IL-2 Regulates SEB Induced Toxic Shock Syndrome in BALB/c Mice

BACKGROUND:Toxic Shock Syndrome (TSS) is characterized by fever, rash, hypotension, constitutional symptoms, and multi-organ involvement and is caused by Staphylococcus aureus enterotoxins such as Staphylococcal Enterotoxin B (SEB). SEB binds to the MHC-IIalpha chain and is recognized by the TCRbeta chain of the Vbeta8 TCR(+) T cells. The binding of SEB to Vbeta chain results in rapid activation of T cells and production of inflammatory cytokines, such as Interleukin-2 (IL-2), Interferon-gamma and Tumor Necrosis Factor-alpha which mediate TSS. Although IL2 was originally identified as the T cell growth factor and was proposed to contribute to T cell differentiation, its role in TSS remains unexplored. METHODOLOGY/PRINCIPAL FINDINGS:Mice were injected with D-Gal (25 mg/mouse). One hour after D-Galactosamine (D-Gal) injection each mouse was injected with SEB (20 microg/mouse. Mice were then observed for 72 hrs and death was recorded at different times. We tested Interleukin-12, IFNgamma, and IL-2 deficient mice (IL-2(-/-)), but only the IL-2 deficient mice were resistant to SEB induced toxic shock syndrome. More importantly reconstitution of IL-2 in IL-2 deficient mice restored the shock. Interestingly, SEB induced IL-2 production from T cells was dependent on p38MAPK activation in macrophages as inhibition of it in macrophages significantly inhibited IL-2 production from T cells. CONCLUSION:This study shows the importance of IL -2 in TSS which has not been previously explored and it also shows that regulating macrophages function can regulate T cells and TSS

Macrophage pretreatment with SB-203580 inhibits SEB induced IL-2 production in T cells and imparts resistance to SEB induced TSS in BALB/mice.

<p>(A) Peritoneal MΦs were preincubated with indicated doses of SB-203580 for 2 hrs and cells were washed and then T cells were added to these macrophage cultures these cultures were then stimulated with SEB (2 µg/ml) for 72 hours and proliferation was measured by adding thymidine at 60 hours for 12 hours. (B) ELISA for IL-2 was done from the supernatant collected at sixty hours from above set. (C) BALB/c mice were injected intraperitoneally (IP) with SB-203580 (25 µg/mouse) and vehicle control (DMSO) (data not shown) one hour before D-Gal injection, then the SEB (20 µg/ml) was injected after one hour after D-Gal injection in the footpad. Mice were bled and ELISA was done to assess serum levels of TNFα (D). Mice were then observed for mortality.</p

SEB induces p38MAPK phosphorylation in peritoneal Macrophages.

<p>(A). PMΦs were stimulated for indicated times and cell lysates were analyzed by western blotting using an anti-phospho p38MAPK specific antibody. Equal loading was confirmed by reprobing the stripped membrane with a p38MAPK specific antibody. (B) Macrophages were stimulated with indicated doses of SEB and cell lysates were analyzed by western blotting using an anti-phospho p38MAPK specific antibody. Equal loading was confirmed by reprobing the stripped membrane with a p38MAPK specific antibody. (C) Macrophages were preincubated with indicated doses of SB-203580 for 2 hrs and stimulated with 2 µg/ml of SEB for 30 minutes, and then the lysates were analyzed by western blotting against phospho p38MAPK. Equal loading was confirmed by reprobing the stripped membrane with a p38MAPK specific antibody.</p

Reconstitution of IL-2 induces shock.

<p>(a) Splenocytes were prepared from IL-2^−/− and WT mice and stimulated with SEB (2 µg/ml) or SEB + rIL-2 (5 ng/ml) and TNFα levels were measured by ELISA. (b) IL-2^−/− mice were injected with SEB (20 µg/mouse), and rIL-2 (500 ng/mouse).Mice were then observed for mortality at indicated times. (c) Above mice were bled one hour after SEB injection and ELISA was done to assess circulating levels of TNF-α in the serum of these mice.</p

Low level of TNFα in IL-2^−/− accounts for the resistance against SEB induced TSS.

<p>(A) IL-2^−/− and BALB/c mice were bled after one hour after SEB (20 µg/mouse) injection and circulating levels of TNFα were assessed by ELISA. (B) IL-2^−/− and BALB/c mice were injected with SEB (20 µg/mouse) in the footpad and spleens were collected after three hours from these mice. Splenocytes were prepared and cultured for 72 hours without further stimulation and proliferation was measured. (C) Spleens were collected from IL-2^−/− and BALB/c mice, splenocytes were prepared and FACS was done for CD4 vs. Vβ8 and CD8 vs. Vβ8.</p

IL-2 knockout mice are resistant to SEB induced TSS.

<p>(A) BALB/c and IFNγ^−/− mice were injected IP with D-Gal (25 mg/mouse) and after one hour mice were injected with SEB (20 µg/mouse) in the footpad and death was recorded at indicated times. (B) IL-12^−/− and BALB/c mice were injected with D-Gal (25 mg/mouse), after one hour mice were injected with SEB (20 µg/ml) and mortality was recorded at indicated times (C) IL-2^−/− mice and BALB/c mice were injected in the footpad with SEB (20 µg/mouse) one hour after D-Gal sensitization and mortality was recorded at indicated times.</p