syndrome via Suppression of Tumor Necrosis Factor Alpha Production

Hongyan Diao1* and Masashi Kohanawa2

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


arrow
ABSTRACT
 
During a Streptococcus pyogenes infection in interleukin-6 (IL-6)-deficient mice, there is elevation of serum tumor necrosis factor alpha (TNF-{alpha}) levels, muscular necrosis, and death compared with infection of C57BL/6 mice. Anti-TNF-{alpha} 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-{alpha} production in S. pyogenes infection.


arrow
TEXT
 
A streptococcal toxic shock syndrome (STSS) due to group A streptococcus (GAS) (Streptococcus pyogenes) was reported in the late 1980s (4). Since then the number of patients with this serious infectious disease has been increasing in North America, Europe, and other parts of the world (5, 11). STSS caused by GAS was characterized as a severe infectious disease which progresses to septic shock and multiple organ failure, with a frequent occurrence of necrotizing fasciitis. STSS is associated with a very high mortality rate and remains a serious concern in the clinical setting (7). It is thought that cytokine production is important in inducing STSS (10). It is recognized that interleukin-6 (IL-6) is detected in the sera of STSS patients (10). Therefore, it is possible that IL-6 plays a crucial role in STSS. IL-6 is known as an proinflammatory cytokine and induces many immunobiological reactions (2). However, it is reported that IL-6 plays a role in both the promotion (3) and the suppression (16) of immunological reactions. On the other hand, IL-6 induces production of acute-phase reactants, which are serum proteins synthesized by hepatocytes (17), and suppresses immune reactions (13). Therefore, the role of IL-6 in infectious diseases is not clear. To clarify the role of IL-6 in S. pyogenes infection, we observed the mortality rates and bacterial growth in the infected sites of IL-6–/– and IL-6+/+mice. Muscular necrosis is one of the peculiar symptoms of invasive streptococcal infection and STSS (7), so we inoculated S. pyogenes into the muscles of mice to induce muscular necrosis.

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-{alpha} (TNF-{alpha}) 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-{alpha} in S. pyogenes infection, we performed an experiment in which an anti-TNF-{alpha} monoclonal antibody (MAb) was administered. A hybridoma cell line secreting a MAb against mouse TNF-{alpha} (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).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 1. IL-6 deficiency renders mice susceptible to S. pyogenes infection. (a) Serum IL-6 levels in IL-6+/+ mice were measured by ELISA after S. pyogenes injection. (b) Survival rates of IL-6+/+ (•) and IL-6–/– ({circ}) 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.

We previously reported that TNF-{alpha} caused deterioration in cases of streptococcal infection (6). Therefore, we investigated serum TNF-{alpha} levels in infected mice. Serum TNF-{alpha} 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-{alpha} (Fig. 2b). For investigation of the role of endogenous TNF-{alpha} in IL-6–/– mice, we performed an anti-TNF-{alpha} MAb administration experiment. The mortality rate and muscular necrosis decreased with anti-TNF-{alpha} MAb treatment (Table 1). NRG, which was injected as a control for the MAb, had no effect on the mortality rate or muscular necrosis.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Kinetics of endogenous TNF-{alpha} production after injection with 5 x 108 CFU of S. pyogenes. (a) The titers of TNF-{alpha} in sera of IL-6–/– ({circ}) and IL-6+/+ (•) mice injected with bacteria were determined. (b) Effects of rmIL-6 on serum TNF-{alpha} levels in IL-6–/– mice after S. pyogenes injection. Each point 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 < 0.01).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of in vivo administration of an anti-TNF-{alpha} MAb on survival rates and muscular necrosis of IL-6–/– mice infected intramuscularly with 5 x 108 CFU/mouse

IL-6 is known as a proinflammatory cytokine in immune reactions. However, until now, it has remained unclear how endogenous IL-6 inhibits endogenous TNF-{alpha} production in streptococcal infection. We investigated whether endogenous IL-6 in streptococcal infection acts directly on immunocompetent cells or not. Titers of TNF-{alpha} 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-{alpha} levels in IL-6–/– mouse splenocytes stimulated with heat-killed S. pyogenes (Fig. 3b). In addition, TNF-{alpha} 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-{alpha} production in IL-6+/+ mouse splenocytes (data not shown). This indicates that IL-6 suppresses TNF-{alpha} production by immunocompetent cells.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 3. TNF-{alpha} production in the culture supernatant from splenocytes stimulated with heat-killed bacteria. (a) Splenocytes from IL-6–/– and IL-6+/+ mice were stimulated with heat-killed bacteria for 24 h. TNF-{alpha} production of the supernatants was measured by ELISA. (b) Effects of rmIL-6 on TNF-{alpha} production in supernatants from splenocytes stimulated with heat-killed bacteria. Splenocytes from IL-6–/– mice were incubated with 0.1 to 1,000 ng/ml rmIL-6. A single asterisk indicates a significant difference from the control (P < 0.05). A double asterisk indicates a significant difference from wild-type mice (P < 0.01).

