Kuschnaroff LM, Goebels J, Valckx D, Heremans H, Matthys P, Waer M. Increased Mortality and Impaired Clonal Deletion After Staphylococcal Enterotoxin B Injection in Old Mice: Relation to Cytokines and Nitric Oxide Production. Scand J Immunol 1997;46:469-478 In the present study peripheral T cell tolerance and the occurrence of shock were evaluated in young and old mice after injection of Staphylococcal enterotoxin B (SEB). In young mice SEB immunization leads to tolerance based on deletion and anergy of SEB-reactive Vb8 þ T cells. With aging, mice developed resistance to SEB-induced deletion of Vb8 þ T cells as well as a high sensitivity to toxic shock. Compared to young mice, older mice injected with SEB showed increased serum levels of interferon-g (IFN-g), interleukin-2 (IL-2) and IL-4. These results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), as splenic mRNA levels taken 2 h after SEB injection showed higher values of IL-2, IL-4, and IFN-g in old mice. In contrast, mRNA levels for FasL and tumour necrosis factor-a (TNF-a) were lower. No difference in IL-10 mRNA was found. Compared to young mice, old mice showed a high, but statistically not significantly different (P ¼ 0.20), production of nitric oxide (NO). Blocking of IFN-g with antibodies or reducing IFN-g by depletion of natural killer (NK) cells resulted, respectively, in a complete or partial protection against mortality in aged mice. Suppressing the NO production by the NO synthase inhibitor N-nitro-L-arginine methylester (L-NAME) increased the mortality in both young and old mice, and abrogated clonal deletion in the surviving young mice. In conclusion, in young mice NO production is a key factor in the protection against mortality and the development of clonal deletion after SEB injection. The higher mortality seen in older mice is mainly related to the elevated production of IFN-g and occurs despite a sufficient production of NO. The decreased clonal deletion of old mice may be related to their decreased expression of Fas ligand or TNF-a after SEB injection.

Kuschnaroff LM, Valckx D, Goebels J, Rutgeerts O, Heremans H, Froyen G, Waer M. Effect of Treatments with Cyclosporin A and Anti-Interferon-g Antibodies on the Mechanisms of Immune Tolerance in Staphylococcal Enterotoxin B Primed Mice. Scand J Immunol 1997;46:459-468 The authors were interested to investigate the effect of Cyclosporin A (CsA), known to block interleukin-2 (IL-2) production, or of anti-interferon-g antibodies (anti-IFN-g Abs) in a model of T cell tolerance induced by the injection of the superantigen Staphylococcal Enterotoxin B (SEB) in BALB/c mice. After SEB immunization, tolerance was mainly achieved through deletion and anergy of SEB-reactive Vb8 þ T cells. Association of CsA treatment with SEB led to a greater decrease of the percentage of Vb8 þ CD4 þ lymphocytes in the spleen and an abolition of clonal anergy. In contrast, treatment of SEB primed mice with anti-IFN-g Abs resulted in an increased percentage of Vb8 þ CD4 þ cells without affecting the induction of clonal anergy. The authors found that 1-2 h after SEB priming, splenic mRNA levels of IFN-g and IL-4 were decreased by either CsA and anti-IFN-g Abs, whereas FasL, Bcl-2, p. 53, and c-myc levels were not influenced by either treatment. However, SEB-induced IL-2 and IL-10 mRNA expression was suppressed only by CsA, whereas tumour necrosis factor-a (TNF-a) was decreased only by anti-IFN-g Abs. To investigate whether the effect of CsA on the tolerance mechanisms was related to suppression of IL-2, CsA was administered together with recombinant IL-2. Whereas anergy was not influenced, the decreased percentage of Vb8 þ CD4 þ cells seen in CsA-treated animals in the second week after SEB injection was partially corrected by the administration of IL-2. Experiments involving bromodeoxiuridine incorporation revealed that the latter effect of IL-2 was mainly due to a correction of the defective proliferation of Vb8 þ T cells after SEB injection in CsA-treated mice. These results suggest that the effect of CsA and anti-IFN-g Abs on tolerance mechanisms are in part explained by their action on cytokines.

After injection of SEB (staphylococcus enterotoxin B), normal adult mice, or thymectomized irradiated mice (TX irr.) reconstituted with lymphocytes taken from normal adult mice became specifically tolerant of SEB. At the same time the percentage of V {38 positive CD4 lymphocytes known to be responsive to SEB was almost 50% decreased, indicating that a high level of clonal deletion was realized. In contrast, mice with an exclusively old T cell compartment (old thymectomized mice, TX irr. mice reconstituted several months previously) became tolerant of SEB without deleting their V {38 + CD4 + cells, indicating that clonal anergy was the major mechanism in play in the induction of tolerance. Finally, TX irr. mice reconstituted with single positive thymocytes known to become recent thymic emigrants developed tolerance for SEB together with a high level (70%) of clonal deletion. Altogether these results indicated that the mechanism involved in peripheral tolerance depended on the age of the lymphocyte: very young lymphocytes underwent mainly clonal deletion whereas long lived lymphocytes underwent predominantly clonal anergy.