It is recognized that IL-6 is a proinflammatory cytokine and augments inflammatory reactions (2). On the other hand, it has been reported that IL-6 inhibits inflammatory reactions in vivo (8, 12) and in vitro (18). Recently, it was also reported that IL-6 acts as an anti-inflammatory cytokine and suppresses TNF-{alpha} production in SOCS3 gene knockout mice (18). Our present study shows that TNF-{alpha} production by splenocytes stimulated with heat-killed S. pyogenes was inhibited by IL-6. This indicates that IL-6 regulates TNF-{alpha} 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.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 0600815, Japan. Phone and fax: 81-11-706-7542. E-mail:diao{at}igm.hokudai.ac.jp. Back

Editor: V. J. DiRita


arrow
REFERENCES
 
    1
  1. Abe, J., J. Forrester, T. Nakahara, J. A. Lafferty, B. L. Kotzin, and D. Y. Leung. 1991. Selective stimulation of human T cells with streptococcal erythrogenic toxins A and B. J. Immunol. 146:3747-3750.[Abstract]
    2. 2
  2. Alonzi, T., E. Fattori, D. Lazzaro, P. Costa, L. Probert, G. Kollias, F. De Benedetti, V. Poli, and G. Ciliberto. 1998. Interleukin 6 is required for the development of collagen-induced arthritis. J. Exp. Med. 187:461-468.[Abstract/Free Full Text]
    3. 3
  3. Cheung, W. C., and B. Van Ness. 2002. Distinct IL-6 signal transduction leads to growth arrest and death in B cells or growth promotion and cell survival in myeloma cells. Leukemia 16:1182-1188.[CrossRef][Medline]
    4. 4
  4. Cone, L. A., D. R. Woodard, P. M. Schlievert, and G. S. Tomory. 1987. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N. Engl. J. Med. 317:146-149.[Medline]
    5. 5
  5. Davies, H. D., A. McGeer, B. Schwartz, K. Green, D. Cann, A. E. Simor, D. E. Low, et al. 1996. Invasive group A streptococcal infections in Ontario, Canada. N. Engl. J. Med. 335:547-554.[Abstract/Free Full Text]
    6. 6
  6. Diao, H., M. Kohanawa, Yimin, H. Nakajima, Y. Sato, T. Minagawa, and A. Nakane. 2002. Lipopolysaccharide triggers invasive streptococcal disease in mice through a tumour necrosis factor-alpha-dependent mechanism. Immunology 105:344-349.[CrossRef][Medline]
    7. 7
  7. Eriksson, B. K., J. Andersson, S. E. Holm, and M. Norgren. 1998. Epidemiological and clinical aspects of invasive group A streptococcal infections and the streptococcal toxic shock syndrome. Clin. Infect. Dis. 27:1428-1436.[Medline]
    8. 8
  8. Mancuso, G., F. Tomasello, M. Migliardo, D. Delfino, J. Cochran, J. A. Cook, and G. Teti. 1994. Beneficial effects of interleukin-6 in neonatal mouse models of group B streptococcal disease. Infect. Immun. 62:4997-5002.[Abstract/Free Full Text]
    9. 9
  9. Nakane, A., T. Minagawa, and K. Kato. 1988. Endogenous tumor necrosis factor (cachectin) is essential to host resistance against Listeria monocytogenes infection. Infect. Immun. 56:2563-2569.[Abstract/Free Full Text]
    10. 10
  10. Norrby-Teglund, A., K. Pauksens, M. Norgren, and S. E. Holm. 1995. Correlation between serum TNF {alpha} and IL6 levels and severity of group A streptococcal infections. Scand. J. Infect. Dis. 27:125-130.[Medline]
    11. 11
  11. O'Brien, K. L., B. Beall, N. L. Barrett, P. R. Cieslak, A. Reingold, M. M. Farley, R. Danila, E. R. Zell, R. Facklam, B. Schwartz, and A. Schuchat. 2002. Epidemiology of invasive group A streptococcus disease in the United States, 1995-1999. Clin. Infect. Dis. 35:268-276.[CrossRef][Medline]
    12. 12
  12. Pallua, N., J. F. Low, and D. von Heimburg. 2003. Pathogenic role of interleukin-6 in the development of sepsis. Part II. Significance of anti-interleukin-6 and anti-soluble interleukin-6 receptor-alpha antibodies in a standardized murine contact burn model. Crit. Care Med. 31:1495-1501.[CrossRef][Medline]
    13. 13
  13. Paul, R., U. Koedel, F. Winkler, B. C. Kieseier, A. Fontana, M. Kopf, H. P. Hartung, and H. W. Pfister. 2003. Lack of IL-6 augments inflammatory response but decreases vascular permeability in bacterial meningitis. Brain 126:1873-1882.[Abstract/Free Full Text]
    14. 14
  14. Schindler, R., J. Mancilla, S. Endres, R. Ghorbani, S. C. Clark, and C. A. Dinarello. 1990. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75:40-47.[Abstract/Free Full Text]
    15. 15
  15. Talkington, D. F., B. Schwartz, C. M. Black, J. K. Todd, J. Elliott, R. F. Breiman, and R. R. Facklam. 1993. Association of phenotypic and genotypic characteristics of invasive Streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome. Infect. Immun. 61:3369-3374.[Abstract/Free Full Text]
    16. 16
  16. Taylor, S., S. Shacks, and Z. Qu. 2001. Effect of anti-IL-6 and anti-10 monoclonal antibodies on the suppression of the normal T lymphocyte mitogenic response by steady state sickle cell disease sera. Immunol. Investig. 30:209-219.[CrossRef][Medline]
    17. 17
  17. Wu, X., D. N. Herndon, and S. E. Wolf. 2003. Growth hormone down-regulation of interleukin-1ß and interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3. Shock 19:314-320.[CrossRef][Medline]
    18. 18
  18. Yasukawa, H., M. Ohishi, H. Mori, M. Murakami, T. Chinen, D. Aki, T. Hanada, K. Takeda, S. Akira, M. Hoshijima, T. Hirano, K. R. Chien, and A. Yoshimura. 2003. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 4:551-556.[CrossRef][Medline]

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.



This article has been cited by other articles:

  • Nowell, M. A., Williams, A. S., Carty, S. A., Scheller, J., Hayes, A. J., Jones, G. W., Richards, P. J., Slinn, S., Ernst, M., Jenkins, B. J., Topley, N., Rose-John, S., Jones, S. A. (2009). Therapeutic Targeting of IL-6 Trans Signaling Counteracts STAT3 Control of Experimental Inflammatory Arthritis. J. Immunol. 182: 613-622 [Abstract] [Full Text]  
  • Puel, A., Picard, C., Lorrot, M., Pons, C., Chrabieh, M., Lorenzo, L., Mamani-Matsuda, M., Jouanguy, E., Gendrel, D., Casanova, J.-L. (2008). Recurrent Staphylococcal Cellulitis and Subcutaneous Abscesses in a Child with Autoantibodies against IL-6. J. Immunol. 180: 647-654 [Abstract] [Full Text]  
  • Rose-John, S., Scheller, J., Elson, G., Jones, S. A. (2006). Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. J. Leukoc. Biol. 80: 227-236 [Abstract] [Full Text]  
  • Yimin, , Kohanawa, M. (2006). A Regulatory Effect of the Balance between TNF-{alpha} and IL-6 in the Granulomatous and Inflammatory Response to Rhodococcus aurantiacus Infection in Mice. J. Immunol. 177: 642-650 [Abstract] [Full Text]  
  • Cavaillon, J.-M., Annane, D. (2006). Invited review: Compartmentalization of the inflammatory response in sepsis and SIRS. Innate Immunity 12: 151-170 [Abstract]  
  • Jones, S. A. (2005). Directing Transition from Innate to Acquired Immunity: Defining a Role for IL-6. J. Immunol. 175: 3463-3468 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Diao, H.
Right arrow Articles by Kohanawa, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Diao, H.
Right arrow Articles by Kohanawa, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»